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Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours.

CD10 is an important cell marker in the diagnosis of acute lymphoblastic leukaemia and of breast myoepithelial (ME) cells in humans. The objective of this study was to assess the value of CD10 as a marker of canine ME cells using immunohistochemistry on routinely processed normal, dysplastic and neoplastic mammary tissue. Five different CD10 positive cell types were identified on the basis of cell morphology, pattern of immunoreactivity, and on the co-expression of additional cell lineage-specific markers. Type 1 cells were typical fusiform cells with a ME cell phenotype (calponin- and cytokeratin [CK] 14-positive, CK8/18-negative). Type 2 cells were typical or atypical polyhedral cells with a luminal epithelial (LE) cell phenotype (calponin- and CK14-negative, CK8/18-positive). Type 3 cells had a type 1 phenotype with variable morphology, and type 4 were atypical neoplastic cells with a mixed ME/LE phenotype. Type 5 cells were typical fusiform cells with a stromal phenotype. Type 1 cells were considered normal ME cells and were found in all sample types; type 2 cells were considered normal or neoplastic LE cells and were also found in all sample types; types 3 and 4 cells were restricted to tumour samples and to malignant tumours, respectively, and type 5 cells were found in all sample types, although predominantly in neoplastic tissue. The findings indicate that the CD10 antigen is a sensitive (although not specific) marker of canine ME cells in normal, dysplastic and neoplastic mammary tissue. Differences in the distribution and staining intensity of CD10-positive cells suggest a number of potential roles for this protein in the pathogenesis of canine mammary neoplasia.

Gliomatosis cerebri (GC) is a rare, diffusely infiltrating, glial cell tumour of neuroepithelial origin. This report describes a case of oligodendroglial GC in a 6-year-old male Poodle with central nervous system symptoms. Computed tomography revealed anomalous parenchyma density and ventricular asymmetry. Cerebrospinal fluid showed elevated protein (30 mg dL(-1)) and nucleated cell count (20 µL(-1)). Presumptive diagnosis of necrotizing meningoencephalitis was made. Because of rapid deterioration of the general condition of the animal, the dog was euthanized. Histologically there was an infiltration of round or ovoid neoplastic cells in the white matter of the left cerebral hemisphere and in leptomeninges. Immunohistochemistry showed that 80% of the neoplastic cells expressed Olig2 and some 50% expressed glial fibrilary acidic protein. On the basis of clinical, histological and immunohistochemical features, a diagnosis of oligodendoglial GC was done. This case represents the first report of a case of oligodendroglial GC in the canid.

Hypomyelination syndrome of the Weimaraner dog is a disease characterised by a reduction or absence of myelin in the axons of the central nervous system (CNS) exclusively. The objective of this study was to analyse the cause of this deficiency of myelin. Tissue samples of the CNS of three Weimaraner dogs with neurological signs were fixed in 10% formalin and embedded in paraffin wax, and histochemical, immunohistochemical and ultrastructural studies were performed. Histochemical staining with haematoxylin and eosin and Kluver-Barrera techniques showed generalised pallor in the peripheral areas of the ventral and lateral funiculi of the spinal cord. Immunohistochemical analysis showed a weak expression of both proteolipid protein (PLP) and myelin basic protein (MBP) and a marked decrease of Olig2(+) cells in the demyelinated areas. The immunohistochemical findings suggested a myelination or remyelination failure because of the smaller population of oligodendrocytes. However, PLP gene mutations may also be the cause of the decrease of PLP expression as described in other species.

