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The equine sarcoid is the most common skin neoplasia of fibroblastic origin in horses, characterized by an excessive accumulation of extracellular matrix produced by sarcoid fibroblasts under hypoxic condition. Neoplastic cells can adapt to hypoxia by using alternative energy sources, particularly those that arise from fatty acid oxidation (FAO). The Carnitine Palmitoyl Transferase 1A (CPT1A) belongs to Carnitine System (CS) and promotes the entrance of fatty acids into the mitochondria for β-oxidation. In this study, CPT1A expression was comparatively addressed in 25 equine sarcoids and 5 normal skin samples using immunohistochemistry (IHC). Specificity of CPT1A antibody was validated by Western Blotting (WB). In normal skin samples IHC staining was weak and mainly confined to basal epidermis and few dermal fibroblasts. Sarcoid fibroblast exhibited a strong cytoplasmic and nuclear signal in 60% of the tumor samples. Cytoplasmic CPT1A expression in sarcoid fibroblasts indicates that the protein is actively involved in metabolic reprogramming processes. Nuclear CPT1A expression suggests that the protein may also be involved in the regulation of neoplastic proliferation., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper, (Copyright © 2024. Published by Elsevier Inc.)

Feline oral squamous cell carcinoma (FOSCC) is characterised by invasive and metastatic behaviour and is poorly responsive to current treatments, hence the need for new therapeutic strategies. FOSCC shares molecular targets with human head and neck squamous cell carcinoma (HNSCC), among these the epidermal growth factor receptor. Cetuximab is an anti-epidermal growth factor receptor monoclonal antibody employed in the therapy of HNSCC and, interestingly, previous work in vitro suggested that it displays cytostatic and cytotoxic properties also against FOSCC. With the present study, we aimed at further investigating the effects of cetuximab on invasion and metastasis pathways proven to be relevant in human patients. To this purpose, FOSCC cell lines SCCF1, SCCF2 and SCCF3 were treated with cetuximab for 48/72 h and subjected to Western blot for matrix metalloproteinases-2/9 (MMP-2/9) and epithelial-mesenchymal transition markers vimentin, E-, P- and N-cadherin. Treatment with cetuximab resulted in downregulation of MMP-2/-9 in all of the three cell lines in a dose-dependent manner. Moreover, cetuximab downregulated vimentin and P-cadherin in SCCF1, upregulated E-cadherin whilst downregulating P-/N-cadherins in SCCF2, and impaired P-/N-cadherins in SCCF3. An in vitro scratch test also demonstrated that cetuximab delayed cell migration in SCCF3. These data suggest that cetuximab mitigates invasion and metastasis processes by impairing MMPs and epithelial-mesenchymal transition pathways in FOSCC, indicating that this monoclonal antibody may help to counteract malignant progression and improve the management of locally invasive disease., (© 2023 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Background: Several studies show that natural foods are a source of compounds with anticancer properties that affect the gut microbiota and its metabolites. In the present study, we investigate the effect of a delactosed buffalo milk whey by-product (DMW) on colorectal carcinogenesis., Methods: The effect of DMW on colorectal carcinoma (CRC) was investigated in the established mouse model of azoxymethane (AOM)-induced colon carcinoma, which closely resembles the human clinical condition of CRC. The effect of DMW on CRC immortalized cell lines was also evaluated to further identify the antineoplastic mechanism of action., Results: Pretreatment of AOM-treated mice with DMW significantly (P < 0.05) reduced the percentage of mice bearing both aberrant crypt foci with more than four crypts (which are early precancerous lesions that progress to CRC) and tumors. In addition, DMW completely counteracted the effect of AOM on protein expression of caspase-9, cleaved caspase-3 and poly ADP-ribose polymerase in colonic tissue. Administration of DMW alone (i.e. without AOM) resulted in changes in the composition of the gut microbiota, leading to enrichment or depletion of genera associated with health and disease, respectively. DMW was also able to restore AOM-induced changes in specific genera of the gut microbiota. Specifically, DMW reduced the genera Atopobiaceae, Ruminococcus 1 and Lachnospiraceae XPB1014 and increased the genera Parabacteroides and Candidatus Saccharimonas, which were increased and reduced, respectively, by AOM. Blood levels of butyric acid and cancer diagnostic markers (5-methylcytidine and glycerophosphocholine), which were increased by AOM treatment, were reduced by DMW. Furthermore, DMW exerted cytotoxic effects on two human CRC cell lines (HCT116 and HT29) and these effects were associated with the induction of apoptotic signaling., Conclusions: Our results suggest that DMW exerts chemopreventive effects and restores the gut microbiota in AOM-induced CRC, and induces cytotoxic effect on CRC cells. DMW could be an important dietary supplement to support a healthy gut microbiota and reduce the prevalence of CRC in humans. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Sirtuin 1 (SIRT1) is a protein involved in aging, cell protection, and energy metabolism in mammals. Recently, SIRT1 has been intensively studied in medical oncology, but the role of SIRT1 is still controversial, as it has been proposed as both an oncogene and a tumor suppressor. The aim of this study is to investigate the expression of SIRT1 by immunohistochemistry in canine mammary tissues, and by Western blot and immunofluorescence analysis in different canine mammary cell lines. Our results showed a decrease in SIRT1 expression from normal mammary gland tissue, and from benign and well-differentiated malignant tumors (G1) to less differentiated ones (G2-G3). Furthermore, a shift in the subcellular localization of SIRT1 from the nucleus to the cytoplasm was observed in less differentiated malignant tumors. However, further studies are needed to investigate the subcellular localization of SIRT1 in canine cancer cells and the role it may play in oncogenesis in animals.

