Leonardo Della Salda

Heat shock protein expression in canine malignant mammary tumours

BackgroundAbnormal levels of Heat Shock Proteins (HSPs) have been observed in many human neoplasms including breast cancer and it has been demonstrated that they have both prognostic and therapeutic implications. In this study, we evaluated immunohistochemical expression of HSPs in normal and neoplastic canine mammary glands and confronted these results with overall survival (OS), in order to understand the role of HSPs in carcinogenesis and to establish their potential prognostic and/or therapeutic value.MethodsImmunohistochemical expression of Hsp27, Hsp72, Hsp73 and Hsp90 was evaluated in 3 normal canine mammary glands and 30 malignant mammary tumours (10 in situ carcinomas, 10 invasive carcinomas limited to local structures without identifiable invasion of blood or lymphatic vessels, 10 carcinomas with invasion of blood or lymphatic vessels and/or metastases to regional lymph nodes). A semi-quantitative method was used for the analysis of the results.ResultsWidespread constitutive expression of Hsp73 and Hsp90 was detected in normal tissue, Hsp72 appeared to be focally distributed and Hsp27 showed a negative to rare weak immunostaining. In mammary tumours, a significant increase in Hsp27 (P < 0.01), Hsp72 (P < 0.05) and Hsp90 (P < 0.01) expression was observed as well as a significant reduction in Hsp73 (P < 0.01) immunoreactivity compared to normal mammary gland tissue. Hsp27 demonstrated a strong positivity in infiltrating tumour cells and metaplastic squamous elements of invasive groups. High Hsp27 expression also appeared to be significantly correlated to a shorter OS (P = 0.00087). Intense immunolabelling of Hsp72 and Hsp73 was frequently detected in infiltrative or inflammatory tumour areas. Hsp90 expression was high in all tumours and, like Hsp73, it also showed an intense positivity in lymphatic emboli.ConclusionThese results suggest that Hsp27, Hsp72 and Hsp90 are involved in canine mammary gland carcinogenesis. In addition, Hsp27 appears to be implicated in tumour invasiveness and its high immunodetection in invasive tumours is indicative of a poorer clinical outcome.

Heat shock proteins in animal neoplasms and human tumours—a comparison

Heat shock proteins (HSPs) are implicated in all phases of cancer from proliferation, impaired apoptosis and sustained angiogenesis to invasion and metastasis. The presence of abnormal HSP levels in several human tumours suggests that these proteins could be used as diagnostic and/or prognostic markers, whilst the direct correlation between HSP expression and drug resistance in neoplastic tissues means they could also be used to predict cancer response to specific treatment. HSPs have also been successfully targeted in clinical trials modifying their expression or chaperone activity. Preliminary studies in veterinary medicine have also demonstrated the presence of altered HSP expression in neoplasms, and the study of carcinogenesis and the role of HSPs in animal models will surely be an additional source of information for clinical cancer research.

PDGFs and PDGFRs in canine osteosarcoma: new targets for innovative therapeutic strategies in comparative oncology.

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70-80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis. Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.

Gross and histopathological changes associated with Aelurostrongylus abstrusus and Troglostrongylus brevior in a kitten.

Aelurostrongylus abstrusus affects the respiratory system of domestic cats worldwide, causing different clinical signs. Recently, Troglostrongylus brevior, a parasite found only in feral and wild animals until 2010, has been regarded as a cause of potentially severe bronchopneumonia in domestic cats. The life cycle of both nematodes involves the same intermediate (i.e. land mollusks) and paratenic hosts (i.e. lizards, birds and small mammals). Biological and epidemiological drivers may cause the spreading of A. abstrusus along with a possible emergence of T. brevior from marginal areas and limited hosts. Given the merit in increasing knowledge on these potentially emerging nematodes, the present paper describes clinical features and the first post mortem gross and histopathological observations in a case of mixed infection by A. abstrusus and T. brevior in a kitten.

A short exposure to polychlorinated biphenyls deregulates cellular autophagy in mammalian blastocyst in vitro

BACKGROUND Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent an important risk factor of reproductive disorders in chronically exposed human populations. However, it is not known whether a short accidental exposure of embryos to PCBs before implantation might influence their further development and whether the effect might be reversible. METHODS AND RESULTS To this aim, in vitro-matured sheep blastocysts were incubated with 2 or 4 µg/ml Aroclor 1254 (A1254), a mixture of 60 PCB congeners for 48 h after which blastocyst proliferation and ability for outgrowth in vitro were assessed. Blastocysts exposed to A1254 showed: (i) reduced proliferation and cell number (particularly in the inner cell mass compartment); (ii) accumulation of vacuoles and lipid droplets, diffused mitochondrial damage and up-regulation of autophagy markers (ATG6 and LC3), all signs indicative of deregulated autophagy, and (iii) massive cell death. Although exposed embryos resumed growth following A1254 removal, their subsequent development remained severely perturbed. CONCLUSIONS These findings indicate that short exposure of blastocysts to PCBs leads to its damage characterized by deregulated autophagy and subsequent cell death.

Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, β-Catenin, P53, Caspase 3) in canine appendicular osteosarcoma

BackgroundOsteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment.Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS).ResultsNuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088).ConclusionsThe present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses.

