Manuela Martano

Bovine Papillomavirus Type 13 Expression in the Urothelial Bladder Tumours of Cattle

Bovine papillomavirus type 13 (BPV-13), a novel Deltapapillomavirus, has been found associated with urothelial tumours of the urinary bladder of cattle grazing on lands infested with bracken fern. BPV-13 was detected in 28 of 39 urothelial tumours. Diagnosis was based on sequencing of L1 and E5 amplicons from tumour samples. The nucleotide sequences generated from these amplicons showed a 100% homology with the sequences of BPV-13 L1 and E5 DNA found in Brazil from a fibropapilloma of the ear in a cow and from equine sarcoids in two horses. GenBank accession number of our representative BPV-13 sequences is JQ798171.1. Furthermore, mRNA encoding BPV-13 E5 oncoprotein was also documented, and its expression was also shown by immunohistochemistry and immunofluorescence in the basal and suprabasal urothelial tumour cells. In twenty-three tumours, BPV-13 was simultaneously found with BPV-2, a Deltapapillomavirus genus, species 4. The latter virus was detected by amplifying and sequencing a 154-bp-sized DNA fragment of BPV-2 E5. In addition, BPV-13 by itself was seen to be expressed in five BPV-2-negative urothelial tumours. This study shows that BPV-13 is present in urothelial tumour cells thus sharing biological properties with BPV-1 and BPV-2. Although further studies are needed, BPV-13 appears to be another worldwide infectious agent responsible for a distressing disease causing severe economic losses in cattle industry.

Innate immune response in human keratinocytes infected by a feline isolate of Malassezia pachydermatis

Malassezia pachydermatis is a normal inhabitant of canine and feline skin that can spread to other pets. The outer layer or epidermis is made up primarily of keratinocytes, which are capable of releasing various factors and expressing receptors that are significantly involved in the immune regulation. Little is known about the mechanism by which M. pachydermatis overcomes the natural barrier of the skin. The aim of this study was to evaluate the direct in vitro interaction between human keratinocytes and a clinical strain of live M. pachydermatis isolated as a pure culture from an otitic cat. Human keratinocytes (HaCat) were infected with M. pachydermatis to analyse the modulation of the innate immune response. Gene expression was analysed by real-time PCR. We demonstrated that M. pachydermatis invaded HaCat cells and modulated the expression of TLR2 after 24h infection, while HBD-2, IL-1β TNF-α, IL-6 and IL-8 were modulated both at 24 and 48 h. Thus, our results demonstrated that M. pachydermatis is able to stimulate the innate immune response in infected human keratinocytes indicating a possible role of this yeast as a human opportunistic pathogen.

Bovine Papillomavirus Type 2 Infection and Microscopic Patterns of Urothelial Tumors of the Urinary Bladder in Water Buffaloes

Microscopic patterns of thirty-four urothelial tumors of the urinary bladder of water buffaloes from the Marmara and Black Sea Regions of Turkey are here described. All the animals grazed on lands rich in bracken fern. Histological diagnosis was assessed using morphological parameters recently suggested for the urinary bladder tumors of cattle. Papillary carcinoma was the most common neoplastic lesion (22/34) observed in this study, and low-grade carcinoma was more common (seventeen cases) than high-grade carcinoma (five cases). Papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and invasive carcinomas were less frequently seen. Carcinoma in situ (CIS) was often detected associated with some papillary and invasive carcinomas. De novo (primary) CIS was rare representing 3% of tumors of this series. A peculiar feature of the most urothelial tumors was the presence in the tumor stroma of immune cells anatomically organized in tertiary lymphoid organs (TLOs). Bovine papillomavirus type-2 (PV-2) E5 oncoprotein was detected by molecular and immunohistochemistry procedures. Early protein, E2, and late protein, L1, were also detected by immunohistochemical studies. Morphological and molecular findings show that BPV-2 infection contributes to the development of urothelial bladder carcinogenesis also in water buffaloes.

Expression of platelet derived growth factor β receptor, its activation and downstream signals in bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are benign skin tumours formed by proliferation of epidermal keratinocytes and dermal fibroblasts caused by bovine papillomaviruses (BPVs). BPV E5 oncoprotein plays a key role in neoplastic cell transformation by specifically binding to the platelet derived growth factor beta receptor (PDGFβR) causing its phosphorylation and activation of proliferation and survival signal transduction pathways, among these phosphatidyl inositol-3-kinase (PI3K)/Akt and Ras-mitogen-activated-protein-kinase-Erk (Ras-MAPK-Erk) pathways. The aim of this study was to investigate the expression of PDGFβR, its phosphorylation status and expression of the downstream molecules phospho-Akt (pAkt) and phospho-Erk (pErk), in naturally occurring bovine cutaneous fibropapillomas. By immunohistochemistry on serial sections we showed cytoplasmic co-expression of the PDGFβR and E5 protein in neoplastic tissue. Western blot analysis revealed that PDGFβR was phosphorylated in higher amount in tumour samples compared to normal skin. pAkt, but not pErk, was also overexpressed in tumour samples. These findings may provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine fibropapillomas and contribute to understanding the molecular scenario underlying BPV induced tumourigenesis.