The Oriental hornet ( Vespa orientalis ) is one of the major predators of honey bees. It has been demonstrated that adults of V. orientalis can harbor honey bee viruses, however the transmission route of infection is still not clear. The aim of this study was to study the possible presence of honey bee viruses in V. orientalis larvae and honey bees collected from the same apiary. Therefore, 29 samples of V. orientalis larvae and 2 pools of honey bee ( Apis mellifera ). samples were analyzed by multiplex PCR to detect the presence of six honeybee viruses: Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Chronic Bee Paralysis Virus (CBPV), Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV) and Sac Brood Virus (SBV). Biomolecular analysis of V. orientalis larvae revealed that DWV was present in 24/29 samples, SBV in 10/29, BQCV in 7/29 samples and ABPV in 5/29 samples, while no sample was found positive for CBPV or KBV. From biomolecular analysis of honey bee samples DWV was the most detected virus, followed by SBV, BQCV, ABPV. No honey bee sample was found positive for CBPV or KBV. Considering the overlapping of positivities between V.orientalis larvae and honey bee samples, and that V.orientalis larvae are fed insect proteins, preferably honey bees, we can suggest the acquisition of viral particles through the ingestion of infected bees. However, future studies are needed to confirm this hypothesis and rule out any other source of infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Power, Martano, Ragusa, Altamura and Maiolino.)

Recent reports of Eucoleus garfiai in wild boars in southern Italy have highlighted the need for collecting epidemiological data on the presence of this parasite and understanding the role of possible interactions between wild boars, E. garfiai , and the environment. This study analyses, using histopathological and biomolecular techniques, the presence of E. garfiai in tongue samples of wild boars hunted in four provinces of the Campania and Latium regions (Italy), in areas located above and below 900 m above sea level (asl). Histopathological examinations revealed the presence of adults and eggs of nematodes, which were subsequently identified as E. garfiai by biomolecular analysis, in the tongue epithelium. The detection of the parasite was more frequent in samples collected from hunting areas located above 900 m asl than in those collected from areas located below 900 m asl (66.67% vs. 38.09%; p < 0.01). Some species of earthworms are intermediate hosts of E. garfiai and it is well known that earthworms are more present in high quality soils. Therefore, we can suggest that the higher prevalence of E. garfiai at higher altitudes is probably linked to a greater presence of earthworms in the soil, due to its higher quality in these areas.

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo ( Bubalus bubalis ) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.

The Oriental hornet ( Vespa orientalis ) is spreading across the Italian territory threatening the health and wellbeing of honeybees by feeding on adult individuals and larvae and by plundering hive resources. Considering the capacity of other hornets in harboring honeybee viruses, the aim of this study was to identify the possible role of the Oriental hornet as a vector for honeybee viruses. Adult hornets were subjected to macroscopical examination to identify the presence of lesions, and to biomolecular investigation to detect the presence of six honeybee viruses: Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Chronic Bee Paralysis Virus (CBPV), Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV), Sac Brood Virus (SBV). No macroscopical alterations were found while biomolecular results showed that DWV was the most detected virus (25/30), followed by ABPV (19/30), BQCV (13/30), KBV (1/30) and SBV (1/30). No sample was found positive for CBPV. In 20/30 samples several co-infections were identified. The most frequent (17/30) was the association between DWV and ABPV, often associated to BQCV (9/17). One sample (1/30) showed the presence of four different viruses namely DWV, ABPV, BQCV and KBV. The detected viruses are the most widespread in apiaries across the Italian territory suggesting the possible passage from honeybees to V. orientalis , by predation of infected adult honeybees and larvae, and cannibalization of their carcasses. However, to date, it is still not clear if these viruses are replicative but we can suggest a role as mechanical vector of V. orientalis in spreading these viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Power, Altamura, Martano and Maiolino.)

