Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Bovine Papillomavirus Type 2 Infection Associated with Papillomatosis of the Amniotic Membrane in Water Buffaloes (Bubalus bubalis).

Multiple papillomatous nodules were observed scattered over the amniotic membrane in six water buffaloes that had recently aborted. Grossly, some of the nodules had multiple villous projections while others appeared as single prominent conical or cylindrical horns. Histology revealed folded hyperplastic and hyperkeratotic epithelium supported by a narrow fibro-vascular stalk. Using PCR, sequences of the bovine Deltapapillomavirus type 2 (BPV-2) E5 gene were amplified from the amniotic papillomas. Furthermore, expression of the E5 gene was detected using reverse transcription (RT)-PCR. Western blotting revealed BPV-2 E5 oncoprotein as well as L1 protein, suggesting both abortive and productive infection. Additionally, a functional complex composed of BPV-2 E5 oncoprotein and the phosphorylated PDGFßR was detected, which is consistent with the activation of PDGFßR by the interaction with BPV-2 E5 oncoprotein. These results demonstrate that BPV-2 can infect the amnion of water buffaloes and suggest that this infection may cause proliferation of the epithelial cells of the amnion. While the precise pathogenesis in uncertain, it is possible that BPV-2 infection of stratified squamous epithelial cells within squamous metaplasia foci and/or amniotic plaques could lead to papilloma formation. Papillomavirus-associated amniotic papillomas have not previously been reported in any species, including humans.

Mitophagy mediated by BNIP3 and BNIP3L/NIX in urothelial cells of the urinary bladder of cattle harbouring bovine papillomavirus infection.

Autophagy is a powerful tool that host cells use to defend against viral infection. Mitophagy, the selective autophagic removal of dysfunctional mitochondria was upregulated in urothelial cancer cells harbouring bovine papillomavirus (BPV) infection, as detected by the expression of BPV E5 protein, the major oncoprotein of bovine Deltapapillomavirus genus. HIF-1α-induced mitophagy receptors, BNIP3 and BNIP3L/Nix, were found to be overexpressed in these cells. The BNIP3 and BNIP3L/Nix receptors were amplified, and amplicon sequencing showed homology between bovine BNPI3 and BNIP3L/Nix sequences deposited in GenBank (accession number: NM_001076366.1 and NM_001034614.2, respectively). The transcripts and protein levels of BNIP3 and BNIP3L/Nix were significantly overexpressed in hypoxic neoplastic cells relative to healthy, non-neoplastic cells. BNIP3 and BNIP3L/Nix interacted with the LC3 protein, a marker of autophagosome (mitophagosome) membrane, ERAS, a small GTPase, and p62, known to be a specific autophagy receptor protein, that plays a role in mitochondrial priming for mitophagy and subsequent elimination. ERAS also interacted with the BPV E5 oncoprotein at mitochondrial level. Furthermore, in anti-Bag3 mitochondrial immunoprecipitates, a complex composed of the Hsc70/Hsp70 chaperone, CHIP co-chaperone, Synpo2, ERAS, LC3, p62, BNPI3, and BNIP3L/Nix was also detected. Bag3 may play a role in mitophagosome formation together with the Synpo2 protein and may be involved in the degradation of Hsc70/Hsp70-bound CHIP-ubiquitinated cargo, in association with its chaperone. ERAS may be involved in mitophagosome maturation via the PI3K signalling pathway. Ultrastructural findings revealed the presence of mitochondria exhibiting severe fragmentation and loss of cristae, as well as numerous mitochondria-containing autophagosomes.

FUNDC1-mediated mitophagy in bovine papillomavirus-infected urothelial cells.

