L. Della Salda

Review paper: a review of the pathology of abnormal placentae of somatic cell nuclear transfer clone pregnancies in cattle, sheep, and mice.

Cloning of cattle, sheep, and mice by somatic cell nuclear transfer (SCNT) can result in apparently healthy offspring, but the probability of a successful and complete pregnancy is less than 5%. Failures of SCNT pregnancy are associated with placental abnormalities, such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium, and altered basement membrane. The pathogenesis of these changes is poorly understood, but current evidence implicates aberrant reprogramming of donor nuclei by the recipient oocyte cytoplast, resulting in epigenetic modifications of key regulatory genes essential for normal placental development. The purpose of this review is to provide an overview of the anatomic pathology of abnormal placentae of SCNT clones and to summarize current knowledge concerning underlying pathogenetic mechanisms.

Expression of Platelet-derived Growth Factor-β Receptor and Bovine Papillomavirus E5 and E7 Oncoproteins in Equine Sarcoid

Equine sarcoids are benign fibroblastic skin tumours that are recognized throughout the world. Infection with bovine papillomavirus (BPV) types 1 and 2 has been implicated as a major factor in disease development; however, the cellular mechanisms underlying fibroblast transformation remain poorly defined. The present study further characterizes aspects of the association with BPV in 15 equine sarcoids. BPV DNA was demonstrated in 12/15 tumours collected from different areas of Italy. Nine of these 12 tumours expressed the BPV oncoproteins E5 and E7, but these oncoproteins were not expressed by normal equine cells. The BPV E5 protein is known to bind to the platelet-derived growth factor-beta receptor (PDGF-betaR) and this molecule was expressed by 11 of the 12 sarcoids in which E5 was demonstrated. These findings add further weight to the theory that BPV and the PDGF-betaR may have a role in the pathogenesis of this disease.

Activated Platelet-Derived Growth Factor β Receptor Expression, P13K-AKT Pathway Molecular Analysis, and Transforming Signals in Equine Sarcoids

The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor β receptor (PDGFβ-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFβ-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical interaction between the activated receptor and the 85-kDa regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K) is shown by coimmunoprecipitation. The PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of the PDGFβ-r is shown to be expressed, and the amount of the investigated molecules is higher than normal (P < .001), suggesting an activation of these effectors in sarcoids. Further, we demonstrate that phospho-JNK and phospho-JUN are more expressed in sarcoids than in normal skin. Our results provide new insights into the pathogenesis of equine sarcoids and support the validity of this in-vivo model to further characterize the molecular pathways underlying BPV E5-induced carcinogenesis.

High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation

BACKGROUND The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s) AEA had on the development of sheep blastocysts in vitro. METHODS We analysed the expression and activity of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation. RESULTS We show that the AEA signalling system is present in sheep and that high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism. Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium. CONCLUSIONS Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.

Hypertrophy of intestinal smooth muscle in cats

Pathological findings of four cats with severe and diffuse smooth muscle hypertrophy of the small intestine (MHSI) are reported and compared to those of five cats with segmental MHSI secondary to neoplastic obstruction and four controls. Histology demonstrated a constant association between idiopathic MHSI and submucosal fibrosis and chronic lymphoplasmacytic enteritis. Morphometry (gut diameter, thickness and area of muscular layers, number and density of smooth muscle nuclei) and MIB-1-immunolabelling showed that the thickness increase was mostly due to hypertrophy, but hyperplasia was also evident. Microbiology from ileal content samples was performed in two cats with primary MHSI, and Campylobacter spp. were isolated, which were also demonstrated by immunohistochemistry and ultrastructure. The association of chronic enteritis with idiopathic MHSI suggests that factors released in intestinal inflammation may also act as hypertrophy stimuli for smooth muscle cells.