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material., Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples., Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples., Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

Deregulation of fatty acid catabolism provides an alternative energy source to glycolysis for cancer cell survival and proliferation. The regulator enzymes of the carnitine system (CS), responsible for the transport of fatty acids across mitochondrial membranes for β-oxidation are deregulated in tumorigenesis. Recently, we found that Carnitine Palmitoyl Transferase 1 (CPT1), a crucial regulator of CS components, is expressed and dysregulated in canine mammary tumor (CMT) tissues and cells. In this study, we examined the protein expression of the three remaining enzymes of CS (Carnitine Acylcarnitine Translocase (CACT), Carnitine Palmitoyl Transferase 2 (CPT2), Carnitine O-acetyltransferase (CrAT), in canine mammary cells and tissues by Western blot and immunohistochemistry. Protein expression of the components of CS was found in normal mammary glands and a concomitant deregulation of expression in CMT tissues that inversely correlated with the degree of tumor differentiation. Moreover, the expression and a different deregulation of CS-related proteins was also observed in CF33, CMT-U27, CMT-U309, and P114 cell lines used as in vitro model. These results demonstrate for the first time the expression of CS components in CMT tissues and cancer cells; however, further studies are needed to elucidate their roles in dogs as well.

Deformed wing virus (DWV) is capable of infecting honeybees at every stage of development causing symptomatic and asymptomatic infections. To date, very little is known about the histopathological lesions caused by the virus. Therefore, 40 honeybee samples were randomly collected from a naturally DWV infected hive and subjected to anatomopathological examination to discriminate between symptomatic (29) and asymptomatic (11) honeybees. Subsequently, 15 honeybee samples were frozen at -80° and analyzed by PCR and RTqPCR to determinate the presence/absence of the virus and the relative viral load, while 25 honeybee samples were analyzed by histopathological techniques. Biomolecular results showed a fragment of the expected size (69bp) of DWV in all samples and the viral load was higher in symptomatic honeybees compared to the asymptomatic group. Histopathological results showed degenerative alterations of the hypopharyngeal glands (19/25) and flight muscles (6/25) in symptomatic samples while 4/25 asymptomatic samples showed an inflammatory response in the midgut and the hemocele. Results suggest a possible pathogenic action of DWV in both symptomatic and asymptomatic honeybees, and a role of the immune response in keeping under control the virus in asymptomatic individuals.

Papillomavirus (PV) infection is associated with development of epithelial cancer in different species, including domestic cat (Felis catus). Felis catus PV type-2 (FcaPV-2) is considered the causative agent of a proportion of feline cutaneous squamous cell carcinoma (SCC), through the transforming properties of its E6 and E7 oncogenes. However, the possible role of FcaPVs in the aetiology of feline oral SCC (FOSCC) is still unclear. The aim of this study was to assess the presence and gene expression of FcaPV-2 in FOSCC samples. We detected FcaPV-2 DNA in 10/32 (31%) of the analysed FOSCC by the use of PCR methods. Importantly, viral mRNA was detected by RT-PCR in 7/10 (70%) of DNA positive samples. In particular, FcaPV-2 L1, E2 and E6E7 genes were found to be expressed in 5/10 (50%), 3/10 (33%) and 5/10 (50%) samples, respectively. Viral DNA was also detected in non neoplastic oral ulcerative lesions (ULs) (4/11, 36%); qPCR suggested a difference in viral load between ULs and FOSCCs, particularly in those expressing E6E7, although it was not statistically significant. These data suggest, but do not definively prove, a possible role of FcaPV-2 in the development of a proportion of FOSCC. Moreover, L1 and E2 gene expression results indicate that FcaPV-2 infection associated with these tumours may possibly be productive., (© 2020 John Wiley & Sons Ltd.)

