Best Practice Approach for Assessment of Microchip-associated Tumors in Preclinical Safety Studies: Position of the Registry of Industrial Toxicology Animal-data (RITA).

Microchip (passive radio-frequency identification device) implantation is a common and widely employed means of animal identification in laboratory animal facilities. However, these devices have been associated with tumors of the skin and subcutis in rodents. While microchip-associated tumors are rare, they pose a challenge for accurate diagnosis and documentation in preclinical toxicity studies. Documentation of these tumors should differentiate microchip-associated lesions with spontaneously occurring or test article-induced tumors. Standardizing criteria for microchip-associated lesions will aid the diagnostic process and allow for preclinical regulatory standardization. To this end, the Registry of Industrial Toxicology Animal-data have developed clear recommendations for diagnosis and documentation of microchip-associated lesions.

Reduced angiogenic gene expression in morbillivirus-triggered oncolysis in a translational model for histiocytic sarcoma.

Histiocytic sarcoma represents a rare malignant tumour with a short survival time, indicating the need of novel treatment strategies including oncolytic virotherapy. The underlying molecular mechanisms of viral oncolysis are largely unknown. As cancer in companion animals shares striking similarities with human counterparts, we chose a permanent canine histiocytic sarcoma cell line (DH82 cells) to identify global transcriptome changes following infection with canine distemper virus (CDV), a paramyxovirus closely related to human measles virus. Microarray analysis identified 3054 differentially expressed probe sets (DEPs), encoding for 892 up- and 869 down-regulated unique canine genes, respectively, in DH82 cells persistently infected with the vaccine strain Onderstepoort of CDV (DH82-Ond-pi), compared to non-infected DH82 cells. Up-regulated genes were predominantly related to immune processes, as demonstrated by functional enrichment analysis. Moreover, there was substantial enrichment of genes characteristic for classically activated M1 and alternatively activated M2 macrophages in DH82-Ond-pi; however, significant polarization into either of both categories was lacking. 'Angiogenesis' was the dominant enriched functional term for the down-regulated genes, highlighting decreased blood vessel generation as a potential mechanism of paramyxovirus-induced oncolysis in DH82 cells. The anti-angiogenic effect of infection was verified by immunohistochemistry, which revealed a lower blood vessel density in an in vivo mouse model, xenotransplanted with DH82-Ond-pi, compared to mice transplanted with non-infected DH82 cells. Reduction in angiogenesis appears to be an important oncolytic mechanism of CDV in DH82 cells, suggesting that similar mechanisms might account for human histiocytic sarcoma and maybe other tumours in conjunction with measles virus.

Matrix metalloproteinases expression in spontaneous canine histiocytic sarcomas and its xenograft model.

Canine histiocytic sarcoma (HS) represents a malignant neoplastic disorder often with a rapid and progressive clinical course. A better understanding of the interaction between tumor cells and the local microenvironment may provide new insights into mechanisms of tumor growth and metastasis. The influence of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) on tumor angiogenesis, invasion and metastasis has been detailed in previous studies. In addition, inflammatory cells infiltrating neoplasms especially tumor associated macrophages (TAM) may contribute significantly to tumor progression. Due to the high variability of spontaneously occurring canine HS, standardized models are highly required to investigate tumor progression and interaction with its microenvironment. Therefore, the present study comparatively characterized the intratumoral macrophage infiltration as well as the expression of MMP-2, MMP-9, MMP-14 and TIMP-1 in spontaneous canine HS and its murine model. In spontaneous canine HS, scattered MAC 387-positive macrophages were randomly found in tumor center and periphery, whereas tumor cells were negative for this marker. Interestingly, quantitative analysis revealed that MMPs and TIMP-1 were mainly expressed at the invasive front while tumor centers exhibited significantly reduced immunoreactivity. Similar findings were obtained in xenotransplanted HS. Interestingly, murine tumor associated macrophages (TAM), characterized by Mac3 expression (CD107b/LAMP2), which was not present in xenotransplanted histiocytic sarcoma cells, strongly express MMPs and TIMP-1. In addition, MMPs are known to regulate angiogenesis and a positive correlation between MMP-14 expression and microvessel density was demonstrated in xenotransplanted histiocytic sarcomas. Summarized similar findings with respect to MMP and TIMP distribution and the role of macrophages in spontaneously-occurring and xenotransplanted HS indicate the high suitability of this murine model to further investigate HS under standardized conditions. Moreover results indicate that MMP expression contributes to tumor progression and invasion and TAMs seem to be major players in the interaction between neoplastic cells, the microenvironment and vessel formation indicating that therapeutic approaches modulating TAM associated molecules might represent promising future treatment options.

