Salvatore Pirino

Spontaneous Mammary Intraepithelial Lesions in Dogs—A Model of Breast Cancer

Mammary intraepithelial lesions (IEL) are nowadays frequently diagnosed as a result of the success of mammographic screening, education programs, and awareness by women. Establishment of an animal model for these lesions to test treatment or preventive modalities is a prerequisite for human clinical trials. A model for spontaneous IELs, especially for estrogen receptor (ER)-negative lesions, does not exist. This study describes the histologic and immunohistochemical similarity between human and canine mammary IELs. Mammary tumors from 200 dogs were classified and histologic sections of the excisional specimens were evaluated for IELs. IELs, found in specimens from 60 dogs, were categorized as adenosis, sclerosing adenosis, intraductal papilloma, sclerosing papilloma, ductal hyperplasia, atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS; high, intermediate, and low grade). Most proliferative IELs without atypia were associated with benign tumors, whereas IELs with atypia (ADH and DCIS) were generally associated with mammary cancer. ER-α expression was significantly low or absent in most ADH and DCIS lesions as well as in their associated tumors. Ki67 expression was significantly higher in high-grade DCIS than in hyperplasia or low-grade DCIS. Two thirds of high-grade DCIS lesions were positive for HER-2. Canine mammary IELs were strikingly similar to those of the human breast. The frequency of IELs in the dog, their association with spontaneous mammary cancer, their pattern of ER-α and HER-2 expression, and their histologic resemblance to human IELs may make the dog an ideal model to study human ER-negative (both HER-2 positive and negative) breast cancer progression as well as prevention and treatment. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2247–56)

Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions

BackgroundBreast cancer is the most frequently diagnosed cancer in women. Intraepithelial lesions (IELs), such as usual ductal hyperplasia (UH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors that predict a womans chance of developing invasive breast cancer. Therefore, a comparative study that establishes an animal model of pre-invasive lesions is needed for the development of preventative measures and effective treatment for both mammary IELs and tumors. The purpose of this study was to characterize the histologic and molecular features of feline mammary IELs and compare them with those in women.MethodsFormalin-fixed, paraffin-embedded specimens (n = 205) from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN), and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine (Sassari, Italy). Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were compared to those of humans. Immunohistochemistry for estrogen receptor (ER-alpha), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu) and Ki-67 was performed in IELs and adjacent tumor tissues.ResultsIntraepithelial lesions were found in 57 of 203 (28%) feline mammary specimens and were categorized as UH (27%), ADH (29%), and DCIS (44%). Most IELs with atypia (ADH and DCIS) were associated with mammary cancer (91%), whereas UH was associated with benign lesions in 53% of cases. Feline IELs were remarkably similar to human IELs. No ER or PR immunoreactivity was detected in intermediate-grade or high-grade DCIS or their associated malignant tumors. HER-2 protein overexpression was found in 27% of IELs.ConclusionThe remarkable similarity of feline mammary IELs to those of humans, with the tendency to lose hormone receptor expression in atypical IELs, supports the cat as a possible model to study ER- and PR-negative breast lesions.

Ovis aries Papillomavirus 3: A prototype of a novel genus in the family Papillomaviridae associated with ovine squamous cell carcinoma

Papillomaviruses play an important role in human cancer development, and have been isolated from a number of animal malignancies. However, the association of papillomaviruses with tumors has been poorly investigated in sheep. In this study, a novel ovine Papillomavirus, OaPV3, was cloned from sheep squamous cell carcinoma. Unlike the already known ovine papillomaviruses, belonging to the Delta genus, OaPV3 lacks the E5 open reading frame and maintains the conserved retinoblastoma motif in the E7 gene. OaPV3 infects exclusively epithelial cells, and was found in skin of healthy sheep of geographically separated flocks located in Sardinia (Italy). This new virus is transcriptionally active in tumors and shares low homology with all the other papillomaviruses, establishing a new genus. Taken together, the co-occurrence of OaPV3 and tumors, its cell and tissue tropism, and its gene repertoire, suggests a role for this virus in development of sheep squamous cell carcinoma.