E5 protein, the major oncoprotein of the bovine Deltapapillomavirus genus, has been detected in 17 of the 19 urothelial cancers by molecular and morphological procedures. In 10 urothelial cancers, the oxygen sensitive subunit HIF-1α, which is upregulated by hypoxia, was overexpressed. Mitophagy, the selective autophagic removal of dysfunctional mitochondria, was upregulated in hypoxic neoplastic cells infected by BPVs which was mediated by FUNDC1, a mitochondrial outer-membrane protein. The FUNDC1 receptor was amplified by PCR, and amplicon sequencing showed a 100% homology with bovine FUNDC1 sequences deposited in GenBank (accession number: NM_001104982). Both transcripts and protein levels of FUNDC1 were significantly decreased in hypoxic neoplastic cells relative to healthy, non-neoplastic cells. FUNDC1 interacted with the LC3 protein, a marker of autophagosome (mitophagosome) membrane, the Hsc70/Hsp70 chaperone, and Bag3 co-chaperone. Bag3 may play a role in mitophagosome formation together with the Synpo2 protein, and may be involved in the degradation of Hsc70/Hsp70-bound CHIP-ubiquitinated cargoes, in association with its chaperone. Ultrastructural findings revealed the presence of mitochondria exhibiting severe fragmentation and loss of cristae, as well as numerous mitochondria-containing autophagosomes. Total and phosphorylated GTPase dynamin-related protein 1 (DRP1), which plays a crucial role in mitochondrial fission, a pre-requisite for mitophagy, was overexpressed at the mitochondrial level. Total and phosphorylated mitochondrial fission factor (Mff), mitochondrial fission protein 1 (Fis1), mitochondrial dynamics 51 (MiD51), and MiD49, which are DRP1 receptors responsible and/or co-responsible for its mitochondrial recruitment were overexpressed.

Bovine papillomavirus E5 oncoprotein expression and its association with an interactor network in aggresome-autophagy pathway.

E5 protein, the major oncoprotein of bovine Deltapapillomavirus (BPV), was found to be expressed in 18 of 21 examined urothelial cancers of cattle. E5 oncoprotein was found to interact with p62 which was degraded through the autophagosome-lysosome pathway as well as LC3-II and appeared to be involved in the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α). Autophagy was morphologically documented by transmission electron microscope (TEM) through the detection of double-membrane autophagosomes and autolysosomes. Overexpression of Bag3 known to mediate selective autophagy was also demonstrated. Furthermore, Bag3 and BPV E5 oncoprotein were seen to co-localize with dynein and 14-3-3γ, which suggested that Bag3 could be involved in inducing the retrograde transport of BPV E5 along microtubules to aggresomes, perinuclear sites with high autophagic flux. Electron dense perinuclear structures consistent with aggresomes were also documented by TEM in urothelial cancer cells. Finally, Bag3 was found to also interact with synaptopodin 2 (Synpo2), which would seem to contribute to cargo degradation as it has been shown to facilitate autophagosome formation. This study provides mechanistic insights into the potential role(s) of autophagy in BPV disease, which can help to develop future treatment and control measures for BPV infection. Activation of autophagy correlates positively with BPV infection and may play a role in biological behavior of bladder cancer as urothelial carcinomas of cattle are known to be characterized by a relatively low rate of metastasis.

Congenital papillomavirus infection in cattle: Evidence for transplacental transmission.

Vertical transmission of bovine papillomavirus (BPV) infection was investigated on livers and kidneys of four foetuses from cows suffering from BPV-2-associated urothelial cancers of the urinary bladder. PCR analysis revealed the presence of BPV-2 E5 DNA in the livers and kidneys of two foetuses. Amplified DNA fragments, composed of 502 bp, showed a 100% homology with BPV-2 sequences (GenBank accession number: M20219.1). BPV-2 was found to be transcriptionally active. Indeed, reverse transcriptase (RT)-PCR showed BPV-2 E5 transcripts. Sequencing of amplified cDNA, composed of 154 bp, showed a 100% identity with BPV-2 E5 sequences (GenBank accession number: M20219.1). Western blot analysis revealed the presence of dimers of E5 oncoprotein. Furthermore, a statistically significant increase of the phosphorylated (activated) form of the platelet-derived growth factor ß receptor (PDGFßR) was also detected in the fetal tissues. PDGFßR is believed to form the most important interaction with the E5 oncoprotein, thus regulating biological activity of virus protein. The strong concordance between virus found in fetal organs with virus detected in infected mothers provides evidence that BPV-2 can spread through blood and vertical infection occurs via transplacental transmission. Finally, molecular findings of this study raise unsolved questions about the potential role of BPVs in reproductive disorders. The presence of E5 oncoprotein, as in adult organs, may also activate the constitutive receptor PDGFßR in foetal organs, which plays a pivotal role in angiogenesis and embryonic development. Therefore, abnormal phosphorylation of PDGFßR may be involved in vascular and organogenesis abnormalities other than cancer.

