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NLRP3 inflammasome is a multiprotein complex that can sense several stimuli such as autophagy dysregulation and increased reactive oxygen species production stimulating inflammation by priming the maturation of proinflammatory cytokines interleukin-1β and interleukin-18 in their active form. In the aging brain, these cytokines can mediate the innate immunity response priming microglial activation. Here, we describe the results of immunohistochemical and molecular analysis carried out on bovine brains. Our results support the hypothesis that the age-related impairment in cellular housekeeping mechanisms and the increased oxidative stress can trigger the inflammatory danger sensor NLRP3. Moreover, according to the recent scientific literature, we demonstrate the presence of an age-related proinflammatory environment in aged brains consisting in an upregulation of interleukin-1β, an increased microglial activation and increased NLRP3 expression. Finally, we suggest that bovine may potentially be a pivotal animal model for brain aging studies.

Carcinoma in situ of the breast is a well-known entity in humans. In veterinary medicine, particularly in canine and feline mammary literature, there is no agreement whether the term in situ should be used to indicate a specific carcinoma histotype or the noninvasive status of a carcinoma of any histotype. Moreover, in the most recent histologic classification of mammary tumors published by the Davis-Thompson Foundation, it is suggested to abandon the term carcinoma in situ given the lack of standardized criteria defining this entity, replacing it with epitheliosis or ductal/lobular hyperplasia with severe atypia. This publication presents a critical review of the term in situ in human and veterinary medicine considering the evolution of the term over the years and its heterogeneous use by different authors, including variations in immunohistochemical markers for classification. This review aims to point out the lack of uniformity in the nomenclature and classification issues in veterinary medicine regarding the use of the term in situ, laying the ground for a process of standardization in future publications.

White striping (WS) is an emerging myopathy of broiler chickens characterized by white striation of muscle. Despite the recent advances, the pathomechanism underlying the WS remains elusive.The aim of this study was to characterize morphological and molecular features of WS in broiler chickens. 50 pectoralis muscles were collected from 55 days old ROSS 308 broiler chickens with a mean weight of 3.5 kg. Samples were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Real-time-PCR was used to evaluate the expression of different cytokines.Histological lesions were observed in all examined animals, both with and without macroscopic evidence of WS. WS muscles showed endomysial and perivascular inflammatory infiltrates of macrophages and cluster of differentiation (CD)8-positive T lymphocytes with severe myofiber atrophy, necrosis, fibrosis and replacement by adipose tissue. There was diffuse sarcoplasmic and sarcolemmal overexpression of the major histocompatibility complex class I (MHC I). The severity of the histologic lesions was positively correlated with the macroscopic degree of white striations. IL-6, IL-17 and lipopolysaccharide-induced TNF-α factor (LITAF) were overexpressed in severe lesions of WS. The presence of the CD8/MHC I complexes, together with the higher expression of IL-6, IL-17 and LITAF in severe degree of WS, suggest that the immune response may be involved in the progression of this myopathy and can be consistent with a hypoxia-induced inflammatory myopathy. These results help to understand the pathomechanism of WS contributing to the reduction of economic losses and improving poultry welfare.

Background and objectives Laparoscopy is the preferred method when operating in the abdomen. In this study, we evaluated systemic and morphological peritoneal cytokine modifications (RANTES/CCL5 and MCP-1/CCL2) due to CO2 pneumoperitoneum in rats. Methods Twenty-five prepubertal Sprague-Dawley rats were randomized into three groups. Pneumoperitoneum lasting 30 minutes, was induced with a flow of 0.5 L/min, in two groups (S1 and S2, n = 20), at a P/CO2 of 6 and 10 mm Hg, respectively. In the control group (C, n = 5), only anesthesia was carried out. All animals were sacrificed after 24 hours. The serum of the rats was collected for ELISA, and the levels of the cytokines RANTES and MCP-1 were investigated. An immunohistochemical analysis of RANTES and MCP-1 was performed on samples of the peritoneum, and the morphological evaluation was conducted with a blinded evaluation by two independent, experienced pathologists by using a grading system (0, 1+, 2+, 3+: no, faint, moderate, and strong reactivity, respectively). Results RANTES mean levels were significantly different in the S1, S2, and C groups (70.3 ± 2.26, 58.23 ± 4.32, 29.66 ± 4.03, respectively, P = .0001). The levels of MCP-1 were 32.1 ± 1.63 in the S1 group, 27.0 ± 9.26 in the S2 group, and 16.4 ± 9.55 in the C group (P = .159). Normal control peritoneum showed little reactivity, whereas a moderate to strong cytoplasmic reaction to anti-CCL5/CCL2 antibodies was observed in mesothelial and inflammatory cells in the S1 and S2 groups. Conclusion CO2 pneumoperitoneum evokes an inflammatory response by modifying plasma RANTES levels and peritoneal CCL5/CCL2 expression.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

