Punica granatum L. Extract Shows Cardioprotective Effects Measured by Oxidative Stress Markers and Biomarkers of Heart Failure in an Animal Model of Metabolic Syndrome.

Metabolic syndrome (MetS) significantly increases the risk of cardiovascular diseases (CVD), a leading cause of death globally. The presented study investigated the cardioprotective role of dietary polyphenols found in pomegranate peels in an animal model of metabolic syndrome. Zucker diabetic fatty rats (ZDF, MetS rats, fa/fa) were supplemented with polyphenol-rich pomegranate peel extract (EPP) at two dosages: 100 mg/kg BW and 200 mg/kg BW. The extract was administered for 8 weeks. The effect of ethanolic peel extract on the concentration of oxidative stress markers (CAT, SOD, MnSOD, GR, GST, GPx, TOS, SH, and MDA), biomarkers of heart failure (cTnI, GAL-3), and alternations in tissue architecture was assessed. The results showed a significant increase in SH concentration mediated via EPP supplementation (p < 0.001). Treatment with a 100 mg/kg BW dosage reduced the TOS level more efficiently than the higher dose. Interestingly, the CAT and GST activities were relevantly higher in the MetS 100 group (p < 0.001) compared to the MetS control. The rats administered EPP at a dose of 200 mg/kg BW did not follow a similar trend. No differences in the GR (p = 0.063), SOD (p = 0.455), MnSOD (p = 0.155), and MDA (p = 0.790) concentration were observed after exposure to the pomegranate peel extract. The administration of EPP did not influence the cTnI and GAL-3 levels. Histology analysis of the heart and aorta sections revealed no toxic changes in phenolic-treated rats. The findings of this study prove that the extract from pomegranate peels possesses free radical scavenging properties in the myocardium. The effect on alleviating ventricular remodeling and cardiomyocyte necrosis was not confirmed and requires further investigation.

Expression of Foxp3 in Fibromas and Fibrosarcomas of Skin and Subcutaneous Tissue in Dogs.

BACKGROUND/AIM: Foxp3 is a transcription factor responsible for the formation of T regulatory lymphocytes. Foxp3 expression can be associated with either neoplastic progression or regression. The aim of the study was to evaluate Foxp3 expression in soft tissue tumours (fibromas and fibrosarcomas) of skin and subcutaneous tissue in dogs and to describe its relationship with tumour malignancy grade. MATERIALS AND METHODS: The study was conducted on 71 skin and subcutaneous tumours including 31 fibromas and 40 fibrosarcomas. The samples underwent histological and immunohistochemical evaluation using anti-Foxp3, anti-Ki, and vimentin antibodies. RESULTS: Cytoplasmic expression of Foxp3 protein in the cutaneous and subcutaneous fibrosarcomas in dogs was confirmed. Moreover, a positive relationship between the expression of Foxp3 and tumour malignancy grade and between Foxp3 and Ki-67 expression was noted. CONCLUSION: A positive correlation between the Foxp3 expression intensity and malignancy grade suggests a significant role of Foxp3 in the carcinogenesis of skin and subcutaneous fibrosarcomas in dogs. Increased expression of Foxp3 may have a positive effect on the progression of cancer.

Prevalence of Tumours and Tumour-like Lesions in Domestic and Exotic Animals from Lower Silesia and its Surrounds in Poland in 2014-2017.

