Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

Strongylus vulgaris (Looss, 1900) in horses in Italy: is it still a problem?

A post-mortem survey was carried out on 46 Sardinian horses to evaluate the presence of Strongylus vulgaris and associated pathology. Horses were from local farms and had been treated with broad-spectrum anthelmintics at least 3 times a year. Examination of the cranial mesenteric arterial system (CMAS) showed parasite-induced lesions in all horses. S. vulgaris larvae were found in 39% of examined arteries, while their detection rate in coprocultures was 4%. Histology, carried out on 26 horses, showed mainly chronic and chronic-active lesions. Histometry showed a significant increase in thickness of the arterial wall, in particular of the intima tunic and adventitia tunic of the ileocolic artery and its colic branch. MCV, MCHC and alpha2, beta and gamma globulins were increased in horses with S. vulgaris larvae in the arteries, while the albumin/globulin ratio was decreased. Horses that were positive on faecal examination showed decreased values for RBC, PCV and the albumin/globulin ratio. Although several studies have shown a dramatic decrease of S. vulgaris infection worldwide, our data show that this parasite continues to exert its pathogenic role, even when its detection rate is quite low within the strongyle population infecting horses.

Mammography and ultrasound imaging of preinvasive and invasive canine spontaneous mammary cancer and their similarities to human breast cancer.

Understanding the evolution of proliferative breast disease such as atypical hyperplasia and carcinoma in situ is essential for clinical management of women diagnosed with these lesions. Therefore, an animal model that faithfully represents human breast disease in every aspect from spontaneity of dysplasia onset, histopathologic features, and genetics to clinical outcome is needed. Previously, we studied canine spontaneous atypical hyperplasia and ductal carcinoma in situ (low, intermediate, and high grade) and reported their similarities to human lesions in histopathologic and molecular features as well as prevalence. To further validate the resemblance of these lesions to humans, we examined their mammographic and sonographic characteristics in comparison with those of human's as well as the potential of the human Breast Imaging Reporting and Data System (BI-RADS) to predict canine disease. Nonlesional, benign, and malignant mammary glands of dogs presented to Sassari Veterinary Hospital were imaged using mammography and ultrasonography. The images where then analyzed and statistically correlated with histopathologic findings and to their similarities to humans. Our results showed that canine mammary preinvasive lesions, benign, and malignant tumors have mammographic abnormalities, including the presence, pattern, and distribution of macrocalcification and microcalcification, similar to their human counterparts. BI-RADS categorization is an accurate predictor of mammary malignancy in canine, with 90% sensitivity and 82.8% specificity. The similarities of mammographic images and the ability of BI-RADS to predict canine mammary malignances with high specificity and sensitivity further confirm and strengthen the value of dog as a model to study human breast premalignancies for the development of prognostic biomarkers.

Immunohistochemical localisation and molecular expression of the steroidogenic enzyme cytochrome P450 17α-hydroxylase /C(17,20)-lyase in the vestibular nuclei of adult male rats.

Many biologically active neurosteroids, including dehydroepiandrosterone (DHEA), are synthesised in the brain. DHEA is a potent endogenous modulator of several neuronal functions, and alterations of DHEA are correlated with various neurobiological deficits. The cytochrome P450 17α-hydroxylase/C(17,20)-lyase (P450C(17) ) plays a pivotal role in the synthesis of DHEA from pregnenolone and progesterone. We investigated the immunohistochemical localisation and molecular expression of P450C(17) in the superior, lateral, medial and inferior vestibular nuclei (VCN) of adult male rats by western blotting and indirect immunofluorescence analysis. Immunoreactive P450C(17) was widely distributed in all VCN and the expression of P450C(17) was confirmed by western blot analysis. The present study demonstrates, for the first time, the presence and anatomical distribution of P450C(17) in the VCN. Given that neurosteroids can modulate neuronal activities in the medial vestibular nucleus, DHEA synthesised in the VCN may play an important role in the control of specific activities at this level.

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species.

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.

Bilateral ovarian malignant mixed Mullerian tumor in a dog.

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital tract. We report a case of bilateral ovarian MMMT in a 10-year-old female dog. Ovaries were only moderately enlarged with a papillary surface and firm nodules. Multiple metastases were observed in the abdominal cavity and pulmonary parenchyma. Histologically, both ovaries had intermingled carcinomatous and sarcomatous components with cartilage and bone. Metastatic lesions were not mixed. The peritoneal metastases were carcinomatous; pulmonary metastases were sarcomatous. Carcinomatous elements of the MMMT were immunohistochemically positive for cytokeratin (CK) AE1/AE3, CK7, and vimentin and estrogen receptors. Conversely, the sarcomatous cells were positive for vimentin, but negative for CKs. Chondrocytes also expressed S-100 protein. On the basis of similarities to human ovarian MMMT, the diagnosis was heterologous malignant mixed Müllerian tumor of the ovary.

Association of jaagsiekte sheep retrovirus with pulmonary carcinoma in Sardinian moufflon (Ovis musimon).

Bronchiolo-alveolar carcinoma has been described in man and in several animal species, including cattle, dogs, opossums, goats and sheep. In sheep, a bronchiolo-alveolar carcinoma, known as ovine pulmonary carcinoma (OPC), is caused by jaagsiekte sheep retrovirus (JSRV), an exogenous type D retrovirus. In the mid-1980s, a severe outbreak of a disease resembling OPC was described in captive Sardinian moufflon (Ovis musimon). In the present study, the use of polymerase chain reaction (PCR) amplification of nucleic acids extracted from archival material established that JSRV was associated with OPC in affected moufflon. JSRV was detected in the lungs and mediastinal lymph nodes. Immunohistochemical and in-situ PCR demonstrated that in the lungs, JSRV proviral DNA was localized in transformed and untransformed type II pneumocytes and in the alveolar macrophages. In the mediastinal lymph nodes, JSRV DNA was mainly located in the cortical follicles and paracortex. These data suggest that JSRV is the cause of OPC in Sardinian moufflon, as it is in Sardinian sheep.

Cadmium chronic administration to lactating ewes: reproductive performance, cadmium tissue accumulation and placental transfer.

20 lactating ewes were allotted to two groups: 10 subjects received orally 100 mg/day of CdCl2 for 108 consecutive days, and the remaining 10 acted as control. Reproductive performance in ewes and cadmium tissue accumulation, both in ewes and their lambs, were investigated. The results showed that in ewes: 1) the regular cadmium intestinal intake negatively influences all reproductive parameters; 2) cadmium is particularly accumulated in kidney and liver, but also in mammary gland, although at a distinctly lower level; 3) chronic administration does not increase cadmium placental transfer in lactating pregnant subjects.

Dermatophilosis in sheep: first report in Italy, experimental reproduction and evaluation of immune response.

A severe exudative-crustous and proliferous dermatitis in a 2 year old sheep caused by Dermatophilus congolensis (observed for the first time in Italy), is reported. The disease was reproduced experimentally in sheep, goats, rabbits and guinea pigs, whose skin was treated in different ways before infection. E.L.I.S.A. and Immunoblotting tests carried out in experimentally infected sheep, showed the antigenic complexity of the pathogen and the existence of cross-immunity to the protein components. Intradermoreaction tests were carried out in all animals. The development of a positive reaction only in rabbits and guinea pigs, confirmed that these animals have a cellular immunity against Dermatophilus congolensis.