The transgenic LmnaG609G progeric mouse represents an outstanding animal model for studying the human Hutchinson-Gilford Progeria Syndrome (HGPS) caused by a mutation in the LMNA gene, coding for the nuclear envelope protein Lamin A/C, and, as an important, more general scope, for studying the complex process governing physiological aging in humans. Here we give a comprehensive description of the peculiarities related to the breeding of LmnaG609G mice over a prolonged period of time, and of many features observed in a large colony for a 2-years period. We describe the breeding and housing conditions underlining the possible interference of the genetic background on the phenotype expression. This information represents a useful tool when planning and interpreting studies on the LmnaG609G mouse model, complementing any specific data already reported in the literature about this model since its production. It is also particularly relevant for the heterozygous mouse, which mirrors the genotype of the human pathology however requires an extended time to manifest symptoms and to be carefully studied.

Sarcopenia is defined as the age-related loss of skeletal muscle mass, quality, and strength. The pathophysiological mechanisms underlying sarcopenia are still not completely understood. The aim of this work was to evaluate, for the first time, the expression of NLRP3 inflammasome in bovine skeletal muscle in order to investigate the hypothesis that inflammasome activation may trigger and sustain a pro-inflammatory environment leading to sarcopenia. Samples of skeletal muscle were collected from 60 cattle belonging to three age-based groups. Morphologic, immunohistochemical and molecular analysis were performed to assess the presence of age-related pathologic changes and chronic inflammation, the expression of NLRP3 inflammasome and to determine the levels of interleukin-1β, interleukin-18 and tumor necrosis factor alpha in muscle tissue. Our results revealed the presence of morphologic sarcopenia hallmark, chronic lymphocytic inflammation and a type II fibers-selective NLRP3 expression associated to a significant decreased number of immunolabeled-fibers in aged animals. Moreover, we found a statistically significant age-related increase of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 suggesting the activation of NLRP3 inflammasome. Taken together, our data suggest that NLRP3 inflammasome components may be normally expressed in skeletal muscle, but its priming and activation during aging may contribute to enhance a pro-inflammatory environment altering normal muscular anabolism and metabolism.

Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoaNeospora caninum,Trypanosoma cruzi,Babesia canis,andHepatozoon canis. However, although cardiac disease due toLeishmaniainfection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma.Leishmaniaamastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated withLeishmaniainfection.

The CDK inhibitor, p27

We have previously reported a critical role of HMGA proteins in pituitary tumorigenesis since either the Hmga1 or Hmga2 gene overexpression/activation induces the development of mixed growth hormone/prolactin cell pituitary adenomas by activating the E2F transcription factor 1, and then enhancing the G1/S transition of the cell cycle. Consistently, amplification and overexpression of the HMGA2 gene was found in human pituitary prolactinomas. Since impairment of the cell cycle control represents a feature of experimental and human pituitary adenomas, we have investigated the possible synergism between the alterations of other cell cycle regulators, such as p27 deficiency or Cdk4(R24C) mutation, with Hmga2 overexpression in pituitary tumorigenesis. Therefore, we crossed the Hmga2/T mice, overexpressing the truncated/active form of the Hmga2 gene, either with the knockout mice for p27(kip1), or with the knockin mice for the Cdk4(R24C) mutation, both developing pituitary adenomas. Increased incidence and decreased latency in the development of pituitary lesions appeared in double mutant Hmga2/T;Cdk4(R24C) mice, and increased features of invasiveness and atypia were observed in pituitary tumors of both Hmga2/T;p27-ko and Hmga2/T;Cdk4(R24C) double mutant mice as compared with single mutant compounds. Interestingly, most of these mice develop pituitary adenomas with high Ki67 index, extrasellar expansion and brain tissue infiltration, representing good mouse models for human aggressive pituitary adenomas. Taken together, the results reported here indicate a cooperation between HMGA2 overexpression and either p27(kip1) or CDK4 impairment in promoting pituitary tumor development and progression.