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.

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.

Predictive value of antral follicle count and anti-Mullerian hormone for follicle and oocyte developmental competence during the early prepubertal period in a sheep model

Circulating anti-Müllerian hormone (AMH) and antral follicle count (AFC) are addressed as suitable markers of oocyte quantity and quality during adulthood. To investigate whether AFC and circulating AMH could predict follicle development and oocyte quality during the prepubertal period we used 40-day-old ewe lambs with high, intermediate and low AFC (≥30, 16-29 and≤15 follicles respectively). The analysis of the response to the exogenous FSH ovarian reserve test showed a positive correlation between AFC, AMH plasma levels, total follicle number and the number of large follicles (≥3mm) grown after exogenous FSH administration. The incorporation of abattoir-derived oocytes collected from ovaries with different AFC in an in vitro embryo production system showed that a high AFC can predict oocyte quality in prepubertal ovaries, reflecting an ovarian status suitable for follicular development. The histological quantification of the ovarian reserve evidenced that AFC was not predictive of differences in either the number of healthy follicles or the size of the primordial follicle pool in prepubertal ovaries. Further studies are needed to investigate the implication on the reproductive performance of the significant inter-individual differences found in the present study in AFC and circulating AMH in the early prepubertal period.

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 humans 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. Cancer Prev Res; 4(11); 1790–8. ©2011 AACR.

Evaluations of Testicular Biopsy by Tru-cut in the Stallion

Testicular biopsy is one of the supplementary examinations performed in the course of andrological testing. In veterinary medicine, it is seldom used in low-fertility or nsterility testing, or in the investigation of suspected cancer. Biopsies are discouraged in horses in particular, since they may result in testicular sclerosis and atrophy n(Galina, 1971; Smith, 1974; Marusi and Corradi, 1989; Varner, 1991; Del Vento et al ., 1992; Threlfall and Lopate, 1993; Roser, 2000). In humans (Foresta and Varotto, n1992; Foresta et al ., 1992; Kessaris et al ., 1995; Harrington et al., 1996) good results are currently being reported with the use of tru-cut needles (needle assemblies with an inner recess that cuts and traps the tissue sample). These can be used to remove a fragment of parenchymal tissue with minimal invasiveness. Our aim was to study the use of this instrument in stallions; to assess the adequacy of the biopsy sample in relation to the size of the needle and sample retrieval mechanism used (either springloaded or manual); and to analyse the suitability for histology of the specimen. We were also interested in the short- and long-term outcomes of the procedure.

Map Lesions: In Situ Investigation on Samples of Multibacillar and Paucibacillar Ovine PTBC

Paratuberculosis (PTBC) is a chronic inflammation of the intestines of ruminants but it can also strike other species, including primates. Ovine paratuberculosis lesions have been the subject of authoritative observations (Perez et al ., 1996). The macroscopic picture shows a thickening and wrinkling of the intestinal mucous membrane , regional lymphangitis and lymphandenopathy, principally found in the terminal part of the ileum but also in the colon, jejunum and duodenum. Histomorphological pictures are characterised by chronic granulomatous enterocolitis, whose typical clinical symptom is the loss of weight due to profuse diarrhea. The etiological agent of PTBC is the bacterium Mycobacterium avium subsp. paratuberculosis (MAP) (Marek, 1910); its presence gives rise to a persistent infection of the intestinal macrophages which appear foamy, in a small granuloma within the interfollicular-basal area of the Peyer’s plaques of the ileal tract. In some cases, even in the presence of marked lesions and clear, clinical symptomatology, coloration of the Ziehl-Nielsen (ZN) produces a negative result. DNA analysis of MAP has identified a peculiar insertion sequence, IS900, present in 15–20 pairs per genome (Green et al ., 1989).