It is well known that factors acting on the decrease of population of honeybees, can act on the male and female reproductive system, compromising the fertility of queens and drones. While there are many studies on female fertility, only a few studies have focused on male fertility and the possible alterations of the reproductive system. The testes of 25 samples of adult drones of Apis mellifera ligustica were analyzed by histopathology using an innovative histological processing technique and the alterations that were found are here described. Most of the samples showed unaltered testes but, in some cases, samples showed degenerated seminiferous tubules, while others appeared immature. Although a limited number of samples were analyzed, the results obtained displayed that histopathological alterations of the testes exist also in honeybees and that more interest should be put to the matter, as honeybees could be considered as bioindicators for endocrine disruptors. Future studies on a larger number of samples are necessary to analyze how different environmental factors can act and induce alterations in the honeybee reproductive system.

Due to high rates of proliferation and DNA synthesis, neoplastic cells have higher requirements of iron than normal cells. For that reason, neoplastic cells have remodelled iron metabolism pathways, over-expressing genes encoding for iron uptake proteins, among which Transferrin Receptor-1 (TFR-1). Accumulating evidence has proven that overexpression of TFR-1 and high Iron concentration, are both widespread condition of cancer cells, both essential to tumour onset and progression. We studied TFR-1 and PCNA immunohistochemical expression in fifteen (15) Canine osteoblastic osteosarcomas (COS). After immunohistochemical staining, counting of TFR-1 positive cells by two independent observers showed that 85%-95% of neoplastic cells were strongly labelled at cytoplasmic level by anti-TFR-1 antibody in all examined COS. Furthermore, 70%-80% of neoplastic cells were positively labelled at the nuclear level by PCNA. Surprisingly, about 100% of intratumour vascular endothelial cells were also positive, whereas extratumour vascular endothelial cells were negative. The latter is an interesting finding, as TFR-1 is usually not expressed in normal vasculature, with the exception of normal brain vascular endothelium, where it allows transport of transferrin, and thus iron, into tissues, suggesting a similar function here to support cancer growth. The early results presented highlight the relevance of TFR-1 expression in canine OS, suggesting therapies involving both TFR-1 and Iron metabolisms in dogs with osteosarcoma should be developed., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Telomerase activity contributes to cell immortalization by avoiding telomere shortening at each cell division; indeed, its catalytic subunit telomerase reverse transcriptase (TERT) is overexpressed in many tumors, including human oral squamous cell carcinoma (hOSCC). In these tumors, matrix metalloproteinases (MMPs), a group of zinc-dependent endopeptidases involved in cell migration, contribute to invasive potential of cancer cells. A proportion of hOSCC is associated with infection by high-risk human papillomavirus (HR-HPVs), whose E6 oncogene enhances TERT and MMPs expression, thus promoting cancer progression. Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor with highly invasive phenotype; however, studies on telomerase activity, TERT, and MMPs expression are scarce. In this study, we demonstrate telomerase activity, expression of TERT, and its transcriptional activator cMyc along with expression of MMP-1, -2, and -9 in FOSCC-derived cell lines SCCF2 and SCCF3, suggesting a contribution by these pathways in cell immortalization and invasion in these tumors. Recent studies suggest that a sub-group of FOSCC as well as SCCF2 and SCCF3 are associated with Felis catus PV type-2 (FcaPV-2) infection. However, in this work, FcaPV-2 E6 gene knock-down caused no shift in either TERT, cMyc, or MMPs levels, suggesting that, unlike its human counterpart, the viral oncogene plays no role in their regulation., (Copyright © 2020 Altamura, Martano, Licenziato, Maiolino and Borzacchiello.)