Sebaceous gland oestrogen alpha (ERalpha) and progesterone (PR) receptor expression was examined immunohistochemically in 26 and 32 dogs respectively with sebaceous gland hyperplasia/adenomas, epitheliomas and carcinomas, and in the glands of 10 healthy controls. The mean percentage of ERalpha positive nuclei in control sebaceous glands was 21.31% compared with 11.5% in hyperplasia/adenoma-type lesions, although these values were not statistically different. In sebaceous gland epitheliomas and carcinomas, positive basal cells represented 7.86% and 3.53% of neoplastic cells respectively and these mean percentages were significantly lower in epitheliomas (P0.024) and carcinomas (P0.015) than in controls. The mean percentage of PR-positive nuclei in control sebaceous glands was 23.96%, similar to the 22.07% found in hyperplasia/adenoma-type lesions. In sebaceous gland epitheliomas and carcinomas, positive cells were scarce and represented 13.5% and 4.06% of neoplastic cells respectively. Differences in the percentage of positive cells between normal and pathological glands reached statistical significance for carcinomas (P0.043). In the control group there was greater PR (P0.001) and ERalpha expression (P0.014) in sebaceous glands in female dogs. The PR and ERalpha immunoreactivity in each category of neoplastic lesions could not be analysed because sample size was too small but when all the sebaceous gland tumours were grouped and analysed, no sex difference was found. The results suggest that oestrogen and progesterone receptor expression is reduced in some canine sebaceous gland tumours. These changes may represent a contributing factor for tumour growth or simply be a consequence of tumour progression.

BACKGROUND We attempted to define the effect of estrogen receptor (ER)alpha activation on gonadotroph progesterone receptor (PR) expression (mRNA and protein) and action (GnRH-stimulated and GnRH self-priming) in short- and long-term ovariectomized (OVX) rats. METHODS Two weeks or 1 year after OVX, rats were injected over 3 days with 125 microg/kg of estradiol benzoate (EB), 7.5 mg/kg of the selective ERalpha agonist propylpyrazole triol (PPT), or 15 mg/kg of the selective ER modulator tamoxifen (TX). Controls were given 0.2 ml oil. The last day of ER analog treatment, half of the rats in each group received 25 mg/kg of progesterone (P). The next day, anterior pituitaries were removed and analyzed for PR-AB mRNA and protein. Gonadotrophin secretion in incubated pituitaries was also measured. RESULTS (i) PR mRNA expression was higher in young than in middle-aged OVX rats although PR protein was absent in pituitaries from both groups of OVX rats; (ii) activation of ERalpha reduced gonadotroph hypertrophy and increased PR mRNA and protein expression (EB > PPT > TX) more efficiently in young than in middle-aged rats, (iii) ER agonists elicited GnRH-stimulated LH and FSH secretion in young but only FSH secretion in middle-aged OVX rats, (iv) evaluated by peak LH concentrations, GnRH self-priming was observed in both groups of OVX rats and (v) P down-regulated PR protein expression in young, and to a lesser extent, in middle-aged OVX rats, in close association with PR-dependent GnRH self-priming. CONCLUSIONS Middle-aged OVX rats exhibited clear-cut LH, but not FSH, secretory defects in pituitary sensitivity to estrogen and P.

Calponin is a 34-kDa smooth muscle-specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin-biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin-positive cells were identified: (1) Type 1: cytokeratin-14-positive pre-existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin-14-positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin-14-positive atypical cells indistinguishable from non-reactive atypical cells, which should have never been detected in haematoxylin and eosin-stained sections and (4) Type 4: cytokeratin-14-negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin-negative and cytokeratin-14-positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high-molecular-weight cytokeratins in luminal epithelial-type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.

A 12-year-old, entire, nulliparous crossbreed female dog was presented with a history of vulval bleeding, bulging of the perineum and faecal tenesmus. A firm, non-painful perineal mass, measuring 9.11x5.4 cm, with erythema was detected. Abdominal radiography showed compression and elevation of the rectal ampulla. A dose of 10 mg/kg aglepristone was administered subcutaneously on days 1, 2, 8, 15, 28 and 35. An incision biopsy was taken on day 15 and immunohistochemical analysis showed that the majority of neoplastic cells expressed progesterone receptors. Both the cutaneous erythema and the faecal tenesmus had resolved by day 28. A 50 per cent reduction in size was observed by day 60 (surgical excision). This study shows that benign tumours of the vagina of the dog that contain progesterone receptors can be reduced in size in a palliative or neoadjuvant setting using the progesterone receptor antagonist aglepristone.