Introduction: An analysis of samples originating from domestic and exotic animals from Lower Silesia but also from other parts of Poland was carried out based on research conducted in 2014-2017. Material and Methods: The materials used for the study were 11,338 tumour samples taken in vivo during surgery and as biopsies and posthumously during necropsies. They were sent to the Department of Pathology at Wroclaw University of Environmental and Life Sciences for histopathological diagnosis. Results: The largest group were canine tumours, of which there were 9,745 (85.95%), followed by feline tumours, totalling 1,397 (12.32%), tumours from exotic animals (186; 1.64%), from horses (7; 0.06%), and from cows (2; 0.02%). A significant number of cases of animals suffering from more than one tumour were also found, which had not been frequently diagnosed previously. Conclusion: The number of neoplasms diagnosed in pets and exotic animals is increasing every year. The average animal age at diagnosis continues to fall. The greatest number of neoplasms were diagnosed in mixed-breed dogs and cats, and the number of tumours in a pure breed strictly correlated with breed's popularity in the research period. Mesenchymal tumours are still the most prevalent type of tumours in dogs, while in cats epithelial tumours predominate. The neoplasm case pattern in animals conforms to that in humans in the same area.

In Vitro Studies on the Influence of Meloxicam on Cytotoxic Activity Induced by Risedronate Sodium in Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines.

The study describes the cytotoxic effect against human and canine osteosarcoma (U-2 OS and D-17) cell lines induced by risedronate sodium and meloxicam per se and in combination. Both cell lines were prepared according to standard procedures for cell cultures studies. The cell viability was estimated in both cell lines treated with chosen concentrations of risedronate sodium and meloxicam. The apoptosis assessment was carried out using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. EC50 values, computed for risedronate sodium and meloxicam cytotoxicity, showed comparable effects against the canine OS cell line in similar concentration of both drugs. In case of human OS, the stronger cytotoxic effect of risedronate sodium was proved. The EC50 values for meloxicam in both cell lines were, statistically, significantly different (* p < 0.05). Moreover, the cytotoxic effect of a combined administration of meloxicam and risedronate sodium in doses 100 µg/mL, compared with the negative control showed statistically significant differences. The human OS cell line was more resistant to both compounds than the canine OS cell line. The apoptotic effect in canine and human osteosarcoma triggered by risedronate sodium and meloxicam was statistically significant (p < 0.05). The cytotoxic effect induced with 100 µg/mL of risedronate sodium proved statistically significant differences between both tested cell lines compared to negative control. The results obtained with 10 and 100 µg/mL of meloxicam were not statistically significant. The study showed the synergic mechanism of action of risedronate sodium and meloxicam, but the concentrations used in vitro will not be possible to achieve in in vivo. Therefore, our results serve as basis only to design future studies on the tissue level.

TRPV1 Receptor Identification in Bovine and Canine Mitral Valvular Interstitial Cells.

Myxomatous mitral valve degeneration (MMVD) is the most common acquired cardiac disease in canine species, and valvular interstitial cells (VICs) are considered the main responsible for the development of this pathology. The scientific interest is focused on isolating and characterizing these cells. The aims of the present study were to verify a novel VICs mechanical isolation method and to characterize isolated cells using immunocytochemistry and immunofluorescence, with parallel histological and immunohistochemistry assays on bovine and canine healthy and MMVD mitral valves. Antibodies against vimentin (VIM), smooth muscle actin (SMA), von Willebrand (vW) factor, Transforming Growth Factor (TGF) ß1, and Transient Receptor Potential Vanilloid 1 (TRPV1) were used. The isolation method was considered reliable and able to isolate only VICs. The different assays demonstrated a different expression of SMA in healthy and MMVD mitral valves, and TRPV1 was isolated for the first time from bovine and canine VICs and the correspondent mitral valve leaflets. The novelties of the present study are the new isolation method, that may allow correlations between laboratory and clinical conditions, and the identification of TRPV1, which will lead to further investigations to understand its function and possible role in the etiology of MMVD and to the design of new therapeutic strategies.

Endosialin (CD248) Expression in Fibromas and Soft-tissue Fibrosarcomas in Dogs.