Horses affected by chronic piroplasmosis may develop poor performance and muscle atrophy. Here we investigate the pathological and immunopathological aspects of myopathy occurring in chronic equine piroplasmosis. The study included 16 horses serologically positive for equine piroplasms presenting with clinical signs and supporting serum biochemical evidence of a myopathy. Skeletal muscle was evaluated by histopathology, immunohistochemistry, indirect immunofluorescence, and molecular detection of piroplasms and inflammatory cytokines in skeletal muscle. Histologic lesions included muscle fiber atrophy (100% of cases), degenerative changes (13/16, 81%), and perivascular perimysial and endomysial lymphocytic infiltrates (81% of cases). In 15 cases (94%), muscle fibers had strong immunostaining for major histocompatibility complex classes I and II. T lymphocyte populations were mainly CD3+, CD8+, and CD4+ in equal proportions, with a lower number of CD79α+ cells. The serum from affected horses was tested by indirect immunofluorescence for binding of IgG, IgM, or IgA to sections of normal equine muscle to detect circulating autoantibodies against muscle antigen(s). In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Reverse transcription polymerase chain reaction (RT-PCR) testing of muscles from affected animals revealed a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor–α gene expression compared to healthy controls. Theileria equi or Babesia caballi was not detected in samples of affected muscle by RT-PCR. Thus, inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration.

Background Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system. Dysregulation of autophagy is implicated in the pathogenesis of many neurodegenerative diseases and integrity of the autophagosomal - lysosomal network appears to be critical in the progression of aging. Our aim was to survey the expression of autophagy markers and Amyloid precursor protein (APP) in aged bovine brains. For our study, we collected samples from the brain of old (aged 11–20 years) and young (aged 1–5 years) Podolic dairy cows. Formalin-fixed and paraffin embedded sections were stained with routine and special staining techniques. Primary antibodies for APP and autophagy markers such as Beclin-1 and LC3 were used to perform immunofluorescence and Western blot analysis. Results Histologically, the most consistent morphological finding was the age-related accumulation of intraneuronal lipofuscin. Furthermore, in aged bovine brains, immunofluorescence detected a strongly positive immunoreaction to APP and LC3. Beclin-1 immunoreaction was weak or absent. In young controls, the immunoreaction for Beclin-1 and LC3 was mild while the immunoreaction for APP was absent. Western blot analysis confirmed an increased APP expression and LC3-II/LC3-I ratio and a decreased expression of Beclin-1 in aged cows. Conclusions These data suggest that, in aged bovine, autophagy is significantly impaired if compared to young animals and they confirm that intraneuronal APP deposition increases with age.

A 3-year-old, male Labrador retriever dog was presented with clinical signs of progressive exercise intolerance, bilateral elbow extension, rigidity of the forelimbs, hindlimb flexion and kyphosis. Microscopical examination of muscle tissue showed marked variability in myofibre size, replacement of muscle with mature adipose tissue and degeneration/regeneration of muscle fibres, consistent with muscular dystrophy. Immunohistochemical examination for dystrophin showed markedly reduced labelling with monoclonal antibodies specific for the rod domain and the carboxy-terminal of dystrophin, while expression of β-sarcoglycan, γ-sarcoglycan and β-dystroglycan was normal. Immunoblotting revealed a truncated dystrophin protein of approximately 135 kDa. These findings supported a diagnosis of congenital canine muscular dystrophy resembling Becker muscular dystrophy in man.