Genetic alterations and/or epigenetic modifications occur frequently in the majority of cancer cells. In addition to playing a crucial role as promoters of tumorigenesis, these processes can also generate metabolic pathways that are different from those in normal cells. Besides the Warburg effect, an alteration in lipid metabolism is also found in cancer cells. Thus, elucidation of the regulators involved in this metabolic reprogramming might provide tools for diagnosis, prognosis, and ultimately treatment of canine mammary tumours (CMTs) in particular. One such regulator is carnitine palmitoyltransferase 1A (CPT1A), which is involved in transportation of long-chain fatty acids into the mitochondrial matrix for beta-oxidation, thereby providing an alternative pathway for the generation of energy for tumour growth and development. In this study, the canine cell lines MDCK, CMT-U309, CMT-U27, and P114 were used as in vitro models for western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Furthermore, western blot and immunohistochemistry were carried out to evaluate CPT1A protein expression in the CMT specimens. The CPT1A protein and mRNA expression levels were increased in the CMT cell lines relative to their levels in normal epithelial cells. Moreover, increased CPT1A expression levels were found in the CMT tissues, being inversely correlated with the tumour differentiation grade. However, additional studies are required to further specify the role of CPT1A in CMTs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: equine sarcoids are the most frequent skin tumors in equidae worldwide. It is well known that delta bovine papillomaviruses are their causative agents. We have recently shown the presence in equine sarcoids of abnormal vessel structures, which could cause a hypoxic condition. The aim of this study was to analyze the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in a subset of BPV positive equine sarcoids and explore the relationship with vascular endothelial growth factor (VEGF) expression., Results: 80% of equine sarcoids showed strong cytoplasmic staining in >60% of neoplastic fibroblasts, while 20% of samples showed a moderate cytoplasmic staining in 40-60% of neoplastic fibroblasts for HIF-1α. Results of Western blotting (WB) were consistent with immunohistochemistry (IHC). Moreover, a positive correlation between HIF-1α and VEGF expression (r = 0.60, p < 0.01) was observed., Conclusion: we have shown that HIF-1α was strongly expressed in equine sarcoid. The upregulation of HIF-1α has been described in numerous tumors and can be modulated by many proteins encoded by transforming viruses. Thus, it is also possible that BPV could have a relevant role in HIF-1α pathway regulation, contributing to the development of equine sarcoids by promoting HIF-1α/VEGF mediated tumor angiogenesis., Competing Interests: The authors declare no conflict of interest.

Buffalo mastitis is an important economic problem in southern Italy, causing qualitative/quantitative alterations in milk and resulting in economic losses due to the sub-clinical course and chronic evolution. We investigated 50 udders of slaughtered buffaloes and subjected them to effectual microbiological screening to evaluate macro and microscopic mammary gland changes, immune-characterize the cell infiltrates, and compare the degree of tissue inflammation with somatic cell counts. Numerous Gram-positive and Gram-negative bacteria were isolated from all samples, majority of which were environmental mastitis pathogens. Histological features referable to chronic mastitis were observed in 92% udders. Lymphocytes, plasma cells and macrophages were found to evolve into aggregates in 48% udders, which often organized to form tertiary lymphoid structures (TLSs). A predominance of interstitial CD8+ over CD4+ lymphocytes and, in TLSs, scattered CD8+ lymphocytes in the mantle cells and CD79+ lymphocytes in germinal centers, were evidenced. Environmental pathogens are known to persist and cause chronic inflammatory changes in buffaloes, where CD8+ lymphocytes play an important role by controlling the local immune response. Moreover, the TLSs evidenced here for the first time in buffalo mastitis, could play a role in maintaining immune responses against persistent antigens, thereby contributing in determining the chronic course of mastitis., Competing Interests: The authors declare no conflicts of interest., (© 2019 The Korean Society of Veterinary Science.)

E6 from high risk human papillomaviruses (HR HPVs) promotes ubiquitination and degradation of p53 tumour suppressor by mediating its binding to ubiquitin ligase E6AP in a ternary complex, contributing to cell transformation in cervical cancer. We have previously shown that Felis catus papillomavirus type -2 (FcaPV-2) E6 is expressed in feline squamous cell carcinoma (SCC) and displays the ability to bind p53 and decrease its protein levels in transfected CRFK cells. However, the mechanism of p53 downregulation has not yet been characterized. Here we show that FcaPV-2 E6 bound to E6AP, which in turn was bound by p53 exclusively in cells expressing the viral oncoprotein (CRFKE6). Furthermore, p53 was highly poly-ubiquitinated and underwent accumulation upon E6AP gene knockdown in CRFKE6. Half-life experiments and proteasome inhibition treatments indicated that down-regulation of p53 protein in CRFKE6 was due to accelerated proteasomal degradation. E6AP/p53 binding was also demonstrated in two feline SCC cell lines expressing FcaPV-2 E6, where p53 protein levels and poly-ubiquitination degree were proportional to E6 mRNA levels. The data obtained in both artificial and spontaneous in vitro models suggest that FcaPV-2 E6 degrades p53 through a molecular mechanism similar to HR HPVs, possibly contributing to the development of feline SCC.

Background: Sarcoids 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., Results: All 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., Conclusions: This 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.