BACKGROUND/AIM: Endosialin is present in human fibrosarcoma neoplastic cells. This study aimed to analyse the expression of selected cellular proteins found in fibrosarcomas and soft-tissue fibroids in dogs. MATERIALS AND METHODS: A total of 71 skin tumours obtained from dogs were used. The samples included 31 fibromas and 40 fibrosarcomas. Histopathological evaluation was performed according to World Health Organization guidelines. Immunohistochemistry was performed with anti-endosialin, Ki-67, cyclo-oxygenase 2 and vimentin antibodies and assessed using the semi-quantitative scale. RESULTS: Endosialin expression was observed in 82.5% of fibrosarcomas and in 35% of fibromas. A significant positive correlation was found between the expression of endosialin in fibrosarcoma neoplastic cells and the degree of histological malignancy and the expression of the Ki-67 and cyclo-oxygenase 2 antigen. Expression of vimentin confirmed mesenchymal origin of this tumours. CONCLUSION: The results of our research suggest that endosialin is involved in the carcinogenesis of fibrosarcoma in dogs.

Urothelial cancer of the prostate gland. Immunohistochemical analysis of two cases of rare canine cancer.

INTRODUCTION: The study aimed to diagnose urothelial cancer of prostate gland (UCPG) in dogs as the primary focus and in its metastasis based on the expression of specific proteins used in immunohistochemical diagnosis of prostate cancer in men. MATERIAL AND METHODS: The study was carried out on specimens collected during a post-mortem examination from macroscopic lesions of the prostate glands from two dogs. The immunoexpression of the following proteins was verified: prostate-specific antigen (PSA), high molecular weight cytokeratins (HMWCK), cytokeratin 7 and 20 (CK-7,-20), E-cadherin, von Willebrand factor, cyclooxygenase-2 (COX-2), microsomal PGE2-1 synthase (mPGES-1) and component of the minichromosome 7 maintenance complex (MCM7). RESULTS: All markers, except for PSA, were expressed both at the primary tumour site and in the metastasis. CONCLUSIONS: The immunohistochemical approach was more useful for the diagnosis of UCPG in dog than typical histopathological staining methods because it allowed for precise determination of features, type and grade of the tumour that may affect its early detection and treatment.

Expression of Periostin in Mammary Cancer Cells of Female Dogs.

BACKGROUND/AIM: Periostin (POSTN) has a significant role in proliferation and migration of tumour cells as well as tumour progression. This study aimed to determinate POSTN expression in cancer cells in malignant and benign tumours of the mammary gland in female dogs. MATERIALS AND METHODS: All together 83 cancers, 24 adenomas and 7 unchanged fragments of the mammary glands of bitches were investigated. Immunohistochemistry was performed using anti-POSTN, Ki-67 and HER2 antibodies. RESULTS: POSTN expression was observed in cancer cells in 31.3% of malignancies and 12.5% of benign tumours. A significantly positive correlation between expression of POSTN in cancer cells and the degree of histological malignancy, expression of Ki-67 antigen and expression of POSTN in CAFs was found. CONCLUSION: The obtained results suggest the possible participation of POSTN in the process of carcinogenesis and progression of mammary tumors in bitches.

Expression of Periostin in Cancer-associated Fibroblasts in Mammary Cancer in Female Dogs.

BACKGROUND/AIM: Mammary neoplasms, like breast neoplasms in women, are one of the most common tumours in female dogs. Cancer-associated fibroblasts (CAFs) found in the tumour stroma play a role in angiogenesis and increase cell migration, contributing to tumour growth and progression, as well as metastasis. The aim of our work was to determine the level of periostin (POSTN) expression in CAFs in mammary tumours of female dogs. MATERIALS AND METHODS: The research material consisted of 77 carcinomas and 24 adenomas of the mammary ridge in female dogs. Immunohistochemistry tests were performed using antibodies directed against the antigens POSTN, Ki-67, ERB-B2 receptor tyrosine kinase 2 (HER2), vimentin, and alpha smooth muscle actin (αSMA). Expression of POSTN at the mRNA level was determined using real-time polymerase chain reaction methods in 20 cases of mammary neoplasms. RESULTS: Expression of POSTN in CAFs was observed in 92% of mammary cancer samples and in 25% of mammary adenoma samples in female dogs. A statistically significant increase in POSNT expression in CAFs was found in the carcinomas compared with mammary adenomas in female dogs. Expression of POSTN in CAFs in mammary carcinomas in female dogs positively correlated with the histological malignancy grade of tumours and the expression of Ki-67 proliferative antigen. CONCLUSION: Our results suggest a role of POSTN on the pathogenesis of mammary tumours in female dogs. Moreover, POSTN may prove to be a useful marker in the evaluation of cancerous stroma of mammary tumours in female dogs, and may have prognostic significance.