Nitrate and nitrite are toxicants that have become increasingly significant environmental chemicals. Increase in environmental distribution of nitrogenous compounds, especially in surface and ground water, has been attributed to the intensive use of nitrate as agricultural fertilizers and to increasing amounts of nitrogenous wastes produced by municipalities, industries, and feedlots. The purpose of this study is to illustrate a fatal nitrate toxicosis in cattle associated with the consumption of fennels (Foeniculum vulgare). Fifteen cows from the same farm suddenly developed weakness, muscular tremors, respiratory distress, and finally convulsions. The affected animals died within 24 to 48 h from the onset of the clinical signs. Five cows underwent a complete post-mortem examination. In all examined animals, gross lesions included presence of dark unclotted blood around the nostrils and the anal region, moderate inflammation of the gastrointestinal mucosa, and brown discoloration of the skeletal muscles and kidneys. The histological examination showed tubular degeneration and congestion of glomerular vessels in the kidney. Toxicological analysis detected nitrates at 4 672.2 ppm in the fennels used to feed the animals. The source of exposure to nitrates was identified in the fennels. The fennels were grown in a polluted area of the Campania region in southern Italy and distributed in a public market for human consumption. The waste from the sale of the fennels was fed to the cows. The accumulation of nitrates in some vegetables poses a risk not only for animal health but also for human and environmental safety.

Sarcopenia, the age-related loss of muscle mass and strength, is a multifactorial condition that represents a major healthcare concern for the elderly population. Although its morphologic features have been extensively studied in humans, animal models, and domestic and wild animals, only a few reports about spontaneous sarcopenia exist in other long-lived animals. In this work, muscle samples from 60 healthy Podolica-breed old cows (aged 15–23 years) were examined and compared with muscle samples from 10 young cows (3–6 years old). Frozen sections were studied through standard histologic and histoenzymatic procedures, as well as by immunohistochemistry, immunofluorescence, and Western blot analysis. The most prominent age-related myopathic features seen in the studied material included angular fiber atrophy (90% of cases), mitochondrial alterations (ragged red fibers, 70%; COX-negative fibers, 60%), presence of vacuolated fibers (75%), lymphocytic (predominantly CD8+) inflammation (40%), and type II selective fiber atrophy (40%). Immunohistochemistry revealed increased expression of major histocompatibility complex I in 36 cases (60%) and sarcoplasmic accumulations of β-amyloid precursor protein–positive material in 18 cases (30%). In aged cows, muscle atrophy was associated with accumulation of myostatin. Western blot analysis indicated increased amount of both proteins—myostatin and β-amyloid precursor protein—in muscles of aged animals compared with controls. These findings confirm the presence of age-related morphologic changes in cows similar to human sarcopenia and underline the possible role of amyloid deposition and subsequent inflammation in muscle senescence.

The hotspot AKT1(E17K) mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. Recently, we have demonstrated that AKT1(E17K) transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1(E17)K mice) we demonstrate that AKT1(E17K) is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1(E17K) induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1(E17K) induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.

Four hundred bovine urothelial tumours and tumour-like lesions were classified in accordance with the 2004 World Health Organization (WHO) morphological classification for human urothelial tumours. The spectrum of neoplastic lesions of the urinary bladder of cattle is becoming wider and bovine urothelial tumours share striking morphological features with their human counterparts. A classification system based on the WHO scheme would also be appropriate for the classification of bovine bladder tumours. Bovine urothelial tumours are most often multiple. Four distinct growth patterns of bovine urothelial tumours and tumour-like lesions are recognized: flat, exophytic or papillary, endophytic and invasive. Carcinoma in situ (CIS) is the most common flat urothelial lesion, accounting for approximately 4% of urothelial tumours. CIS is detected adjacent to papillary and invasive tumours in 80-90% of cases. Approximately 3% of papillary lesions are papillomas and approximately 5% are 'papillary urothelial neoplasms of low malignant potential' (PUNLMP). Low-grade carcinoma is the most common urothelial tumour of cattle. High-grade carcinomas, and low and high-grade invasive tumours, are less commonly seen. Bovine papillomavirus (BPV) infection and ingestion of bracken fern both play a central role in carcinogenesis of these lesions.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. PPAR-gamma is expressed in multiple normal and neoplastic tissues, such as the breast, colon, lung, ovary and placenta. In addition to adipogenic and anti-inflammatory effects, PPAR-gamma activation has been shown to be anti-proliferative by its differentiation-promoting effect, suggesting that activation of PPAR-gamma may be useful in slowing or arresting the proliferation of de-differentiated tumour cells. In this study, we investigated the expression of PPAR-gamma in normal and neoplastic canine nasal epithelium. Twenty-five samples composed of five normal nasal epithelia and 20 canine nasal carcinomas, were immunohistochemically stained for PPAR-gamma. The specificity of the antibody was verified by Western Blot analysis. Confocal laser scanning microscopical investigation was also performed. In normal epithelium, the staining pattern was cytoplasmic and polarized at the cellular free edge. In carcinomas, the neoplastic cells showed mainly strong cytoplasmatic PPAR-gamma expression; moreover, perinuclear immunoreactivity was also detected and few neoplastic cells exhibited a nuclear positivity. Our results demonstrate different patterns of PPAR-gamma expression in normal canine nasal epithelium when compared with canine nasal carcinoma. The importance of this transcription factor in the pathophysiology of several different tumours has stimulated much research in this field and has opened new opportunities for the treatment of the tumours.

