Dissecting aortitis in a goat associated with Pasteurella multocida and Staphylococcus spp infection.

Reports of primary cardiovascular disease in goats are rare and most commonly include ventricular septal defect, valvular endocarditis, traumatic pericarditis, ionophore poisoning and nutritional cardiomyopathies. We now report the pathological findings in a 67 kg, 6-year-old, adult female Boer goat that presented with neurological signs (ie, head pressing, unsteadiness and paddling) and hyperthermia 2 days prior to death. Lack of therapeutic response to meloxicam and penicillin‒streptomycin and poor prognosis led to euthanasia of the animal. At necropsy, the main findings included severe aortic dissection with luminal thrombosis and stenosis, and pulmonary congestion and oedema. Histological examination of the aorta revealed severe chronic granulomatous and fibrosing dissecting aortitis with mineralization. Bacterial culture of the affected aortic segment resulted in isolation of a profuse growth of Pasteurella multocida and a moderate growth of Staphylococcus spp. Histopathological findings in the central nervous system were consistent with neurolisteriosis.

Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

Fatal cerebrovascular accident in a captive red panda (Ailurus fulgens fulgens) with concurrent amdoparvovirus infection.

We report the pathological and molecular findings in an adult male Himalayan red panda (Ailurus fulgens fulgens) whose death was attributed to parenchymal brain haemorrhage (PBH) of the thalamus. Post-mortem examination revealed severe, acute PBH and intraventricular haemorrhage with major involvement of the thalamus, as well as scattered chronic microinfarctions. Vascular disease in the brain and other organs was suggestive of systemic hypertension. Histological lesions included arteriolar hyalinosis and varying degrees of arteriosclerosis, arterial tunica media hypertrophy and hyperplasia and infiltration of arterial walls by lipid-laden macrophages. Other relevant findings included marked myocardial fibrosis, lymphoplasmacytic tubulointerstitial nephritis, lymphoplasmacytic meningoencephalitis and chronic mitral valve degeneration. The changes in the cerebral vasculature were consistent with hypertensive encephalopathy and a cerebrovascular accident, specifically PBH, which has not been previously reported in this species. Additionally, polymerase chain reaction analysis for red panda amdoparvovirus (RPAV) was positive in the brain and kidneys. Preceded by hypertensive vascular changes and brain microinfarctions, sudden death in this animal likely resulted from fatal PBH with intraventricular haemorrhage. The clinicopathological role of RPAV infection is unknown in this case, although its contribution to the chronic renal disease is considered possible in the context of our current understanding of RPAV-associated pathology.

Investigating a Prognostic Factor for Canine Hepatocellular Carcinoma: Analysis of Different Histological Grading Systems and the Role of PIVKA-II.

BACKGROUND: Hepatocellular carcinoma (HCC) in dogs is uncommon and often associated with a good prognosis, although some cases prove to be aggressive. In human oncology HCC is often very aggressive and diagnostic methods and prognostic factors are widely used to predict its biological behaviour. These include the expression of PIVKA-II. METHODS: in order to identify a prognostic factor for canine HCC, we applied different methods of histological grading and investigated PIVKA-II expression in 22 HCC of dogs treated surgically and followed clinically for at least 2 years. RESULTS: Nineteen patients analysed have passed the observation period without tumour recurrence, while 3 died following the development of metastases. PIVKA-II was positive in 15/22 cases without correlation with prognosis or tumoural grading even if a trend of PIVKA-II negativity in low WHO grades as well as increased number of PIVKA-II positive cases in higher WHO grades weres observed. CONCLUSIONS: This work showed that, PIVKA-II cannot be considered either as a marker of malignancy or as a prognostic marker for canine HCC. The poor prognosis depends usually on the clinical presentation. Thus prognostic parameters in canine HCC able to predict its aggressive behaviour through histological examination are still missing. The most promising method, limited to our study, seems to be the WHO histological grading.

The mitotic regulator polo-like kinase 1 as a potential therapeutic target for c-Myc-overexpressing canine osteosarcomas.

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo-like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c-Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self-renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c-Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c-Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c-Myc and PLK1-overexpressing cOSA cell line (D17). PLK1 and c-Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c-Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT-qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5-15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G2 /M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c-Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c-Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c-Myc.

Genomic and Transcriptomic Characterization of Canine Osteosarcoma Cell Lines: A Valuable Resource in Translational Medicine.