Expression of the feline leukemia virus subgroup C receptors in normal and neoplastic urothelium of the urinary bladder of cattle associated with bovine papillomavirus infection.

The feline leukemia virus subgroup C receptors (FLVCRs) were originally cloned as virus receptors, but are now believed to function also as heme transporters and are expressed in a broad range of tissues in a wide range of mammalian species. Expression of FLVCR1 and FLVCR2 was investigated in 19 bovine papillomavirus-associated urinary bladder cancers and in 15 non-neoplastic samples of bladder from cattle. E5 oncoprotein of bovine Deltapapillomaviruses (δPVs) was detected in 17 of the 19 bladder cancers. Flvcr1 and Flvcr2 were amplified and sequenced both in neoplastic and non-neoplastic samples showing a 100% identity with bovine Flvcr1 and Flvcr2 mRNA sequences present in GenBank database (accession numbers: NM_001206019.1 and NM_001192143.1, respectively). Reverse transcription (RT)-PCR showed that Flvcr1 and Flvcr2 were overexpressed in 4 and 5 out of 19 urothelial cancers, respectively, but in none of the non-neoplastic samples. In addition, western blot analysis detected higher levels of FLVCR1 and FLVCR2 in samples in which transcripts were not increased, suggesting post-translational changes to these proteins. Increased FLCVR1 and FLVCR2 was also observed immunohistochemically in the neoplastic cells. Immunolabeling for FLVCR1 was seen in the cytoplasm and plasm membrane of urothelial cancer cells, wheras immunolabeling for FLVCR2 was present within the nucleus. This is the first time that FLVCR expression has been investigated in bovine tissues and the first to suggest that expression could be increased in cancers. Additional studies are required to define the role, if any, of FLVCR in papillomavirus-associated cancer cells.

Oral fibropapillomatosis and epidermal hyperplasia of the lip in newborn lambs associated with bovine Deltapapillomavirus.

Congenital fibropapillomatosis of the gingiva and oral mucosa and epidermal hyperplasia of the lip are described, for the first time, in two newborn lambs. Expression of the E5 oncoprotein of bovine deltapapillomavirus types 2 (BPV-2) and -13 (BPV-13) was detected in both fibropapillomas and the hyperplastic epidermal cells suggesting the BPV infection was the cause of the proliferative lesions. No DNA sequences of BPV-1 and BPV-14 were detected. Both BPV-2 and BPV-13 DNA were also amplified from peripheral blood mononuclear cells (PBMCs) of the newborn lambs' dams. The concordance between BPV genotypes detected in the blood of dam and the oral and skin pathological samples of their offspring suggests that a vertical hematogeneous transmission was most likely source of BPV infection. Immunoblotting revealed the presence of E5 dimers allowing the viral protein to be biologically active. E5 dimers bind and activate the platelet derived growth factor ß receptor (PDGFßR), a major molecular mechanism contributing to disease. The detection of E5 protein within the proliferating cells therefore adds further evidence that the BPV infection was the cause of the proliferative lesions seen in these lambs. This is the first evidence of vertical transmission of BPVs in sheep resulting in a clinical disease.

Chaperone-assisted selective autophagy in healthy and papillomavirus-associated neoplastic urothelium of cattle.