Studies in human immune-mediated inflammatory myopathies have documented expression of major histocompatibility complex class I (MHC class I) and class II (MHC class II) antigens on muscle fiber membranes in the presence or absence of cellular infiltration. Here we evaluate the presence and distribution of these antigens in canine masticatory muscle myositis, an immune-mediated inflammatory myopathy. Twelve samples of temporalis and masseter muscles from dogs with a clinical diagnosis of canine masticatory muscle myositis were examined by immunohistochemistry and double-immunofluorescence confocal microscopy. MHC class I and class II antigens were expressed in muscle fibers independent of inflammatory cell infiltration. Furthermore MHC class I and class II antigens were expressed on the sarcolemma and co-localized with dystrophin. Our results suggest that MHC class I and class II expression in canine masticatory muscle myositis may play a role in the initiation and maintenance of the pathological condition, rather than just a consequence of a preceding local inflammation.

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/ PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels.

// Vincenza Leone 1 , Concetta Langella 1 , Francesco Esposito 1 , Claudio Arra 2 , Giuseppe Palma 2 , Domenica Rea 2 , Orlando Paciello 3 , Francesco Merolla 1 , Davide De Biase 3 , Serenella Papparella 3 , Angela Celetti 1 , Alfredo Fusco 1, 4 1 Istituto per l’Endocrinologia ed Oncologia Sperimentale del CNR e/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita degli Studi di Napoli “Federico II”, Naples, Italy 2 Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy 3 Department of Pathology and Animal Health, University of Naples “Federico II”, Naples, Italy 4 Instituto Nacional de Câncer - INCA, Rua Andre Cavalcanti, Rio de Janeiro, CEP RJ, Brazil Correspondence to: Alfredo Fusco, e-mail: alfusco@unina.it Keywords: Ccdc6, thyroid, CREB1, knock-in mice Received: February 3, 2015 Accepted: April 14, 2015 Published: April 27, 2015 ABSTRACT CCDC6 was originally identified upon rearrangement with RET in human thyroid papillary carcinomas generating the RET/PTC1 oncogene. We have previously reported that CCDC6 interacts with CREB1 and represses its transcriptional activity. Since the function of at least one allele of CCDC6 is lost following RET/PTC1 rearrangements, we aimed at the generation of mice, carrying a CCDC6 mutant gene. Previous studies suggested that the coiled-coil domain of CCDC6, mainly encoded by human exon 2, is required for the protein function. Therefore, we engineered a murine Ccdc6 construct, carrying a deletion of the exon 2, that was able to exert only a mild repression on CREB1 transcriptional activity, with respect to the wild type Ccdc6. Subsequently, we generated Ccdc6 -ex2 knock-in mice. These mice developed thyroid hyperplasia associated with an enhanced CREB1 activity and an increased expression of the CREB-1 regulated genes. These results strongly support a CCDC6 promoting role, ascribed to its functional impairment, in the development of thyroid papillary carcinomas harboring the RET/PTC1 oncogene.

