M. Massimini

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 μM, 1 μM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

HSP32 and HSP90 Immunoexpression, in Relation to Kit Pattern, Grading, and Mitotic Count in Canine Cutaneous Mast Cell Tumors.

Literature data indicate heat shock protein (Hsp) 32 and 90 as potential molecular targets in canine neoplastic mast cells (MCs). However, their immunoexpression patterns in canine mast cell tumors (MCTs) have not been investigated. Thus, the aim of this study was to evaluate the immunohistochemical expression of Hsp32 and Hsp90 in 22 canine cutaneous MCTs, in relation to KIT immunolabeling pattern, histological grade, and mitotic count. All cases showed cytoplasmic labeling of Hsp90, variably associated with nuclear and/or membranous labeling. Relationships of Hsp90 or Hsp32 immunolabeling with KIT pattern, mitotic count, and tumor grade were not observed. However, the reduced Hsp32 immunoexpression observed in most grade III/high-grade MCTs suggests a tendency toward a loss of immunosignal in poorly differentiated MCs. The great heterogeneity in extent and distribution of Hsp90 immunoexpression among the different MCT cases may also partially explain the difficulties in predicting the in vivo biologic activity of Hsp90 inhibitors on canine MCTs.

Survivin and Sox9: Potential Stem Cell Markers in Canine Normal, Hyperplastic, and Neoplastic Canine Prostate:

Canine prostatic carcinoma is a relevant model for human prostatic carcinoma. Survivin is proposed as a biomarker of malignancy in human prostatic cancer. Sox9 is a stem cell marker required for prostate development and expressed in several adult tissues. The aims of the present study were to evaluate the patterns and expression levels of 2 putative stem cell markers, survivin and Sox9, in canine benign prostatic hyperplasia (BPH) and prostatic carcinoma to investigate their potential as stem cell markers. Immunohistochemistry with specific antibodies was performed on 3 samples of normal prostate gland, 18 samples of canine BPH, and 16 samples of prostatic carcinoma. The basal cell layer of normal and hyperplastic prostatic lobules had nuclear Sox9 immunolabeling and nuclear and rarely cytoplasmic survivin immunostaining, identifying them as potential stem cell markers. Significantly more frequent survivin and Sox9 expression (≥10% of nuclei) was observed in prostatic carcinoma as compared with BPH. The potential coexpression of survivin with Sox9, androgen receptor, and p63 was also investigated in selected BPH and prostatic carcinoma cases with immunofluorescence, and a partial colocalization was observed. Results indicate that Sox9 and survivin could be considered markers of stemness in canine prostate cells. Given its role in proliferation, cells in the basal cell layer with nuclear survivin expression are likely to be transit-amplifying cells that maintain some stem cell proprieties.

Diffuse Pulmonary Adenocarcinoma with Micropapillary Growth Pattern in a Cat

A 12-year-old female European shorthair cat was presented with severe dyspnoea. Echocardiography revealed hypertrophic cardiomyopathy and pleural effusion. The cat died from acute decompensated left heart failure. At necropsy examination, the lungs were diffusely congested and firm, with multifocal grey areas and sparse haemorrhages. No solid masses were detected. Histopathology revealed a diffuse neoplastic proliferation characterized by irregular growth along alveolar walls with a micropapillary pattern. Tumour cells were large, highly pleomorphic and intensely positive for pan-cytokeratin and CAM 5.2. Tumour growth was obscured by simultaneous lesions related to chronic pulmonary congestion and interstitial lung disease. Histological features were consistent with a diffuse invasive pulmonary adenocarcinoma with a micropapillary pattern of tumour growth. Differential diagnosis included large cell carcinoma, which is usually characterized by rosettes or solid clusters of cells occupying alveolar lumen. Extensive cytokeratin immunolabelling was helpful in the differentiation from histiocytic proliferative disease.

Ovarian teratoma in an adult female Zoogoneticus tequila (Webb & Miller 1998): histological and immunohistochemical features

The Mexican Goodeid, Zoogoneticus tequila (Webb & Miller 1998), is considered nearly extinct in the wild, and it is maintained in captivity by the non-profit international ‘Goodeid Working Group’ represented by zoological institutions and aquarists in North America, Mexico and Europe. The unique, currently existing Italian colony was founded in 2007 by one of the authors (Arbuatti, Della Salda & Romanucci 2013), and it has produced about 240 fish so far. Information concerning the anatomy and biology of Z. tequila is extremely limited (Webb & Miller 1998; De La Vega-Salazar, Avila-Luna & Mac ıas-Garcia 2003). A single pathology survey on the unique Italian colony was also previously published by the authors (Arbuatti et al. 2013), but no other literature reports regarding pathologies of this species are available. Z. tequila is a viviparous species, and females give birth to free-swimming fry after an intraovarian gestation. The present case describes an adult female showing a progressive coelomic distention, suggestive of gestation. However, after a period of anorexia of 2-day duration, the fish died and was immediately submitted for necropsy examination. Macroscopical examination confirmed the abdominal enlargement, associated with the presence of a mucous secretion from the genital pore (Fig. 1a). A large, oval, brownish to grey, multilobulated mass (2 9 1.5 9 1 cm) occupied most of the coelomic cavity (Fig. 1b,c), causing intestinal occlusion by compression of the intestine without infiltration. Samples of the mass and representative tissues of all major organs were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut in 5-lm-thick sections, stained with haematoxylin and eosin (H&E) and examined by light microscopy. Additional sections were also processed for immunohistochemistry (IHC) using the standard avidin–biotin–peroxidase method and specific antibodies against pan-cytokeratin (CK), vimentin, neuron-specific enolase (NSE) (Dako) and glial fibrillary acidic protein (GFAP) (Chemicon International). Negative controls were performed in all instances by omitting the primary antibody from the staining schedule or replacing it by an antibody of irrelevant specificity. Sections of canine skin and brain were used as positive controls for CK and vimentin, and for NSE and GFAP, respectively. Histologically, the mass consisted of a complex variety of tissues derived from the three germ cell layers, suggesting a diagnosis of tridermic teratoma. Structures of ectodermal origin included multifocal, keratinizing squamous epithelium, and large areas of nervous system tissue resembling cerebrum (Figs 2 & 3). Mesenchymal tissues (mesodermal origin) consisted of skeletal muscle, cartilage, adipose tissue and fibrous connective tissue, with multifocal sheets of melanomacrophages Correspondence M Romanucci, Faculty of Veterinary Medicine, University of Teramo, Loc. Piano D’Accio S.P. 18, 64100 Teramo, Italy (e-mail: mromanucci@unite.it)

Unexpected Cardiac Death During Anaesthesia of a Young Rabbit Associated with Fibro-fatty Replacement of the Right Ventricular Myocardium

Summary A 6-month-old female pet rabbit was presented for routine ovariectomy. The pre-anaesthetic evaluation was unremarkable and no anaesthetic complications occurred during the procedure. However, at the end of the surgery, the rabbit suddenly showed acute bradycardia and cardiac death. Necropsy examination revealed marked dilation of the right ventricle, associated with diffuse thinning of the right ventricular free wall. Gross and histopathological findings were suggestive of a congenital dilated cardiomyopathy characterized by fibro-fatty replacement of the right ventricular myocardium. Similar myocardial lesions have not been previously described in rabbits, although they have been documented in myocardial diseases of man, dogs, cats, cattle, horses and chimpanzees.