The specific role of each oestrogen receptor (ER) isoform (alpha and beta ) and site (nucleus and plasma membrane) in LH release was determined in ovariectomized (OVX) rats injected over 6 days (days 15-20 after OVX) with a saturating dose (3 mg/day) of tamoxifen (TX), a selective ER modulator with nuclear ERalpha agonist actions in the absence of oestrogen. This pharmacological effect of TX was demonstrated by the fact that it was blocked by the selective ERalpha antagonist methyl-piperidinopyrazole. Over the past 3 days of the 6-day TX treatment, rats received either 25 microg/day oestradiol benzoate (EB), 1.5 mg/day selective ERalpha agonist propylpyrazole triol (PPT) and the selective ERbeta agonist diarylpropionitrile (DPN), or a single 3 mg injection of the antiprogestin onapristone (ZK299) administered on day 20. Blood samples were taken to determine basal and progesterone receptor (PR)-dependent LH-releasing hormone (LHRH)-stimulated LH secretion and to evaluate LHRH self-priming, the property of LHRH that increases gonadotrope responsiveness to itself. Blood LH concentration was determined by RIA and gonadotrope PR expression by immunohistochemistry. Results showed that i) EB and DPN potentiated the negative feedback of TX on basal LH release; ii) DPN reduced TX-induced PR expression; iii) EB and PPT blocked TX-elicited LHRH self-priming and iv) ZK299 reduced LHRH-stimulated LH secretion and blocked LHRH self-priming. These observations suggest that oestrogen action on LH secretion in the rat is exerted at the classic ERalpha pool and that this action might be modulated by both ERbeta and membrane ERalpha through their effects on PR expression and action respectively.

BackgroundFeline mammary carcinoma has been proposed as a natural model of highly aggressive, hormone-independent human breast cancer. To further explore the utility of the model by adding new similarities between the two diseases, we have analyzed the oncogene HER-2 status at both the protein and the gene levels.MethodsFormalin-fixed, paraffin-embedded tissue samples from 30 invasive carcinomas, 7 benign lesions and two normal mammary glands were analyzed. Tumour features with prognostic value were recorded. The expression of protein HER-2 was analyzed by immunohistochemistry and the number of gene copies by means of DNA chromogenicin situhybridization.ResultsImmunohistochemical HER-2 protein overexpression was found in 40% of feline mammary carcinomas, a percentage higher to that observed in human breast carcinoma. As in women, feline tumours with HER-2 protein overexpression had pathological features of high malignancy. However, amplification of HER-2 was detected in 16% of carcinomas with protein overexpression, a percentage much lower than that observed in their human counterpart.ConclusionFeline mammary carcinoma would be a suitable natural model of that subset of human breast carcinomas with HER-2 protein overexpression without gene amplification.

In the rat, administration of tamoxifen (TX) in the absence of oestrogen (E) induces LHRH self-priming, the progesterone receptor (PR)-dependent property of LHRH that increases gonadotrope responsiveness to itself. The oestrogen-dependent PR can be phosphorylated/activated by progesterone (P4) and, in the absence of the cognate ligand, by intracellular LHRH signals, particularly cAMP/protein kinase A. We have recently found that oestradiol-17β (E2), acting on a putative membrane estrogen receptor-α in the gonadotrope, inhibits this agonist action of TX. This study investigated the mechanism by which E2 inhibits TX-elicited LHRH self-priming using both incubated pituitaries from TX-treated ovariectomized (OVX) rats and anterior pituitary cells from OVX rats cultured with TX. It was found that (1) in addition to the inhibitory effect on TX-elicited LHRH self-priming, E2 blocked P4 and adenylyl cyclase activator forskolin augmentation of LHRH-stimulated LH secretion, and (2) E2 did not affect the increasing action of TX on gonadotrope PR expression or pituitary cAMP content. Furthermore, inhibition of protein phosphatases with okadaic acid suppressed E2 inhibition of TX-elicited LHRH-induced LH secretion, while stimulation of protein phosphatases with ceramide blocked TX-induced LHRH self-priming. Together, these results indicated that membrane ER-mediated E2 inhibition of the TX-stimulated LHRH self-priming pathway involves a blockade of gonadotrope PR phosphorylation/activation, but not a deficient response of PR to phosphorylases. Results also suggested that the inhibitory effect of E2on TX-induced LHRH self-priming is exerted through modulation of cellular protein phosphatase activity in the gonadotrope.