Central core disease is a nonprogressive or slowly progressive congenital myopathy with a variable degree of hypotonia and axial and proximal muscle weakness that is histologically characterized by areas devoid of oxidative enzyme activity, resulting from an absence or low numbers of mitochondria in these regions (central core). A 10-month-old, male, pony foal was examined because of stiff gait, marked contractures of the distal portion of the limbs, flexion deformities of the hooves, and moderate hypotonia that had been present from birth. The foal had increased creatine kinase (282 U/ liter; reference interval 10-135 U/liter), lactate dehydrogenase (1,188 U/liter; reference interval 150–450 U/liter), and aspartate transaminase (377 U/liter; reference interval

Histiocytic diseases in veterinary medicine have been revised in the last few decades, but these are considered relatively rare in horses. This report describes a 9-year-old female horse, Dutch Warmblood, presented for investigation of severe nasal bleeding. A multinodular bilateral mass of 5 cm, reddish to white in color, that invaded and destroyed the surrounding tissues, was observed during a clinical examination of the nostril The morphological features of the tumor cells were represented by cytologically bizarre, highly phagocytic, multinucleated giant cells. These findings, together with immunohistochemical results allowed a diagnosis of histiocytic sarcoma.

Background: Ehlers-Danlos syndrome comprises a group of rare inherited connective tissue diseases characterized by skin hyperextensibility, joint laxity, skin and vessel fragility, and poor wound healing. Objective: The purpose of this report was to describe the clinical, histologic, and ultrastructural findings in 2 dogs with collagenopathies consistent with Ehlers-Danlos syndrome. Methods: Two dogs were examined clinically; skin extensibility index was calculated. Skin biopsies obtained from the dorsum were examined by light and electron microscopy. Results: Both dogs had clinical signs of skin hyperextensibility and fragility, lower skin elasticity, vessel fragility, and poor wound healing. One dog had a hip dislocation, and the other had bilateral medial patellar luxation (grade II), subcutaneous hematomas produced by minimal trauma, and generalized periodontitis. Histologic and ultrastructural examination confirmed abnormalities in the structure and arrangement of collagen fibrils. Fibroblasts were characterized by variable dilatation of the rough endoplasmic reticulum, and anomalous elastic fibers (elaunin fibers) were present in the dermis. Conclusion: Although the primary defects underlying collagenopathies in animals are still unknown, analysis of the ultrastructural changes in collagen fibrils and clinical findings could facilitate better characterization of these disorders in dogs.

Background: Canine angiostrongylosis is a nematode infection in domestic dogs and wild carnivores. The present report focuses on epidemiological, clinical and histopathological findings in a case of fatal disseminated angiostrongylosis in a dog living in southern Italy and provides data on the extent of the spread of Angiostrongylus vasorum in the same area.Case presentation: A 4-year-old female English Setter from the Campania region of southern Italy was referred with a 2-week history of cough and severe respiratory distress that did not respond to antimicrobial therapy. Based on clinical, radiological, echographical and cytological findings (including the presence of larvae), a suspect diagnosis of lungworm infection was performed. After few days the dog died due to progressive clinical aggravation. Complete postmortem examination was conducted within 24 hours from death and samples from lungs, heart, liver, kidney, spleen, stomach and small intestine were fixed in 10% buffered formalin. Grossly, several hemorrhagic foci were observed mostly in the lungs, liver, kidney. Microscopically, the lungs contained numerous, multifocal to coalescing granulomas composed of epitheliod macrophages, multinucleated giant cells and some neutrophils, frequently associated with parasite eggs and larvae. The lungs contained many firm nodules, many adult nematodes approximately 1.5 to 2 cm in length were observed in cut sections and identified as A. vasorum. A subsequent parasitological survey performed with FLOTAC on stray dogs living in the same area showed the presence of A. vasorum larvae in 17 of 1639 stray dogs examined (1.04%).Conclusion: This survey provides new data on distribution of A. vasorum and underlines that canine angiostrongylosis should be considered as differential diagnosis in dogs.