Oral squamous cell carcinoma (OSCC) is one of the most aggressive and deadliest tumors worldwide. The aryl hydrocarbon receptor (AHR) is a nuclear transcription factor known as a dioxin receptor and mediates the toxic effects of industrial contaminants. In addition, AHR has been implicated in multiple cellular processes and its expression has been shown to play a critical role in tumorigenesis, including human oral cancer cell lines. In the present study, we evaluated the expression of AHR/HSP-90 in 25 formalin‑fixed, paraffin-embedded human oral cancer specimens by IHC analysis. CYP1A1 expression was regarded as an AHR reporter gene. The data indicated a complete correlation between AHR expression and cancer grade enabling us to propose AHR as a prognostic marker of oral cancer. Moreover, in OSCC cell line CAL27, we observed the modulatory effect of polydatin, a widespread natural substance and direct precursor of resveratrol, on AHR expression. A computational approach was performed to predict the site of interaction of polydatin on the AHR surface. Our studies confirm the involvement of AHR signaling in the clinicopathological specimens of oral cancer and suggest the use of polydatin for oral cancer prevention.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Equine sarcoids are locally invasive, fibroblastic benign skin tumors. Bovine papillomavirus type-1 (BPV-1) and/or Bovine papillomavirus type-2 (BPV-2) are believed to be the causative agent of sarcoids, although the mechanisms by which the virus induce the tumor are still poorly understood. We hypothesized that in genetically predisposed equines latent BPV infection may be reactivated by immunosoppression and/or mechanical injury leading to a form of pathologic wound which may transform into a sarcoid. In this study, we investigated in 25 equine sarcoids and in five normal skin samples the histological features and evaluated the immunohistochemical and molecular expression of type I and type III Collagen, vimentin (VIM), alfa Smooth Muscle Actin (α-SMA), Matrix Metalloproteinase (MMPs) -2, 9, 14 and tissue inhibitor of metalloproteinase 2 (TIMP-2)., Results: In 64% of investigated sarcoids, type I collagen staining was stronger than that of type III collagen. In 80% of sarcoids, SFs were strongly positive for vimentin and negative for α-SMA; the remaining sarcoid samples (20%) showed 70-80% of SFs labeled for vim and approximately 20-30% labeled for α-SMA. Moreover, all sarcoid specimen showed a variable staining pattern (weak to moderate) for MMP-9 and MMP-14, and a moderate to strong staining for MMP-2 and TIMP-2. Biochemical analysis confirmed immunohistochemical results and showed in sarcoids, for the first time, the cleaved form of MMP9, the 35 KDa active species for MMP-9., Conclusions: This study revealed that in equine sarcoids exhibit an altered turnover of the Extracellular Matrix (ECM) deposition and degradation, as result of an altered expression of MMPs and TIMPs. Therefore, these observations seem to confirm that the basic mechanism for growth of equine sarcoids could be a neoplastic transformation during wound healing.

Angiogenesis is crucial for the growth and metastasis of malignant tumours, and various proangiogenic factors promote this process. One of these factors is vascular endothelial growth factor (VEGF), which appears to play a key role in tumour angiogenesis. The aim of the present study was to assess whether VEGF expression is associated with angiogenesis, disease progression and neoplastic proliferation in canine oral squamous cell carcinoma (OSCC) tissue. VEGF immunoreactivity was quantified by immunohistochemistry in 30 specimens, including normal oral mucosa and OSCC tissues graded as well, moderately or poorly differentiated. VEGF expression was correlated with tumour cell proliferation, as assessed using the proliferating cell nuclear antigen (PCNA) marker and microvessel density (data already published). The present results revealed that VEGF and PCNA expression increased significantly between normal oral tissue and neoplastic tissue, and between well and moderately/poorly differentiated tumours. In addition, VEGF expression was strongly correlated with PCNA expression and microvessel density. It was concluded that VEGF may promote angiogenesis through a paracrine pathway, stimulating endothelial cell proliferation and, similarly, may induce tumour cell proliferation through an autocrine pathway. The present results suggest that the evaluation of VEGF may be a useful additional criterion for estimating malignancy and growth potential in canine OSCCs.