The immunohistochemical expression, tissue-specific and cell-specific distribution patterns of progesterone receptors (PR), growth hormone (GH) and insulin growth factor-I (IGF-I) have been studied in 22 cases of feline fibroadenomatous change (FFAC). PR and GH were detected in all cases and were distributed homogeneously throughout the lesion, while IGF-I was detected in 77% of the cases at the site of ductal budding. The simultaneous expression of PR, GH and IGF-I was detected in epithelial cells in 14 of 22 cases while PR and GH expression only was detected in epithelial cells in 11 cases. Cases that expressed GH and IGF-I without PR expression in the stroma were the most numerous. Double immunohistochemical staining showed the co-localisation of PR and GH in a subset of ductal epithelial cells located between basal/myoepithelial and luminal cells (probably undifferentiated stem cells). These results suggest that ligand-activated progesterone receptors may induce the local synthesis of GH which in turn may exert its proliferative action directly and also indirectly through the production of other growth factors, such as IGF-I, in an autocrine/paracrine manner.

A panel of six monoclonal antibodies (mAbs) recognizing antigenic determinants on canine immunoglobulin (Ig) heavy or light chains was produced and characterized. All monoclonals recognized the IgG(2) subclass, although only two were subclass-specific (CA3H1 and CA4F1). The CA3B8 mAb was found to be specific for an epitope on canine immunoglobulin G heavy chain, (IgG(1) and IgG(2) subclasses). Two mAbs (CA2E9 and CA5B2) reacted with an epitope on the heavy chain of canine IgG and IgM and another, CA4E7, bound to canine IgA, IgG and IgM isotypes; CA4E7 recognized an epitope on canine immunoglobulin light chain. CA4E7, CA4F1 and CA5B2 recognized an epitope in the Fab region. Three mAbs, CA3B8, CA4E7 and CA5B2, showed much lower reactivity with canine IgG by ELISA when IgG was periodate-treated, suggesting that they recognized a carbohydrate determinant. Cross-reactivity analysis of these mAbs with sera from horse, goat, cow, sheep, pig, cat, rabbit, hamster, rat, mouse and human indicated that two mAbs, CA3B8 and CA5B2, recognized a canine IgG-specific epitope; two others, CA3H1 and CA4E7, recognized an epitope also present in rabbit and sheep immunoglobulin respectively; and the remaining two (CA2E9 and CA4F1) recognized an epitope broadly present on the Igs of the species analyzed. This panel of antibodies will be a useful tool for future canine immunodiagnosis tests. With the exception of CA2E9, all mAbs were able to recognize plasma cells on paraffin-embedded tissues, and will thus be useful for immunohistochemical assays.

Myxomas of the joints are extremely rare in domestic animals, only four cases, all in dogs, having been reported previously. This paper describes a myxoma originating from the synovium of the right radiocarpal joint of a mature female Dobermann pinscher with right front limb lameness. The tumour was excised surgically and no recurrence was detected during a 2-year follow-up period. Immunohistochemically, the tumour cells reacted with antibodies to vimentin and S100 protein but not with antibodies to cytokeratins (high and low molecular weight) or human callus keratin. Los mixomas articulares son extremadamente raros en los animales domésticos; sólo se han registrado cuatro casos, todos ellos en perros. En este artículo se describe un mixoma originado en la sinovia de la articulación radiocarpiana derecha de una hembra madura de de una hembra madura de Dobermann pinscher con cojera de la extremidad delantera derecha. El tumor fue extirpado quirúrgicamente y no se detectó ninguna recidiva durante un periodo de seguimiento de 2 años. Inmunohistoquímicamente, las células tumorales reaccionaron con anticuerpos contra la vimentina y la proteína S100, pero no con anticuerpos contra las citoqueratinas (de alto y bajo peso molecular) o a la queratina del callo humano. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Estrogen receptors (ER) were determined by both the biochemical dextran-coated charcoal (DCC-ER) and the immunohistochemical Avidin biotin- peroxidase complex (IHC-ER) methods in proliferative mammary lesions collected from 37 cats: 20 malignant tumors without metastasis at first presentation, seven malignant tumors with lung and/or lymph node metastases and 10 benign tumors and dysplasias. Total number of samples analyzed by both methods was 44. The DCC-ER method was applied to frozen tissue samples and the IHC-ER method was applied to neutral buffered formalin-fixed, paraffin wax-embedded tissue samples by using the NCL-6F11 monoclonal antibody. Biochemically, 21 (47.7%) cases had equal or more than 5 fmol/mg of protein (standard positivity threshold). Immunohistochemically, 11 (25%) cases were scored positive, the percentage of positive nuclei being statistically linked to the intensity of immunostaining. Normal mammary gland tissue (13 cases) and/or dysplastic areas (5 cases) found in the surroundings of the main lesion were IHC-ER positive in 76.9% and 40% of the cases, respectively. Concordance between DCC-ER and IHC-DCC was 72.7% and the results of the DCC and the IHC-ER methods were significantly correlated (P < 0.05) by the χ2 test. Specificity (true negatives) and sensitivity (true positives) of the ICH-ER method were 95.6% and 47.6%, respectively. One out of eleven DCC-ER positive and IHC-ER negative discordant cases (9.09%) was a DCC-ER false positive, because the surrounding normal mammary gland tissue was IHC-ER positive. The remaining 10 cases had ER content values equal or lower than 23 fmol/mg of protein, a figure that could represent the sensitivity threshold of the immunohistochemical method employed.