The expression of the beta1 common chain of the VLA integrin subfamily was evaluated immunohistochemically in a series of five normal and 30 neoplastic canine testicles. The tumours, consisting of seminomas or Sertoli cell tumours, were classified according to WHO criteria as intraductal without signs of invasion, intraductal with signs of invasion, or diffuse. Expression of beta1 integrin decreased progressively from intraductal tumours without signs of invasion, to the diffuse type, in which immunolabelling was generally absent. In a few cases of diffuse neoplasia, groups of neoplastic cells exhibited strong positivity that was not restricted to the basal pole of the cell membrane. These results suggest that the expression of beta1 integrin was related to the histological tumour type, possibly reflecting a specific requirement for a reduction in integrin by neoplastic cells with infiltrative and metastatic potential.

Summary The epithelial intercellular adhesion molecule, E cadherin, was evaluated immunohistochemically in a series of normal and malignant neoplastic canine mammary glands. The normal glands showed strong immunolabelling at the intercellular borders of all the ductal cells. Tubular and papillary mammary adenocarcinomas had reduced intercellular E cadherin positivity with “heterogeneous” or “cytoplasmic” distribution. Solid and squamous cell carcinomas were negative. These results suggest that reduced expression and abnormal distribution of E cadherin may be related to the differentiation grade of these neoplasms.

This report describes the histopathology of two hundred and fifty-three mesenchymal tumors of the urinary bladder in cattle grazing on lands rich in bracken fern. Approximately 80% were hemangiomas and angiosarcomas. Hemangioma (capillary, cavernous, and large vessels) was the most frequent mesenchymal tumor and was more common than angiosarcoma. Although the appearance of endothelial cells can vary remarkably, epithelioid angiosarcomas, often containing multinucleated cells, were the most frequent malignant vascular tumors. Hemangiopericytoma and tumors of muscle and soft connective tissue origin, alone and/or in association with tumor-like lesions, were less frequently seen. Furthermore, forty-five cases of intravascular papillary endothelial hyperplasia (IPEH), a lesion not previously reported in the urinary bladder of cattle, were also described. Bovine papillomavirus type-2 DNA was amplified in tumor samples. Forty vascular tumors were investigated by dual-labeling immunofluorescence, and, for the first time, a coexpression of E5 and platelet-derived growth factorβreceptor (PDGFβR) was shown to occur. The results show that the BPV-2 E5 oncoprotein binds to the activated form of the PDGFβreceptor thus playing an important role in mesenchymal as well as epithelial carcinogenesis of the urinary bladder. Furthermore, these findings demonstrate that BPV-2 infects both epithelial and mesenchymal cells.

Gastrointestinal (GI) strongyle infection remains one of the main constraints to goat production worldwide. Samples of small intestine from 15 Syrian goats naturally infected with Trichostrongylus colubriformis were examined by routine histology, histochemistry and immunohistochemistry to describe the histological changes and the phenotypes of inflammatory cellular components of the mucosa. Results indicated that the immune response to infection by T. colubriformis was characterized by an increased rate of the severity of the histologic lesions, an increase rate of T cell lymphocytes recruitment to the intestinal mucosa and quantitative and qualitative changes in the histochemical composition of mucin in goblet cells.

Introduction We have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status. Material and methods Blood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Results Cytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG > CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog. Conclusions These results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53.

Idiopathic inflammatory myopathies are muscle diseases characterized by inflammation, necrosis, and fibrosis. The neurotransmitter serotonin (5-HT) has been shown to promote fibrosis in many tissues and organs by activating TGF beta-1 signaling. In this study, we evaluated the potential role of 5-HT in the pathogenesis of fibrosis in canine idiopathic inflammatory myopathies. Muscle biopsies from dogs affected by masticatory muscle myositis or polymyositis and from healthy dogs were processed for immunohistochemistry and Western blotting. The immunohistochemical analysis showed a strong expression of 5-HT in muscle tissues of affected dogs, whereas the amine was absent in the muscles of healthy dogs. Biochemical analysis showed increased expression levels of the selective 5-HT2A receptor in the muscle specimens of the most severely affected dogs versus controls. Further, increased phosphorylation levels of the TGF beta-1 signaling mediators SMAD2/3 and ERK1/2 were detected in tissue samples from affected dogs as compared to tissues from healthy clogs. Although further studies are needed, our findings highlight for the first time a potential role of 5-HT in the development of fibrosis in canine idiopathic inflammatory myopathies, thus supporting other evidence that 5-HT pro-fibrotic activity occurs via activation of TGF beta-1 signaling pathway. (C) 2012 Elsevier B.V. All rights reserved.