We previously showed that the innovative radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) improves metabolic dysfunctions in a diabetic mouse model. Here, we compared the in vivo effects of IAC with those of the anti-diabetic drugs pioglitazone (PIO) and exendin-4 (EX-4). Diabetes was induced in C57Bl/6J mice by streptozotocin and nicotinamide administration. Paralleled by healthy controls, diabetic animals (D) were randomly assigned to four groups and treated daily for 7 consecutive weeks: D+saline, ip; D+IAC 30mg/kgb.w., ip; D+PIO 10mg/kgb.w. per os; and D+EX-4, 50μg/kgb.w., ip. Our results show that IAC reduced basal hyperglycemia and improved glucose tolerance better than PIO or EX-4. Interestingly, in the heart of diabetic mice, IAC treatment normalized the increased levels of GSSG/GSH ratio and thiobarbituric acid reactive substances, indexes of oxidative stress and damage, while PIO and EX-4 were less effective. As supported by immunohistochemical data, IAC markedly prevented diabetic islet β-cell reduced density, differently from PIO and EX-4 that had only a moderate effect. Interestingly, in diabetic animals, IAC treatment enhanced the activity of pancreatic-duodenal homeobox 1 (PDX-1), an oxidative stress-sensitive transcription factor essential for maintenance of β-cell function, as evaluated by quantification of its nuclear immunostaining, whereas PIO or EX-4 treatments did not. Altogether, these observations support the improvement of the general redox balance and β-cell function induced by IAC treatment in streptozotocin-nicotinamide diabetic mice. Furthermore, in this model, the correction of diabetic alterations was better obtained by treatment with the radical scavenger IAC than with pioglitazone or exendin-4., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012. Published by Elsevier B.V.)

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.

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.

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 (PDGF β R) was shown to occur. The results show that the BPV-2 E5 oncoprotein binds to the activated form of the PDGF β 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.

Background: The aim of the present study was to evaluate, by immunohistochemical staining, metallothionein expression in normal, benign and malignant canine apocrine gland tissues and to correlate the protein expression with the histological grade of malignancy., Materials and Methods: MT immunostaining was evaluated in 25 formalin-fixed and paraffin-embedded canine apocrine glands (2 normal and 23 neoplastic). Moreover, we evaluated quantitative expression of MT in normal and neoplastic cells by western blotting., Results: a marked increase in MT expression was observed in neoplastic, compared to normal samples as well as in malignant compared to benign tumors. Western blotting analysis revealed one major protein band of approximately 14 kDa in normal, as well as in the neoplastic, tissues., Conclusion: We conclude that MT expression appears to be a potential biomarker for the diagnosis of canine apocrine gland tumors and may also assist in the better understanding of the evolution of this neoplasia.

The particular combination of polydeoxyribonucleotides, l-carnitine, calcium ions, proteolytic enzyme and other ingredients acts in a synergetic way in the regeneration of skin and connective tissues. This new formulation of active principles was tested in vitro as a cell and tissue culture medium and in vivo for various preparations in support of tissue regeneration. In vitro, the new blend allowed the maintenance of skin biopsies for more than 1 year in eutrophic conditions. Immunocytochemical analyses of fibroblasts isolated from these biopsies confirmed a significant increase of the epidermal and connective wound-healing markers such as collagen type I, collagen type IV, cytokeratin 1 (CK1), CK5, CK10 and CK14 versus controls. To examine the effects of the new compound in vivo, we studied impaired wound healing in genetically diabetic db/db mice. At day 18, diabetic mice treated with the new composition showed 100% closure of wounds and faster healing than mice treated with the other solutions. This complex of vital continuity factors or life-keeping factors could be used as a tissue-preserving solution or a cosmetic/drug/medical device to accelerate wound healing in the treatment of patients with deficient wound repair to promote the regeneration of cutaneous and connective tissues (injuries-wound, dermatitis) and prevent the recurrent relapses., (Copyright © 2011 John Wiley & Sons, Ltd.)

The removal of pathologically generated free radicals produced during ischemia, reperfusion and intracranical hemorrhage seems to be a viable approach to neuroprotection. However, at present, no neuroprotective agent has proven effective in focal ischemic stroke phase III trials, despite the encouraging data in animal models. This study aimed to explore the effect of the brain penetrant low molecular weight radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in neurological damage subsequent to ischemia-reperfusion injury in Mongolian gerbils. We examined the intraperitoneal effects of IAC on temporary bilateral common carotid artery occlusion (BCCO) by means of morphological and histological analysis of the hippocampus. Significant dose-dependent protective effects of IAC (1 to 10mg/kg b.w.) against neuropathological and morphological brain changes were seen when administered i.p. 1h before temporary BCCO in Mongolian gerbils. When administered up to 6h after BCCO, IAC actually reverses the neurodegenerative processes (e.g. hippocampal cell viability) induced by ischemia in a dose-dependent fashion. Data show that IAC is highly effective in protecting and preventing oxidative brain damage associated with cerebral flow disturbances. It is also effective even in late treatment of the insult, emphasizing its potential role for the management of ischemic stroke patients.