Viral oncolysis - can insights from measles be transferred to canine distemper virus?

Neoplastic diseases represent one of the most common causes of death among humans and animals. Currently available and applied therapeutic options often remain insufficient and unsatisfactory, therefore new and innovative strategies and approaches are highly needed. Periodically, oncolytic viruses have been in the center of interest since the first anecdotal description of their potential usefulness as an anti-tumor treatment concept. Though first reports referred to an incidental measles virus infection causing tumor regression in a patient suffering from lymphoma several decades ago, no final treatment concept has been developed since then. However, numerous viruses, such as herpes-, adeno- and paramyxoviruses, have been investigated, characterized, and modified with the aim to generate a new anti-cancer treatment option. Among the different viruses, measles virus still represents a highly interesting candidate for such an approach. Numerous different tumors of humans including malignant lymphoma, lung and colorectal adenocarcinoma, mesothelioma, and ovarian cancer, have been studied in vitro and in vivo as potential targets. Moreover, several concepts using different virus preparations are now in clinical trials in humans and may proceed to a new treatment option. Surprisingly, only few studies have investigated viral oncolysis in veterinary medicine. The close relationship between measles virus (MV) and canine distemper virus (CDV), both are morbilliviruses, and the fact that numerous tumors in dogs exhibit similarities to their human counterpart, indicates that both the virus and species dog represent a highly interesting translational model for future research in viral oncolysis. Several recent studies support such an assumption. It is therefore the aim of the present communication to outline the mechanisms of morbillivirus-mediated oncolysis and to stimulate further research in this potentially expanding field of viral oncolysis in a highly suitable translational animal model for the benefit of humans and dogs.

Novel canine bocavirus strain associated with severe enteritis in a dog litter.

Bocaviruses are small non-enveloped viruses with a linear ssDNA genome, that belong to the genus Bocaparvovirus of the subfamiliy Parvovirinae. Bocavirus infections are associated with a wide spectrum of disease in humans and various mammalian species. Here we describe a fatal enteritis associated with infection with a novel strain of canine bocavirus 2 (CaBoV-2), that occurred in a litter of German wirehaired pointers. Necropsy performed on three puppies revealed an enteritis reminiscent of canine parvovirus associated enteritis, accompanied with signs of lymphocytolytic disease in bone marrow, spleen, lymph nodes and thymus. While other major causes of enteritis of young dogs, including canine parvovirus, were excluded, by random PCR in combination with next-generation sequencing, a novel CaBoV-2 strain was detected. Phylogenetic analysis of the genome of this novel canine bocavirus strain indicated that this virus was indeed most closely related to group 2 canine bocaviruses. Infection with canine bocavirus was confirmed by in situ hybridization, which revealed the presence of CaBoV-2 nucleic acid in the intestinal tract and lymphoid tissues of the dogs. In a small-scale retrospective analysis concerning the role of CaBoV-2 no additional cases were identified. The findings of this study provide novel insights into the pathogenicity of canine bocaviruses.

Development of a Bovine leukemia virus polymerase gene-based real-time polymerase chain reaction and comparison with an envelope gene-based assay.

Bovine leukemia virus (BLV) causes a persistent infection with provirus formation in B-lymphocytes. A real-time polymerase chain reaction (PCR) based on the conserved BLV polymerase (BLV pol) gene sequences was developed. Dually labeled probes were used to permit detection by the 5' exonuclease assay. The assay was validated with 350 samples of bovine peripheral blood mononuclear cells including 144 samples from BLV-seropositive animals worldwide (South America, Europe, Middle East, Australia) representing 5 of the recently described 7 BLV envelope-based genotypes. The BLV pol real-time PCR proved to be highly specific and sensitive with the detection of up to 1 copy of an internal control plasmid. The 95% confidence intervals for assay sensitivity and specificity were ≥ 98.27% and ≥ 98.33%, respectively. Restriction fragment length polymorphism and phylogenetic BLV pol-based sequence analysis of the investigated samples were performed and compared with the previous described BLV env-based genotypes. Grouping of the sequences based on the pol gene yielded similar results as the env gene-based assay.