Osteosarcoma (OSA) represents the most common primary bone tumor in dogs and is characterized by a highly aggressive behavior. Cell lines represent one of the most suitable and reproducible pre-clinical models, and therefore the knowledge of their molecular landscape is mandatory to investigate oncogenic mechanisms and drug response. The present study aims at determining variants, putative driver genes, and gene expression aberrations by integrating whole-exome and RNA sequencing. For this purpose, eight canine OSA cell lines and one matched pair of primary tumor and normal tissue were analyzed. Overall, cell lines revealed a mean tumor mutational burden of 9.6 mutations/Mb (range 3.9-16.8). Several known oncogenes and tumor suppressor genes, such as ALK, MYC, and MET, were prioritized as having a likely role in canine OSA. Mutations in eight genes, previously described as human OSA drivers and including TP53, PTCH1, MED12, and PI3KCA, were retrieved in our cell lines. When variants were cross-referenced with human OSA driver mutations, the E273K mutation of TP53 was identified in the Wall cell line and tumor sample. The transcriptome profiling detected two possible p53 inactivation mechanisms in the Wall cell line on the one hand, and in D17 and D22 on the other. Moreover, MET overexpression, potentially leading to MAPK/ERK pathway activation, was observed in D17 and D22 cell lines. In conclusion, our data provide the molecular characterization of a large number of canine OSA cell lines, allowing future investigations on potential therapeutic targets and associated biomarkers. Notably, the Wall cell line represents a valuable model to empower prospective in vitro studies both in human and in dogs, since the TP53 driver mutation was maintained during cell line establishment and was widely reported as a mutation hotspot in several human cancers.

Doxorubicin treatment modulates chemoresistance and affects the cell cycle in two canine mammary tumour cell lines.

BACKGROUND: Doxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC). RESULTS: Both cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis. CONCLUSIONS: DOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.

Evaluation of the neoplastic infiltration of the skin overlying canine subcutaneous soft tissue sarcomas: An explorative study.

Studies regarding the neoplastic infiltration of the skin overlying canine subcutaneous soft tissue sarcoma (sSTS) are lacking. In case of the absence of tumor infiltration, there would be the possibility of leaving this unaffected skin in place, thus simplifying surgery. The aim of the study was to investigate whether the skin overlying sSTSs is infiltrated by neoplastic cells. Dogs with sSTSs treated surgically were prospectively enrolled. After excision, the skin was dissected from the tumor along the natural surgical plane of cleavage and histologically evaluated. Twenty-nine dogs with an sSTS were included (22 grade I, 6 grade II, and 1 grade III). The sSTS-overlying skin was not tumor-infiltrated in 14/29 cases (48.3%). A higher frequency of infiltration was observed in higher grade sSTSs (grades II and III, 100%; P = .006); nevertheless, 8/22 grade I sSTSs (36%) also showed cutaneous infiltration. This infiltration involved the dermis of the skin directly in contact with the tumor (multifocal in 11 and diffuse in four cases). Although the cutaneous tumor infiltration is less frequent in grade I sSTSs and a wide excision may still be the safest treatment for any sSTS for a greater possibility of local control, this study opens the possibility to a less aggressive cutaneous excision, but still with a local curative intent, as only the skin directly in contact with the sSTS has been proven to be tumor-infiltrated. Additional studies are warranted to confirm that excision of only this skin may guarantee a complete local control, especially in lower-grade sSTSs.

CD3-CD20-positive nodal lymphoma with cross-lineage rearrangement in a dog.

A 7-y-old mixed-breed male dog was presented with a history of generalized lymphadenopathy. Fine-needle aspirates of the enlarged peripheral lymph nodes were suggestive of lymphoma. Histologic examination of a retromandibular lymph node was suggestive of high-grade, medium large-cell lymphoma. Immunohistochemistry revealed concurrent expression of CD3 and CD20. The co-localization of the 2 antigens was confirmed by immunofluorescence. PCR for antigen receptor gene rearrangements (PARR) detected clonal rearrangements for both T-cell receptor gamma and B-cell receptor. The final diagnosis was CD3-CD20-positive anaplastic lymphoma with cross-lineage rearrangement.

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models.

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR-2, PDGFRs and c-Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c-Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.

PDGFR-α, PDGFR-ß, VEGFR-2 and CD117 expression in canine mammary tumours and evaluation of the in vitro effects of toceranib phosphate in neoplastic mammary cell lines.

Canine mammary tumours (CMTs) are one of the most common malignancies in bitches. Platelet-derived growth factor receptor (PDGFR) α and ß, vascular endothelial growth factor receptor-2 (VEGFR-2) and CD117 are tyrosine kinase receptors involved in several tumours and represent suitable targets for specific therapy with toceranib phosphate. The purpose of this study was to evaluate the expression of these receptors in the pathogenesis and progression of CMTs. PDGFRα, PDGFRß, VEGFR-2 and CD117 were expressed in 46/83 (55.4 per cent), 33/83 (39.8 per cent), 46/83 (55.4 per cent) and 32/83 (38.5 per cent) of CMTs, respectively. Immunohistochemical results showed a statistically significant loss of PDGFRα and PDGFRß expression in simple carcinomas compared with complex/mixed carcinomas. Protein expression by western blot revealed specific bands corresponding to PDGFRα and VEGFR-2 in 3/7 and in 1/7 cell lines. Moreover, in vitro treatment showed that toceranib phosphate weakly reduced cell proliferation in one canine mammary cell line. Before considering TKR inhibitors for possible therapeutic approaches, actually further studies are necessary to evaluate the effect of these drugs on CMTs in vivo.