Chaperone-assisted selective autophagy (CASA) is a newly-described selective tension-induced macroautophagy pathway mediated by Bag3 that is believed to be essential for mechanotransduction in skeletal muscle and to be an important regulator of the immune system. We investigated CASA machinery both in healthy and in fifteen papillomavirus-associated neoplastic bovine urothelium. The components of CASA complex, that comprises the molecular chaperones HspA8/Hsc70 and Hsp8B/Hsp22 and the cochaperones Bag3 and STUB1/CHIP, were studied by molecular, microscopic and submicroscopic investigations. CASA complex was found to be constitutively expressed in healthy bovine urothelium; its expression increased in urothelial cancers of cattle, namely thirteen papillary carcinomas and two papillary urothelial neoplasm of low malignant potential (PUNLMPs). We suggest that basal levels of CASA are important in the healthy urothelium which interfaces with the community of urinary microbiota thus representing an important epithelial cell-autonomous mechanism of antibacterial defense. Co-immunoprecipitation studies using an antibody against bovine papillomavirus E5 protein revealed that the oncoprotein co-localized with CASA complex in urothelial cancer cells. This suggests that infection by BPV E5 could influence cell behaviour by interfering with basal autophagy processes although this study did not conclusively show that this interaction increased the expression of CASA proteins. In neoplastic urothelium, CASA could be involved in regulating fundamental cellular processes such adhesion, migration, and proliferation and so might influence the biological behaviour of urothelial tumors in cattle.

Expression of hepcidin and ferroportin in full term placenta of pregnant cows.

Hepcidin (HEP) and ferroportin (FPN) play a central role in systemic iron homeostasis. The HEP/FPN axis controls both extracellular iron concentration and total body iron levels. HEP is synthesized mainly by hepatocytes and controls the absorption of dietary iron and the distribution of iron to the various cell types; its synthesis is regulated by both iron and innate immunity. FPN is a membrane protein and the major exporter of iron from mammalian cells, including iron recycling macrophages, iron absorbing duodenal enterocytes, and iron storing hepatocytes. HEP limits the pool of extracellular iron by binding FPN and mediating its degradation, thus preventing its release from intracellular sources. Here we investigated, for the first time, the molecular and morphological expression of HEP and FPN in placenta of pregnant cows at term. Their expression has been evaluated investigating their mRNAs by reverse transcriptase PCR (RT-PCR). Sequencing of related amplicons revealed a 100% identity with HEP and FPN sequences from Bos taurus as reported in the GeneBank (mRNASequence ID: NM_001114508.2 and ID: NM_001077970.1, respectively). HEP and FPN proteins have also been revealed by Western blot analysis and immunohistochemistry. The strongest immunoreactivity for both proteins was observed in the cytoplasm of the trophoblastic cells of the villi and the caruncular crypts of the placentome. Hep mRNA was more representative in caruncular rather cotyledonar areas; on the contrary, Fpn mRNA was more expressed in cotyledonar rather than in caruncular areas. Transcripts of ferritin, transferrin and its receptor have been also documented by real time RT-PCR. HEP and FPN placental proteins may play a dual role. HEP/FPN axis seems to have a central role in infections, with microorganisms within macrophages or that survive in the bloodstream or other cellular spaces. In addition, HEP may be responsible for iron flux regulation as a molecular bridge for iron trafficking and response to infection. FPN may also have a significant role for embryonic development, growth and organogenesis.

ERas is constitutively expressed in full term placenta of pregnant cows.