Background Triple negative breast cancer (TNBC) in humans is defined by the absence of oestrogen receptor (ER), progesterone receptor (PR) and HER2 overexpression. Mammalian target of rapamycin (mTOR) is overexpressed in TNBC and it represents a potential target for the treatment of this aggressive tumour. Feline mammary carcinoma (FMC) is considered to be a model for hormone-independent human breast cancer. This study investigated mTOR and p-mTOR expression in FMC in relation to triple negative (TN) phenotype. Results The expression of mTOR, p-mTOR, ERα, PR and HER2 was evaluated in 58 FMCs by immunohistochemistry and in six FMC cell lines by Western blot analysis. 53.5% of FMC analyzed were ER, PR, HER2 negative (TN-FMC) while 56.9% and 55.2% of cases expressed mTOR and p-mTOR respectively. In this study we found that m-TOR and p-mTOR were more frequently detected in TN-FMC and in HER2 negative samples. Conclusions In this study, we demonstrate that there is also a FMC subset defined as TN FMC, which is characterised by a statistically significant association with m-TOR and p-mTOR expression as demonstrated in human breast cancer.

The distribution of oestrogen and progesterone receptors was analysed in 18 cases of feline fibroadenomatous change (FFAC) using commercially available specific monoclonal antibodies and the Avidin-biotin peroxidase complex technique on formalin-fixed, paraffin-embedded tissue. In all cases of FFAC, progesterone receptors were detected either in epithelial cells (mostly suprabasal) or in epithelial and stromal cells. Oestrogen receptors were detected in approximately half of these cases, in suprabasal or luminal epithelial cells exclusively. Myoepithelial cells lacked both oestrogen and progesterone receptors. The techniques used have identified the specific cellular distribution of steroid hormones receptors in this hormone-dependent lesion of the feline mammary gland. The results confirm those of previous biochemical analyses with respect to progesterone receptors and add new data concerning the possible involvement of oestrogen receptors and stromal fibroblasts in the hormonal control and development of the lesion.

In vitro activities of a series of gold, copper and ruthenium clotrimazole (CTZ, CAS 23593-75-1) and ketoconazole (KTZ, CAS 65277-42-1) derivatives were investigated individually and in combination with human neutrophils (PMNs) against a wild type strain of Saccharomyces cerevisiae. For 11 out of 12 tested metal complexes, the minimal inhibitory concentrations (MICs) at which 100 % of yeast growth was inhibited ranged from 0.75 to 3.0 µmol/L. The complex RuCl 3 (CTZ) 3 · 2CH 3 OH (1f) (MIC = 0.75 µmol/L) was, although modestly, the only one able to increase the fungistatic activity of the parental drug (MIC = 1 µmol/L). On the other hand, at a sub-MIC concentration (0.5 µmol/L), the complexes [Cu(KTZ)Cl 2 ] 2 · 2H 2 O (2c) and RuCl 2 (KTZ) 2 (2e) displayed synergistic fungicidal effects with PMNs whereas phagocytic capacity was enhanced by complexes [Cu(KTZ) 3 Cl 2 ] (2b) and RuCl 2 (KTZ) 2 (2e). The findings suggest that the metal-based agents may give rise to drugs with improved antifungal properties.