Survivin in skin pathologies

Abstract:  Survivin is a member of the inhibitor of apoptosis (IAP) protein family acting at the intersection between proliferation and cell survival. This protein exhibits low or undetectable expression in most adult tissues but is increased in the majority of cancers. Suggested to be one of the most cancer‐specific proteins identified to date, survivin acts as a signalling node in tumour maintenance and, after first promising results, is now attracting increasing attention as a target in anti‐cancer therapy. In the skin, survivin has been implicated in a number of pathological conditions such as psoriasis and tumours of melanocytic and epithelial origin. Its expression can correlate with tumour severity, metastasis and decreased patient survival and has been inversely correlated with the sensitivity to cytotoxic agents used in anti‐cancer therapy. Survivin may also be of importance for normal epidermal homeostasis possibly supporting self‐renewal of epidermal stem cells. In this review, the authors summarize and discuss current data of survivin in skin biology and provide a comprehensive compilation of survivin expression in skin pathologies with focus on future therapeutical use.

Survivin expression in canine epidermis and in canine and human cutaneous squamous cell carcinomas

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is ubiquitously expressed during tissue development, undetectable in most normal tissues, but re-expressed in most cancers, including skin malignancies. Expression of survivin was evaluated retrospectively in 19 canine cutaneous squamous cell carcinomas (SCCs; one in situ; 16 well differentiated; one invasive, one lymph node metastasis) and 19 well differentiated SCCs from human beings. Seven specimens of normal canine skin were included. Immunohistochemical expression of full-length survivin was determined using a commercially available antibody. In addition, apoptotic rate [Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling index (TUNEL) index] and mitotic index (MI), counting mitoses in 10 high power fields (HPF), were determined. Scattered survivin positive nuclei were identified in the epidermal basal cell layer of normal canine skin. Nuclear survivin expression was identified in 18 of 19 human and in all canine SCCs, mainly along the base of the tumour cell population. Cytoplasmic survivin expression was rarely observed in human SCCs and in 84.2% of canine SCCs. The TUNEL index ranged from 0.1 to 2.6 in human beings and from 7.5 to 69.4 in dogs, while MIs ranged from 0 to 4 in human beings and dogs. No correlation was found between survivin expression and apoptotic or mitotic rates. Canine and human tumours showed similar nuclear survivin expression, indicating similar functions of the molecule. We demonstrated survivin expression in normal adult canine epidermis. Increased nuclear survivin expression in pre-neoplastic and neoplastic lesions demonstrates a possible association of survivin with development of SCCs in human beings and dogs.

Oxidative Stress and Protein Quality Control Systems in the Aged Canine Brain as a Model for Human Neurodegenerative Disorders

Aged dogs are considered the most suitable spontaneous animal model for studying normal aging and neurodegenerative diseases. Elderly canines naturally develop cognitive dysfunction and neuropathological hallmarks similar to those seen in humans, especially Alzheimers disease-like pathology. Pet dogs also share similar living conditions and diets to humans. Oxidative damage accumulates in the canine brain during aging, making dogs a valid model for translational antioxidant treatment/prevention studies. Evidence suggests the presence of detective protein quality control systems, involving ubiquitin-proteasome system (UPS) and Heat Shock Proteins (HSPs), in the aged canine brain. Further studies on the canine model are needed to clarify the role of age-related changes in UPS activity and HSP expression in neurodegeneration in order to design novel treatment strategies, such as HSP-based therapies, aimed at improving chaperone defences against proteotoxic stress affecting brain during aging.

Expression of heat shock proteins in premalignant and malignant urothelial lesions of bovine urinary bladder.

Abnormal heat shock protein (HSP) levels have been observed in a number of human tumours, where they are involved in all hallmarks of cancer. Since bovine urothelial tumours share striking morphological and biochemical features with their human counterparts, the aim of this study was to evaluate the immunohistochemical levels of Hsp27, Hsp60, Hsp72, Hsp73 and Hsp90 in 28 normal bovine urinary bladders and 30 bovine papillomavirus-positive urothelial tumours (9 in situ carcinomas, 9 low-grade and 12 high-grade carcinomas) and adjacent premalignant lesions obtained from cows suffering from chronic enzootic haematuria, in order to investigate the role of these proteins in the process of urothelial carcinogenesis. A semi-quantitative method was used for the analysis of the results. Western blot analysis was also used to confirm HSP expression in normal controls. All investigated HSPs were expressed in normal bovine urothelium, showing characteristic patterns of immunolabelling throughout urothelial cell layers, which usually appeared to be conserved in urothelial hyperplasia and dysplasia. On the other hand, gradual loss of Hsp27 immunostaining resulted to be significantly associated with increasing histological grade of malignancy (P < 0.01). As well, a significantly reduced immunosignal of Hsp73 and Hsp90 was observed in high-grade and low-/high-grade carcinomas, respectively (P < 0.01). In contrast, Hsp60 (P < 0.01) and Hsp72 (P < 0.05) immunoreactivity appeared to be significantly increased both in premalignant and malignant lesions when compared to that observed in normal urothelium, thus suggesting an early involvement of these proteins in neoplastic transformation of urinary bladder mucosa.