Iacopo Vannozzi

Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas.

Samples from feline invasive mammary carcinomas (FMCs) were used to determine the prognostic significance of the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD). Forty-eight queens bearing FMCs were included in a 2-year follow-up study. Mammary tumors were classified according to the World Health Organization system and graded on the basis of histologic criteria. Tumor sections were immunostained using anti-VEGF and anti-von Willebrand factor (vWf) antibodies. VEGF expression was quantified on the basis of the percentage of positive cells. MVD of vWf-positive microvessels was determined by both mean microvessel counts and highest microvessel counts. Normal mammary gland tissues showed an inconspicuous VEGF staining. In FMCs the proportion of VEGF-positive cells was significantly higher in papillary and solid carcinomas than in tubular and papillary cystic tumors. An increased number of cells expressing VEGF was also observed in poorly differentiated FMCS. Sixteen (33.3%) of the queens bearing invasive carcinomas were still alive at the end of the 2-year follow-up period, and 32 (66.7%) had died. The VEGF expression was significantly correlated with the clinical outcome, but no correlation was observed with the invasion of lymphatic vessels. A correlation between the higher percentage of VEGF-positive cells and the unfavorable prognosis was demonstrated by the estimation of curves for overall survival (P = 0.03). Univariate analysis showed that MVD did not correlate with the overall survival. The results of our study demonstrated that VEGF expression, although not associated with increased angiogenesis, is a prognostic indicator in feline mammary tumors. In contrast, there is no support for a role of neovascularization as an indicator of survivability.

MIB-1 Labeling Index in Feline Dysplastic and Neoplastic Mammary Lesions and Its Relationship with Postsurgical Prognosis

Samples from feline normal, dysplastic, and neoplastic mammary tissues were used to investigate the usefulness of MIB-1 labeling index (MIB-1 I) as a prognostic indicator. Forty-eight queens bearing invasive carcinomas were included in a 2-year follow-up study. Mammary lesions were classified according to the World Health Organization system, and invasive carcinomas were further graded on the basis of the degree of tubule formation, the degree of nuclear and cellular pleomorphism, and mitotic count. Additional sections were immunostained using MIB-1 antibody, and MIB-1 I was expressed as a percentage of positive nuclei. In normal mammary gland tissues, the mean MIB-1 I was <1%. A low proliferation rate was found in all mammary adenosis and in situ carcinomas, and the highest rates were observed in feline mammary hypertrophy and invasive carcinomas. Twenty-one (43.7%) of the queens bearing invasive carcinomas were still alive at the end of the trial, and 27 (56.2%) had died. The MIB-1 I was not significantly correlated with clinical outcome, age, histologic type, or grading of the tumors, but a borderline correlation was observed with invasion of lymphatic vessels. Univariate analysis showed that high MIB-1 I was also not associated with decreased overall survival, whereas the grading system of the tumors had high predictive value (P = 0.0040) for postsurgery survival. The lack of correlation between MIB-1 I and postsurgery survival suggests that this marker alone is not sufficient to determine a correct prognosis in feline mammary carcinomas, even if it is a useful proliferation marker.

Steroid hormone receptors in normal, dysplastic and neoplastic feline mammary tissues and their prognostic significance

The expression of oestrogen-α and progesterone receptors was determined in 13 normal, 21 dysplastic and 53 neoplastic feline mammary tissues. Expression of the receptors was correlated with cell proliferation, as assessed by the MIB-1 immunolabelling index, and with the clinical course of the disease. The expression of oestrogen receptors was significantly higher in healthy tissues and in adenosis than in neoplastic lesions, and the levels of progesterone receptors increased in fibroadenomatous changes and in ‘in situ’ carcinomas but decreased in invasive carcinomas. The oestrogen and progesterone receptor status of the invasive carcinomas did not correlate either with the histological parameters or with the overall survival of the cats, although the oestrogen receptor-negative tumours had a poor prognosis. Oestrogen receptorpositive neoplasms had a significantly lower MIB-1 immunolabelling index than oestrogen receptor-negative neoplasms.

FERTILITY AT THE FIRST POST PARTUM ESTROUS COMPARED WITH FERTILITY AT THE FOLLOWING ESTROUS CYCLES IN FOALING MARES AND WITH FERTILITY IN NONFOALING MARES

Summary A retrospective study on the reproductive performance of 401 artificially inseminated trotter mares during six breeding seasons is presented. Mares, 279 post partum (PP) and 122 maiden and barren, or nonlactating (NL), were inseminated with fresh semen obtained from four fertile stallions of the same breed. Pregnancy rate (PR) of mares inseminated at the foal heat (182/253, 71.9%) was lower, but not significantly different, than the PR (22/26, 84.6%) of mares inseminated for the first time at the second post partum cycle and similar to the PR at the first and second cycle of NL mares (95/112, 77.8% and 25/33, 75.7%, respectively). PR of mares inseminated at the foal heat was higher, but nonsignificantly different, from PR of the post partum mares not pregnant after artificial insemination (AI) at foal heat and inseminated again at the following estrous cycle. The PR after AI at the foal heat was significantly higher than the PR when the AI was performed at the third or later cycle in NL mares (71.9% vs 22.2%, P