ERas is a new gene recently found in mouse embryonic stem (ES) cells and localized on the X chromosome. It plays a role in mouse ES cell survival and is constitutively active without any mutations. It was also found to be responsible for the maintenance of quiescence of the hepatic stellate cells (HSCs), liver-resident mesenchymal stem cells, the activation of which results in liver fibrosis. This gene was not present in human ES cells. ERas was found to be activated in a significant population of human gastric cancer, where ERAS may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin. ERas gene has been found to be expressed both in ES cells and adult tissues of cynomolgus monkey. Cynomolgus ERAS did not promote cell proliferation or induce tumor formation. ERAS was also detected in normal and neoplastic urothelium of the urinary bladder in cattle, where bovine ERAS formed a constitutive complex with platelet derived growth factor ß receptor (PDGFßR) resulting in the activation of AKT signaling. Here, molecular and morphological findings of ERAS in the full term placenta of pregnant cows have been investigated for the first time. ERAS was studied by reverse transcriptase PCR (RT-PCR). Alignment of the sequence detects a 100% identity with all transcript variant bovine ERas mRNAs, present in the GenBank database (http://www.ncbi.nlm.nih.gov). Furthermore, ERAS was detected by Western blot and investigated by real time PCR that revealed an amount of ERAS more than ERAS found in normal bovine urothelium but less than ERAS present in the liver. Immunohistochemical examination revealed the presence of ERAS protein both at the level of plasma membrane and in cytoplasm of epithelial cells lining caruncular crypts and in trophoblasts of villi. An evident ERAS immunoreactivity was also seen throughout the chorionic and uterine gland epithelium. Although this is not a functional study and further investigations will be warranted, it is conceivable that ERAS may have pleiotropic effects in the placenta, some of which, like normal urothelial cells, might lead to activation of AKT pathway. We speculate that ERAS may play a key role in cellular processes such as cell differentiation and movement. Accordingly, we believe it may be an important factor involved in trophoblast invasiveness via AKT signaling pathway. Therefore, ERas gene is a functional gene which contributes to homeostasis of bovine placenta.

Proteomic analysis of protein purified derivative of Mycobacterium bovis.

BACKGROUND: Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). METHODS: Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. RESULTS: Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. CONCLUSIONS: This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Papillomaviruses: a systematic review

Abstract In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.

Papillomaviruses: a systematic review.

In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.

Papillomaviruses: a systematic review

In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.

Genetics and metabolic deregulation following cancer initiation: A world to explore.

Cancer is a group of highly complex and heterogeneous diseases with several causes. According to the stochastic model, cancer initiates from mutation in somatic cells, leading to genomic instability and cell transformation. This canonical pathway of carcinogenesis is related to the discovery of important mechanisms that regulate cancer initiation. However, there are few studies describing genetic and metabolic alterations that deregulate transformed cells, resulting in epithelial-mesenchymal transition (EMT) and its most dramatic consequence, the metastasis. This review summarizes the main genetics and metabolic changes induced by reactive oxygen species (ROS) that lead to EMT.

Detection of bovine papillomavirus type 14 DNA sequences in urinary bladder tumors in cattle.

Bovine papillomavirus type 14 (BPV-14) is a novel Deltapapillomavirus (δPV) which is most closely related to BPV-1, -2, and -13, well-known members of the δPV genus. So far BPV-14 has been detected in cutaneous neoplastic lesions in cattle and in feline sarcoids. As BPV-14 may share biological and pathological properties with BPV-1, -2 and -13, it has been hypothesized that, like other δPVs, BPV-14 could be associated with bovine bladder neoplasia. In this study, 50 tumors of the urinary bladder of cattle were diagnosed. DNA was extracted from all tumor samples as well as from 25 normal bladder samples and submitted to BPV-14 L1 PCR and subsequent amplicon sequencing analysis. BPV-14 L1 DNA sequences of specific 195bp amplicons were obtained from 17 of 50 (34%) tumor DNA isolates; no BPV-14 DNA was detected from 25 normal samples. Amplicons revealed a 99% homology with the corresponding BPV-14 L1 DNA region (GenBank accession number KP276343.1). Co-infections by two or three δPV types were also seen. This study reveals the presence of BPV-14 DNA alone or in combination with other δPV DNA in bovine bladder tumors alone and suggests that BPV-14 could also be involved in bladder neoplasia as its E5 oncoprotein has the potential to induce cell proliferation. Furthermore, this is the first study to show the presence of BPV-14 in Europe, suggesting that BPV-14, like other δPVs, has a worldwide distribution.