Apoptotic fraction in lymphoid tissue of FIV-infected SPF cats

In the present study the apoptotic fraction has been investigated in peripheral blood mononuclear cells (PBMCs) and in lymphoid tissue of six clinically asymptomatic serologically positive specific pathogen free (SPF) FIV-infected cats with a decline in peripheral blood CD4+ lymphocytes, compared to five FIV- SPF controls. Apoptosis in PBMCs was scored in relation to cell cycle phases judged by the integrating cytometric measure of DNA content with 3H-thymidine and 3H-leucine incorporation. Apoptosis in lymphoid tissue was revealed with the ApopTag-peroxidase kit, quantified by image analysis and expressed as apoptotic index (number of apoptosis per 100 cells). The high percentage of apoptotic death in lymphocytes from FIV+ cats was chronologically related to the entrance of cells in the S phase of the cell cycle (p < 0.0001). No difference in the apoptotic index was revealed comparing the follicular, cortical + paracortical and medullary compartments in lymph nodes of FIV+ and FIV- cats. In each group of cats a similar pattern of apoptosis expression was revealed in lymph nodes: significantly higher in follicular vs. both cortical + paracortical and medullary compartments (p < 0.001). In the thymus a significant increase in apoptotic index was revealed in the cortical compartment of the FIV+ cats compared to FIV- (p < 0.001), while in the spleen both the red and white pulp expressed a higher value in FIV+ cats compared to FIV-(p < 0.05) and the former showed a pattern of expression as follows: in the red pulp significantly higher than in the white pulp (p < 0.001). This investigation suggests that the priming signals for apoptosis in FIV infection parallels the S phase of the cell cycle and peripheral blood changes could follow both thymic and splenic modifications in apoptotic expression.

β-catenin in canine skin: immunohistochemical pattern of expression in normal skin and cutaneous epithelial tumours.

In normal adult skin, β-catenin is a structural component of the intercellular junction and the Wnt/β-catenin pathway plays a key role in the regulation of cutaneous homeostasis, particularly in the maintenance of hair follicle stem cells. No data are available on the expression pattern of β-catenin in normal canine skin and in canine cutaneous epidermal and follicular tumours. The present study used immunohistochemistry to determine β-catenin expression in four samples of normal canine skin and 62 cutaneous epithelial tumours (14 epidermal, 30 follicular and 18 glandular). β-catenin expression was localized to the nucleus of matrical and dermal papilla cells in anagen hair follicles and was also found in scattered cells of the outer root sheath, suggesting that these follicular epithelial cells may have a high proliferative potential. Nuclear labelling, considered a hallmark of activation of the Wnt/β-catenin signalling pathway, was observed in canine follicular tumours with matrical differentiation (100% of cases of trichoepithelioma and pilomatricoma), suggesting that a possible mutation of the canine CTNBB1 gene may underlie these tumours. In contrast, malignant tumours (squamous cell carcinoma, basal cell carcinoma, sebaceous and apocrine gland carcinoma and epithelioma) were characterized by reduction/loss of β-catenin membrane labelling compared with normal cutaneous epithelial cells and benign tumours, suggesting that reduction/loss of β-catenin expression is important in the acquisition of the malignant phenotype and may have a role in the infiltration and metastasis of these tumours.

Congenital and acquired pathology of ovary and tubular genital organs in ewes: a review

Advances in our understanding of ovarian cyclicity, pathogenesis of subfertility and/or infertility and reproductive pathology in food animals have frequently entailed examination of abattoir material. Despite the fact that most lesions in ewes are likely to be of relatively minor significance to fertility, results of previous studies suggest that lesions of the female reproductive system may represent a significant source of loss to sheep husbandry. The objective of this paper is to review the pathophysiology, the effects on reproductive efficiency and the key gross and histological diagnostic features of congenital and acquired pathology of ovary and tubular genital organs in ewes.

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.

Use of Electron Microscopy to Classify Canine Perivascular Wall Tumors

The histologic classification of canine perivascular wall tumors (PWTs) is controversial. Many PWTs are still classified as hemangiopericytomas (HEPs), and the distinction from peripheral nerve sheath tumors (PNSTs) is still under debate. A recent histologic classification of canine soft tissue sarcomas included most histologic types of PWT but omitted those that were termed undifferentiated. Twelve cases of undifferentiated canine PWTs were evaluated by transmission electron microscopy. The ultrastructural findings supported a perivascular wall origin for all cases with 4 categories of differentiation: myopericytic (n = 4), myofibroblastic (n = 1), fibroblastic (n = 2), and undifferentiated (n = 5). A PNST was considered unlikely in each case based on immunohistochemical expression of desmin and/or the lack of typical ultrastructural features, such as basal lamina. Electron microscopy was pivotal for the subclassification of canine PWTs, and the results support the hypothesis that canine PWTs represent a continuum paralleling the phenotypic plasticity of vascular mural cells. The hypothesis that a subgroup of PWTs could arise from a pluripotent mesenchymal perivascular wall cell was also considered and may explain the diverse differentiation of canine PWTs.