Retrospective study of factors affecting multiple ovulations, embryo recovery, quality, and diameter in a commercial equine embryo transfer program

In this study, 198 donor mares of different breeds, ages, and reproductive category were inseminated with fresh, cooled and frozen or frozen and cooled semen at the embryo transfer station or in private artificial insemination centers during 10 breeding seasons. The results of this activity were retrospectively analyzed by Pearson Chi-square test and logistic regression to evaluate factors affecting multiple ovulations, embryo recovery, embryo quality, and embryo diameter. Out of the 661 cycles, 937 ovulations were recorded (mean ovulations/cycle: 1.42 ± 0.58). Ovulation rate and incidence of multiple ovulations were significantly affected by age, breed, and reproductive category. Uterine flushings for embryo recovery were performed between 7 and 10 days after ovulation and resulted in the recovery of 338 embryos (51.1% embryos/cycle and 36.1% embryos/ovulation, respectively). At least one embryo was recovered in 298 flushings (45.1%). The factors affecting embryo recovery were age, breed, reproductive category, type of semen, number of ovulations, and location of artificial insemination. Flushing protocol and day of flushing had no effect on embryo recovery. Age, type of semen, number of ovulations, and day of flushing had a significant influence on embryo diameter (N = 215). None of the factors included in the model had an effect on embryo quality distribution.

HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome

The proto-oncogene HER-2/neu (c-erbB-2) encodes a transmembrane receptor protein with tyrosine-kinase activity. Previous studies have shown that HER-2 protein over-expression is present in canine mammary tumours, however, possible prognostic and predictive analogies between protein over-expression patterns in canine and human species are still controversial. Thirty-five canine mammary carcinomas, 11 mammary adenomas, and normal, hyperplastic or dysplastic tissues taken at the marginal area of the tumours were evaluated by immunohistochemistry (IHC) for HER-2 expression, using the Hercept Test® system scoring guidelines. HER-2 over-expression was detected in 3/11 adenomas and 10/35 carcinomas. Normal, hyperplastic and dysplastic mammary tissues were also found to be positive. The correlations between HER-2 expression and tumour histological grading, mitotic index, the presence of lymphatic invasion, and overall survival (OS) were evaluated. In carcinomas, HER-2 positive status only correlated with the mitotic index. A positive correlation was also found between HER-2 positive status and the presence of HER-2 over-expression in normal, hyperplastic or/and dysplastic mammary tissues surrounding the tumours. The percentage of HER-2 over-expressing tumours was similar to the percentage previously observed in canine benign and malignant mammary tumours. However an investigation regarding morphologically normal and hyperplastic or dysplastic tissues surrounding neoplastic lesions also showed HER-2 over-expression. In contrast with human mammary tumours, this study confirmed that in canine species, HER-2 over-expression does not identify a subgroup of tumours with a poor prognosis. In fact, we found HER-2 over-expression in morphologically non-neoplastic mammary tissues, surrounding hyperplastic and neoplastic lesions.

Clinical use of twice daily injections of buserelin acetate to induce ovulation in the mare.

The difficulties in the prediction of ovulation time in the mare, together with the increasing use of cooled and frozen semen for artificial insemination, have stimulated investigators to search for methods for induction of ovulation. These methods are essentially based on exogenous administration of one of three hormones directly or indirectly involved in the mechanism of ovulation: hCG (Human Chorionic Gonadotrophin), CEG (Crude Equine Gonadotrophin) (Duchamp et al., 1987), or GnRH (Gonadotrophin-releasing hormone). Repeated administration of hCG, which is a heterologous glycoprotein for mares, may stimulate an immune response able to neutralise the effect of the hormone (Roser et al., 1979). Although CEG has not shown this negative side effect, it is not found on the market, and purified extracts are produced and utilised only by few research groups. GnRH, physiologically released in a pulsatile manner, and its analogues have been tested for induction of ovulation in oestrous mares either by repeated injections at given time intervals (Palmer and Quellier, 1988) or as slow-release subcutaneous implants. For this purpose, a slowrelease subcutaneous implant containing the synthetic GnRH analogue deslorelin acetate (OvuplantA) is successfully used in United States and Australia (Meinert et al., 1993; Meyers et al., 1997). The response to OvuplantA administration was similar to that obtained using hCG. However, this formulation is currently not available in the Italian and European market. The efficacy of buserelin, a GnRH analogue also commercialised in Italy, has been recently investigated for induction of ovulation in the mare. The administration of a single dose has not yielded encouraging results (Vidament et al., 1992). On the other hand, Battut et al. (2001) observed that most mares treated twice daily with intravenous (IV) administration of 20 or 40 mg of buserelin ovulated within 48 h. The aim of our study was to evaluate the clinical efficacy of repeated administration of 40 mg buserelin for induction of ovulation in the mare, as compared to a single administration of 2500 IU hCG or a placebo.