ERas protein is overexpressed and binds to the activated platelet-derived growth factor ß receptor in bovine urothelial tumour cells associated with papillomavirus infection.

Embryonic stem cell-expressed Ras (ERas) encodes a constitutively active form of guanosine triphosphatase (GTPase) that binds to and activates phosphatidylinositol 3 kinase (PI3K), which in turn phosphorylates and activates downstream targets such as Akt. The current study evaluated ERas regulation and expression in papillomavirus-associated urothelial tumours in cattle grazing on lands rich in bracken fern. ERas was found upregulated and overexpressed by PCR, real time PCR and Western blot. Furthermore, protein overexpression was also confirmed by immunohistochemistry. ERas was found to interact physically and colocalise with the activated platelet derived growth factor ß receptor (PDGFßR) by coimmunoprecipitation and laser scanning confocal investigations. Phosphorylation of Akt, a downstream effector both of ERas and PDGFßR, appeared to be increased in urothelial tumour cells. Altogether, these data indicate that ERas/PDGFßR complex could play a role in the pathogenesis of bovine papillomavirus-associated bladder neoplasia.

Mincle, an Innate Immune Receptor, Is Expressed in Urothelial Cancer Cells of Papillomavirus-Associated Urothelial Tumors of Cattle.

BACKGROUND: Mincle, macrophage-inducible C-type lectin, is a member of C-type lectin receptors. It plays an important role in anti-mycobacterial and anti-fungal immunity. Furthermore it senses dead cells through its primary ligand SAP130. MATERIALS AND FINDINGS: We examined ten urothelial tumors of the urinary bladder of cattle. Eight of them expressed E5 cDNA of bovine papillomaviruses type 2 (BPV-2) and type 13 (BPV-13) that belong to Deltapapillomavirus genus. Two of them were not examined for detection of E5 cDNA. Mincle expression appeared to occur in urothelial neoplastic cells only. No mincle expression was detected in urothelial cells from healthy cattle. Mincle expression was characterized by a membranous pattern in papillary urothelial cancers; isolated and/or clustered urothelial cells showing a strong cytoplasmic immunoreactivity were primarily seen in invasive urothelial cancers. CONCLUSION: This is the first study about the expression of mincle in veterinary oncology and the first report which describes the expression of functional mincle receptor in neoplastic cells in medical literature. As it has been shown that urothelial cancer cells have the ability to function as antigen-presenting cells (APCs), it is conceivable that mincle expression is involved in the presentation of cancer cell antigens to cells of the immune system. Furthermore, since expression of mincle contributes to the control of Mycobacterium bovis BCG infection, this study has exciting clinical implications in comparative medicine keeping in mind that Bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment of non-muscle invasive bladder cancer in man. Mincle expression in urothelial tumor cells warrants further study to better understand the role, if any, of this receptor in bladder cancer. Future studies will provide insights in the role of mincle receptor of urothelial cancer cells in antitumor immunotherapy.

Ptaquiloside, the major carcinogen of bracken fern, in the pooled raw milk of healthy sheep and goats: an underestimated, global concern of food safety.

Bracken fern (Pteridium aquilinum) is a worldwide plant containing toxic substances, which represent an important chemical hazard for animals, including humans. Ptaquiloside, 1, a norsesquiterpenoid glucoside, is the major carcinogen of bracken detected in the food chain, particularly in the milk from farm animals. To date, ptaquiloside has been shown in the milk of cows feeding on a diet containing bracken fern. This is the first study that shows the systematic detection of ptaquiloside, 1, and reports its direct quantitation in pooled raw milk of healthy sheep and goats grazing on bracken. Ptaquiloside, 1, was detected by a sensitive method based on the chemical conversion of ptaquiloside, 1, into bromopterosine, 4, following gas chromatography-mass spectrometry (GC-MS) analysis. The presence of ptaquiloside, 1, possibly carcinogenic to humans, in the milk of healthy animals is an unknown potential health risk, thus representing a harmful and potential global concern of food safety.