Expression of Podoplanin in Mammary Cancers in Female Dogs.

BACKGROUND/AIM: Mammary neoplasms are very common tumours in female dogs. Cancer-associated fibroblasts (CAFs) play an important role in the oncogenesis process. One of the useful proteins used in the diagnostics of CAFs cells is podoplanin (PDPN). The aim of our study was to assess the expression of PDPN in mammary cancer in female dogs. MATERIALS AND METHODS: Our study cohort included 61 cancers and 21 adenomas of the mammary tumour in bitches. Expression of podoplanin, Ki-67 and HER2 was determined using the Immunohistochemical (IHC) method. PDPN expression at the mRNA level was determined using real-time PCR. RESULTS: Expression of PDPN in CAFs was observed in 22.9% of cases of mammary cancers in bitches, with no PDPN expression in adenomas. A positive correlation was found between the expression of PDPN in CAFs and the grade of histological malignancy and expression of Ki-67. CONCLUSION: PDPN plays a significant role during the process of carcinogenesis of mammary tumours in female dogs.

Cardiomyocyte marker expression in dogs with left atrial enlargement due to dilated cardiomyopathy or myxomatous mitral valve disease.

INTRODUCTION: Dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD) are common heart conditions in dogs. They have different etiology and pathogenesis and although other studies focused on changes in the left ventricles of the affected hearts, the aim of our study was to assess the expressions of some intrinsic proteins in the enlarged left atria. MATERIAL AND METHODS: We performed an immunohistochemical analysis of left atrial specimens obtained from 15 dogs with DCM, 35 dogs with MMVD and six control dogs. We assessed the expression of following proteins: SERCA1, SERCA2, sarcomeric actinin, smooth muscle actin, and dystrophin. RESULTS: We noted a higher percentage of SERCA1-positive cells in the MMVD group and lower percentage of dystrophin-positive cells in the DCM group as compared to control group. The expression of other proteins was similar in the hearts of control dogs and dogs with heart diseases. CONCLUSIONS: The observed changes in the expression patterns of some proteins in the atria of dogs with DCM and MMVD suggest that atrial enlargement relies not only on volume overload, but also alterations of the intrinsic proteins can contribute to the pathogenesis of dilated cardiomyopathy.

The immunohistochemical evaluation of selected markers in the left atrium of dogs with end-stage dilated cardiomyopathy and myxomatous mitral valve disease - a preliminary study.

BACKGROUND: Dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD) are the most common diseases noted in dogs. Although their pathogenesis varies, both include a significant enlargement of the left atrium. The study was carried out on left atrial specimens obtained from 56 dogs, including those from 34 dogs with clinically diagnosed MMVD, 15 dogs with DCM and 7 dogs without heart disease (control group). Dogs in the MMVD and the DCM groups presented with left atrial enlargement and stage D heart failure. The specimens underwent immunohistochemical examination using desmin, vimentin, periostin and caspase-3 antibodies. RESULTS: There were alterations in the expression of the studied proteins in the study groups compared to the control group. The changes included: irregularity of desmin cross-striation and desmosomes, a higher amount of vimentin-positive cells, a change in the periostin expression pattern from cytoplasmic to extracellular, and a lower expression of caspase-3. The alterations were more pronounced in the DCM group than in the MMVD group. CONCLUSIONS: During heart failure, the pattern of desmin, vimentin, periostin and caspase-3 expression alters in the left atrium, regardless of the cause. The changes are more pronounced in dogs with DCM than in dogs with MMVD and similar left atrial enlargement, suggesting that volume overload may not be the only cause of myocardial changes in DCM.