Preliminary report on the expression of leptin and leptin receptor (ObR) in normal, hyperplastic and neoplastic canine mammary tissues.

Leptin and its receptor (ObR) expression were investigated by immunohistochemistry in normal, hyperplastic and neoplastic canine mammary tissues and related to clinical-pathological features. Leptin expression was detected in healthy mammary tissues, adenosis and in benign mammary tumours and was lower in ductal hyperplasias and malignant tumours. A high percentage of ObR-positive cells were present in adenosis, benign tumours and in complex carcinomas, while ObR expression was lower in healthy mammary tissues, in ductal hyperplasias and in a large part of invasive mammary carcinomas. Our data demonstrated that cancer cells expressed at low level leptin and ObR in canine mammary tumours with a more aggressive behaviour, as well as in lymph node metastases. Consequently, leptin and ObR expressions in this species resulted to be not associated with a reduced overall survival.

Effect of a hydrocolloid dressing on first intention healing surgical wounds in the dog: a pilot study

OBJECTIVES To evaluate the efficacy of a hydrocolloid dressing for the treatment of surgical wounds in dogs. METHODS Six healthy young female dogs of medium size and different breed underwent ovariohysterectomy. Histological evaluation was performed on biopsies taken from the edges of the wounds at day 7. The dressing was applied on one half of the wound according to manufacturers instructions; the second half served as control. Biopsy specimens were fixed in a 10% formalin buffered solution pH 7.4, paraffin embedded and stained with haematoxylin and eosin. For clinical assessment, the presence and quality of exudate, erythema of the surrounding area, swelling and correct apposition of the wound margins were evaluated. RESULTS The hydrocolloid dressing was easy to use. The clinical quality of the treated skin wounds was superior to the non-treated ones. Comparison of histological features between treated and untreated wounds showed a more regular organisation of the granulation tissue in the treated wounds, with fibroblasts being aligned parallel to the overlying epidermis. The number of inflammatory cells and the extension of granulation tissue were less prominent and less widespread in treated compared to untreated wounds. CONCLUSION The dressing performed very well in terms of adhesiveness and flexibility. It was useful in the management of surgical wounds to avoid contamination and ameliorate the epithelialisation rate and granulation tissue morphology of the surgical scar.

Pancreatic Islet Cell Tumor Secreting Insulin-Like Growth Factor Type-II in a Dog

7-year-old, intact female, Gordon Setter was examined for a 6-month history of progressive weakness and ataxia without loss of appetite or change in weight. The owner reported a slight improvement in the signs after food consumption. The dog was kept indoors, regularly vaccinated, and fed a commercial maintenance diet. Physical examination revealed weakness, difficulty in holding quadrupedal posture, and mild muscle hypotrophy. No abnormalities were detected in the CBC, morphologic evaluation of the smear, and coagulation profile. Biochemical profile showed moderate hypoglycemia on fasting (57 mg/dL; range 80–120 mg/dL). Abdominal ultrasound showed an hypoechoic pancreatic lesion of 23 mm in diameter, with indistinct margins and poorly contrasting with adjoining structures. Eco-guided fine needle biopsy of the lesion was performed. The cytologic specimen contained a large number of naked nuclei on a cytoplasmic background with indistinct margins, occasionally acinar structures with moderate anisokaryosis. The cytologic pattern, together with clinical signs, suggested neuroendocrine tumor. Abnormalities were not detected on chest X-ray in 3 standard projections. Serum concentration of insulin was 0.5 mIU/mL (range 4–16 mIU/mL). Serum concentration of insulin-like growth factor type II (IGF-II) was evaluated by immunoradiometric assay (IRMA) after chromatographic separation. Five hundred microliters of serum were obtained from the dog and gel-filtered by fast protein liquid chromatography on HyPrep Sephacryl S-200 High Resolution column (GE Healthcare, Amersham Place, Little Chalfont, Buckinghamshire, UK) in a buffer containing 50 mM NaH2PO4, 0.15 M NaCl, 0.02% NaN3 ,p H 7.2. Samples were eluted at 0.8 mL/min and collected at 3minute intervals. 1 The 44 fractions collected were pooled and tested for IGF-II immunoreactivity as follows: fractions 8–11 corresponding to the 150 kDa ternary complex, fractions 12–16 corresponding to the 45 kDa binary complex, and fractions 24–27 corresponding to the free form of IGF-II. The fraction of IGF-II bound to insulin-like growth factor binding protein (IGFBP)-3 and acid labile subunit (ALS) forming a 150-kDa complex was 0.8 ng/mL. Similarly to normal dogs, IGF-II was only measurable in the 150 kDa region and undetectable in the others. IGF-II was measured by IRMA with reagents kit provided by DSL, a on acid ethanol pretreated samples. 2 The sensitivity of the assay was 0.13 ng/mL; the intra-assay and interassay coefficients of variation were 5.3 and 8.7%, respectively. No detectable cross-reactivity was found against IGF-I, up to 480,000 ng/mL, proinsulin, up to 2mg/mL, and insulin, up to 4.3mg/mL.