Background: Chronic exposure to high free fatty acids (FFA) can lead to irreversible damage of beta-cell accounting for impaired insulin secretion. Multiple mechanisms concur in generating the damage, but activation of oxidative stress may contribute to the final toxic effect. To better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 24-h pre-culture with 1.0 mmol/l FFA on the function, survival and mRNA expression of several enzymes involved in the generation and scavenging of reactive oxygen species (ROS)., Material and Methods: Human islets, prepared by collagenase digestion and density gradient purification from 9 pancreases of multiorgan donors, were incubated for 24-h in the presence 1.0 mmol/l long-chain mixture (oleate:palmitate, 2:1) FFA, with or without 100 micromol/l IAC, a non-peptidyl low molecular weight radical scavenger. At the end of incubation period, insulin secretion was measured by static incubation, and mRNA expression of insulin, Cu/Zn-SOD, Mn-SOD, Catalase, Glutathione peroxidase (GSH-px) and HO-1 by quantitative Real-Time RT-PCR. Nitrotyrosine levels were determined by an ELISA technique., Results: As compared to control incubation (Ctrl, no FFA), exposure to FFA was associated with impaired insulin release and reduced insulin mRNA expression. The presence of IAC in the incubation medium increased insulin release significantly and prevented changes in mRNA expression. Exposure to FFA was associated with oxidative stress as indicated by a significant accumulation of nitrotyrosine and IAC restrained such an increase. mRNA expression of Cu/Zn-SOD, Mn-SOD, Catalase, GSH-Px, and HO-1 were all modified after FFA exposure. These changes were partially prevented in the presence of IAC., Conclusions: In human islets 24-h exposure to high FFA causes oxidative stress associated with changes of several enzymes involved in ROS scavenging. These effects were prevented by the use of an antioxidant molecule.

The present study aimed at evaluating, through immunohistochemical staining, E-cadherin, beta-catenin and adenomatous polyposis coli (APC) expression and distribution in normal, dysplastic and neoplastic canine colon and rectum, and at correlating the protein expression with the histological grade of malignancy. In order to obtain a more thorough evaluation of the APC/beta catenin complex, both proteins were also assessed for colocalization in neoplastic cells through double immunofluorescence staining. Immunohistochemical investigation showed a marked decrease of E-cadherin and APC expression in malignant tumours, while a lack of membranous beta-catenin distribution and a cytoplasmic positivity, rather than a decrease of expression, was observed in less differentiated carcinomas. Confocal laser microscopical observation showed cytoplasmic beta-catenin distribution solely in APC-negative cells, demonstrating that the correct beta-catenin distribution on the cell membrane can be APC dependent, as in human colorectal carcinomas. Therefore, the changes in adhesion molecules can play a very important role both in colorectal carcinogenesis and in malignant progression; moreover, these proteins can also be considered powerful prognostic tools in veterinary oncology.

Background: Mammary tumors are a very common neoplasm in the dog and show histological features and biological behaviour similar to human mammary carcinomas. Recently, a pathway named Wnt-1, involving beta-catenin and APC protein, has emerged as an important player in many human tumor types, including mammary neoplasms., Materials and Methods: Thirty-five samples of canine mammary tumors (10 benign and 25 malignant) were studied in order to evaluate the co-expression of beta-catenin, APC protein and E cadherin with confocal laser microscopical observation, by western blot analysis and by correlating data obtained with the histological grade of tumours., Results: A progressive decrease of E-cadherin together with disruption of beta-catenin expression was observed in less differentiated malignant tumors. In addition, a loss of beta-catenin membranous distribution and a cytoplasmic accumulation was often coexpressed with disrupted expression of APC protein. Western blot analysis showed a progressive increase of beta-catenin in malignant tumors, which could be the expression of disrupted /-catenin catabolism leading to cytoplasmic accumulation. In some less differentiated malignant tumors, a marked beta-catenin decrease was also observed. This feature could be linked to mutations in beta-catenin gene coding for a truncated and lighter protein., Conclusion: These results may indicate the multifunctional role played by beta-catenin in canine mammary oncogenesis.