Epidemiological and pathological features of primary cardiac tumours in dogs from Poland in 1970-2014.

Primary heart tumours affect less than 1% of dogs. Due to their rare incidence, every research showing the frequency of cardiac tumours is valuable. Routine diagnostics is often complemented with immunohistochemical analysis. This study was conducted on 110 patient records from all veterinary faculties in Poland from dogs diagnosed with heart tumours between 1970 and 2014. The dogs' age, breed and sex with tumour localisation and histopathological diagnosis were analysed. Because of its most common incidence, samples of haemangiosarcoma underwent further examination with assessment of the expression of cell markers that have not been evaluated earlier (i.e. minichromosome maintenance proteins and beta-catenin). We noted 111 tumours including 88.3% malignant and 10.8% benign ones. Haemangiosarcoma and aortic body tumour were the most frequent cardiac neoplasms in the dogs examined (45.9% and 27.9% of all tumours, respectively). Immunohistochemical analysis of haemangiosarcoma showed a positive expression of all markers examined. CD31, vimentin, and beta-catenin showed a positive reaction in all 11 samples examined. At least one proliferative marker (Ki-67, MCM-3 or MCM-7) showed a positive reaction in each sample. MCM-3 showed a higher expression than the two other proliferative markers (P = 0.006), but only Ki-67 showed a positive correlation with the mitotic index (P > 0.05, r = 0.89). Although beta-catenin, MCM-3 and MCM-7 showed a positive reaction in the haemangiosarcomas examined, their usefulness as diagnostic and prognostic factors should be a topic of further research.

Surgical resection of peripheral odontogenic fibromas in African pygmy hedgehog (Atelerix albiventris): a case study.

BACKGROUND: Neoplastic lesions of the mammary gland, lymph nodes, or oral cavity in African pygmy hedgehogs (Atelerix albiventris) are common in captive animals. Chemotherapy and radiotherapy protocols have not yet been established for the African pygmy hedgehog. Thus, surgical resection is the current treatment of choice in this species. CASE PRESENTATION: A 5-year-old male African pygmy hedgehog showed multiple erythematous, round small tumors located in the oral cavity, on both sides of maxilla. The treatment of choice was surgical resection of tumors using a surgical knife under general anesthesia. Excised neoplastic lesions were diagnosed as peripheral odontogenic fibroma by histopathology. Six months after surgery relapse of tumors in the oral cavity was not observed. CONCLUSIONS: The treatment adopted in this case report is safe for the patient and provides the best solution for mild proliferative lesions of the oral cavity. To our knowledge this is the first report of surgical resection of oral tumors (peripheral odontogenic fibroma) in the African pygmy hedgehog.

Useful immunohistochemical indicators in canine mast cell tumours.

Morphological and immunohistochemical analysis of 45 canine mast cell tumours was performed to determine whether the proteins examined are useful for a more precise description of tumour morphology and a more reliable determination of the prognosis in patients. Tissue sections were stained according to the standard haematoxylin and eosin (HE) technique and with toluidine blue to demonstrate cytoplasmic granules. Immunohistochemical studies were performed, using the cell markers CD117 (c-kit), p16 and von Willebrand factor (FVIII). In CD117 three different staining patterns were observed: (1) membranous reaction, (2) intense staining of cytoplasm, and (3) a diffuse, delicate cytoplasmic reaction. Von Willebrand antibody was evaluated on the basis of the number of blood vessels stained. p16 expression was evaluated by scoring positive nuclear reaction. Positive expression was demonstrated for all examined antigens, but their level of expression differed depending on the grades of tumour malignancy. Statistical analysis of the results documented a pronounced positive correlation between the markers studied and the grade of tumour malignancy (P < 0.001). It was shown that each of the cell markers examined represents a useful prognostic indicator for patients with mast cell tumours. The calculated correlation coefficients demonstrate a strong association between the expressions of CD117, FVIII and p16, and the histological malignancy of a tumour.