Bovine Papillomavirus Type 2 Infection and a Series of Mesenchymal Tumors of the Urinary Bladder in Cattle

This report describes the histopathology of two hundred and fifty-three mesenchymal tumors of the urinary bladder in cattle grazing on lands rich in bracken fern. Approximately 80% were hemangiomas and angiosarcomas. Hemangioma (capillary, cavernous, and large vessels) was the most frequent mesenchymal tumor and was more common than angiosarcoma. Although the appearance of endothelial cells can vary remarkably, epithelioid angiosarcomas, often containing multinucleated cells, were the most frequent malignant vascular tumors. Hemangiopericytoma and tumors of muscle and soft connective tissue origin, alone and/or in association with tumor-like lesions, were less frequently seen. Furthermore, forty-five cases of intravascular papillary endothelial hyperplasia (IPEH), a lesion not previously reported in the urinary bladder of cattle, were also described. Bovine papillomavirus type-2 DNA was amplified in tumor samples. Forty vascular tumors were investigated by dual-labeling immunofluorescence, and, for the first time, a coexpression of E5 and platelet-derived growth factor β receptor (PDGFβR) was shown to occur. The results show that the BPV-2 E5 oncoprotein binds to the activated form of the PDGFβ receptor thus playing an important role in mesenchymal as well as epithelial carcinogenesis of the urinary bladder. Furthermore, these findings demonstrate that BPV-2 infects both epithelial and mesenchymal cells.

Histopathological findings of the midgut in European honey bee (Apis Mellifera L.) naturally infected by Nosema spp.

Abstract Background: Nosemosis is an infection of ventricular epithelial cells of adult European honey bees (Apis mellifera), caused by Nosema species. Until recently, it was thought to be caused only

Malignant pilomatricoma with multiple bone metastases in a dog: Histological and immunohistochemical study.

An eleven year-old mongrel dog was referred with a history of left forelimb lameness and an ulcerated mass on the neck. Histologically, the cutaneous neoplasm revealed cystic lobules composed of basaloid cells with abrupt transition to central keratotic material, containing pycnotic and shadow cells. Approximately 3 months after primary diagnosis, a lesion of the cortical bone on the left humerus was observed using X-ray. Samples obtained from the humerus were processed for histopathological examination and the neoplastic tissue was observed to be similar to the type identified in the neck. Based on these findings, the tumor was diagnosed as a malignant pilomatricoma (MP) with bone metastasis. MP is a rare skin tumor that originates from hair matrix cells. To date, only nine reports have been presented in dogs. In the present study, we discuss the cytological and histological patterns of MP, confirmed by immunohistochemistry using β catenin antibody.

Expression and activation of platelet-derived growth factor β receptor, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in canine mammary tumours

Canine mammary tumours are frequent neoplasms mostly affecting intact female dogs, for which no 100% efficient therapy is available. Platelet derived growth factor β receptor (PDGFβR) is a tyrosine kinase receptor (TKR) with a potential role in human breast cancer and a series of canine tumours. In this study we demonstrated, for the first time, expression of PDGFβR and its downstream transduction molecules, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), as well as their activated forms in canine mammary tumours by both biochemical analysis and immunohistochemistry. PDGFβR was expressed and hyperphosphorylated in the majority of tumour samples and tumour derived cell lines. Additionally, both MEK and ERK were expressed and activated in cell lines as well as biopsies. TKR inhibitors (TKRi) are currently under investigation as possible therapy in human breast and several canine tumours, thus our in vivo and in vitro findings pave the way for future studies aimed at establishing a potential therapeutic employment of TKRi for the treatment of canine mammary cancer.

Expression of endothelin-1 and endothelin-1 receptor A in canine mammary tumours.

Endothelins and their receptors have been implicated in numerous diseases and have recently emerged as relevant players in a variety of malignancies. Tumours overexpress the Endothelin-1 (ET-1) and the Endothelin-A receptors (ETAR) and their interaction enhances tumour growth and metastasis by promoting tumour cell survival, proliferation and angiogenesis. In this study we have evaluated the expression of ET-1 and ETAR in 50 canine mammary tumours, compared to normal controls. Results demonstrated a progressive increase in ET-1 and ETAR expression from benign tumour to grade 1 and to grade 2 malignant mammary tumours with a decrease of expression in grade 3 carcinomas. Co-localization of ET-1 and ETAR was observed in benign mammary tumours and in G1 and G2 carcinomas, while absent in G3 carcinomas. Concluding, ET-1/ETAR can be considered reliable markers for evaluating malignancy of canine mammary tumours and could have importance for the development of specific anticancer therapies.

Expression of vascular endothelial growth factor (VEGF) in equine sarcoid

BackgroundSarcoids are the mostcommon skin tumors in horses, characterized by rare regression, invasiveness and high recurrence following surgical intervention and Delta Papillomaviruses are widely recognized as the causative agents of the disease. In order to gain new insights into equine sarcoid development, we have evaluated, in 25 equine sarcoids, by immunohistochemistry and western blotting analysis, the expression levels of VEGF, Ki67 and bcl-2. Moreover, we have measured microvessel density and specific vessel parameters.ResultsAll sarcoid samples showed a strong and finely granular cytoplasmatic staining for VEGF in the majority (90%) of keratinocytes, sarcoid fibroblasts and endothelial cells. Numerous small blood vessels, immunostained with Von Willebrand factor, often appeared irregular in shape and without a distinct lumen, with mean values of microvessel area and perimeter lower than normal. Moreover, in all sarcoid samples, Ki67 immunoreactivity was moderately positive in 5–10% of dermal sarcoid fibroblasts, while Bcl2 immunoreactivity was detected in 52% of the sarcoid samples, with a weak staining in 20–50% of dermal sarcoid fibroblasts. Biochemical analysis was consistent with immunohistochemical results.ConclusionsThis study has provided evidence that in equine sarcoid: VEGF was strongly expressed; the increased number of vessels was not associated with their complete maturation, probably leading to a hypoxic condition, which could increase VEGF synthesis; the levels of sarcoid fibroblasts proliferation were very low. Concluding, VEGF may have a role in equine sarcoid development, not only through the increase of angiogenesis, but also through the control of sarcoid fibroblast activity.