Inhibin-α, E-cadherin, calretinin and Ki-67 antigen in the immunohistochemical evaluation of canine and human testicular neoplasms.

INTRODUCTION: The steady increase of dogs with diagnosed testicular neoplasms observed in recent years prompted us to carry out immunohistochemical (IHC) studies for their better characterization. The aim of the study was to analyze most common canine testicular neoplasms (seminomas, Leydig cell and Sertoli cell tumors) with selected IHC markers and to compare the expressions of these proteins in corresponding canine and human testicular tumors. MATERIAL AND METHODS: Studies were carried out on testicular canine tumors: 40 cases of seminoma, 40 cases of Leydig cell tumor and 40 cases of Sertoli cell tumor. Moreover, 15 cases of human seminomas and 5 cases of human Leydig cell tumors were also analyzed. Immunohistochemistry was performed on paraffin sections by standard technique using monoclonal anti-human antibodies against E-cadherin, inhibin-α, calretinin and Ki-67. The slides were subjected to computer-aided image analysis and the intensity of the immunoreactivity was assessed by a semi-quantitative scoring system. RESULTS: Due to the very low prevalence of the Sertoli cell-derived tumors in the human population, we were able to examine the markers' expression only in the canine gonadal tumors. We revealed that, apart from E-cadherin in Leydig cell tumors and calretinin in seminomas, the expression of all the analyzed markers in canine and human testicular tumors was similar. E.g. there was no immunoexpression of inhibin-α in 75% of canine and 100% of human cases of seminoma. The immunoreactivity of Ki-67 was intense in 40% of canine and 60% of human seminomas. Immunoexpression of inhibin-α in Leydig cell tumor was intense in 70% of canine and 100% of human cases, respectively. Also the immunoreactivity of calretinin was intense in 75% of cases of canine and 100% of human Leydig cell tumors. In 50% of canine and 40% of human Leydig cell tumors, the immunoexpression of Ki-67 was weak. CONCLUSIONS: The applied anti-human monoclonal antibodies against common antigens and markers of human testicular neoplasms could be routinely used for the immunohistochemical evaluation of canine testicular tumors.

Myocarditis in dogs: etiology, clinical and histopathological features (11 cases: 2007-2013).

BACKGROUND: Myocarditis is a disease caused by numerous etiological factors and characterized by a non-specific course. The only method allowing for precise characterization of inflammatory changes is the histopathological examination of heart muscle specimens. The study was conducted on heart muscle preparations from 11 dogs with ante-mortem diagnosis of cardiac disease. Animals presented with a poor response to an applied treatment or had suspected sudden cardiac death. The heart specimens were taken post-mortem, preserved and stained with haematoxylin and eosin. Subsequently, the presence and intensity of changes, i.e. inflammatory infiltration, the amount of connective tissue and features of cardiomyocyte degeneration were estimated. The specimens from dogs suspected of having a myocarditis of bacteriological etiology underwent additional bacteriological and immunohistochemical examination. RESULTS: The examination revealed an inflammatory infiltration of variable intensity combined with the degenerative changes in all dogs. There were vegetative and abnormal cystic forms of Borrelia burgdorferi sensu lato in 6 dogs. A Staphylococcus aureus infection was confirmed in one dog and an acute coronary syndrome with neutrophil infiltration was revealed in another one. CONCLUSIONS: Although the clinical pattern in patients with myocarditis is diverse, the definitive morphological diagnosis is made based on the histopathological examination. This examination can lead to a better understanding of the pathogenesis of the disease. To the best of our knowledge, this is the first description of myocarditis combined with the presence of spore forms of Borrelia burgdorferi sensu lato in the heart specimens of dogs.