The Silent Corticotroph Adenoma (SCA) is a pituitary adenoma variant characterized by the immunoreactivity for adrenocorticotropic hormone (ACTH) and related peptides, without the clinical signs of Cushing's disease. SCA has been postulated to either secrete structurally abnormal ACTH that is inactive but detectable by immunohistochemistry or radioimmunoassay, or to secrete ACTH intermittently or at low levels continuously. Excess of ACTH has been associated to type II muscle atrophy. We describe a case of type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog. The dog showed moderate to severe proximal muscle wasting and weakness with normal levels of muscle-associated enzymes. In the limb muscle biopsies, type II fibers were uniformly smaller than type I fibers. In temporalis muscles, there were few atrophic fibers, and several irregular areas of loss of enzymatic activity observed in NADH, SDH and COX stains. The tumour showed a trabecular growth pattern and immunohistochemical analysis demonstrated the presence of cytoplasmic immunoreactivity for ACTH. The muscle atrophy was considered to be related to an excess of inactive ACTH. Studying spontaneous occurring rare diseases in animals could help to understand the mechanism of similar diseases in human has well.

Idiopathic inflammatory myopathies (IIMs) are inflammatory disorders of unknown origin. On the basis of clinical, histopathological, and immunological features, they can be differentiated into three major and distinct subsets: dermatomyositis; polymyositis; and inclusion-body myositis. Although a few animal models for IIM are currently available, they lack several characteristic aspects of IIMs. The aim of our study was to examine skeletal muscle involvement in an experimental animal model of visceral leishmaniasis, a disseminated infection caused by the protozoan parasite Leishmania infantum, and to compare features of associated inflammation with those of human IIM. Syrian hamsters infected intraperitoneally with amastigotes of L. infantum were killed at 3 or 4 months post-infection, and the skeletal muscles were studied. Focal inflammation was predominantly observed in the endomysium and, to a lesser extent, in perivascular areas. Degenerating muscle fibers were also found, as well as myonecrosis. Immunofluorescence with confocal laser scanning microscopy was used to characterize the phenotype of inflammatory infiltrates and the distribution of MHC class I and II in muscle biopsies. The infiltrating inflammatory cells consisted mainly of T cells, and CD8(+) T cells were found in non-necrotic muscle fibers that expressed MHC class I on the sarcolemma. In addition to T cells, several macrophages were present. The model we are proposing closely resembles polymyositis and may be useful in studying certain aspects of this disease such as the role of T cells in muscle inflammation and myocytotoxicity, while also providing novel therapeutic targets.

The broad category of neuromuscular diseases covers conditions that involve the weakness or wasting of the body muscles. These problems may occur in the spinal cord, the peripheral nerves or the muscle fibers. Some may be hereditary, while others are acquired. Commonly recognized conditions fall into the categories of myopathies, which are diseases of the muscle like muscular dystrophy, disorders of the junction where the nerve impulses are transmitted to the muscle like myasthenia gravis, and neuropathies, which are diseases of the peripheral nervous system. The diagnosis of most neuromuscular diseases rest on careful clinical evaluation of the patient, electromyography, the muscle biopsy, and in some instances, molecular genetic studies. Muscle biopsy, associated to histochemical and immunohistological techniques, plays a key role in diagnosis of many neuromuscular disorders. A number of morphological abnormalities of muscle can be recognized on histological stains such as haematoxylin and eosin and Engel trichrome. Histochemical techniques are essential for the study of muscle biopsies for four main reasons. First, they demonstrate the non-uniform nature of the muscle highlighting the different biochemical properties of specific fibre type and their selective involvement in certain disease processes. Second, they may show an absences of a particular enzyme. Third, an excess of a particular substrate can be demonstrated. Fourth, they may show structural changes in the muscle which would not be apparent with routine histological stains, such as the enzyme-deficient cores in central core disease "mouth-eaten" fibers, and abnormalities in the distribution of mitochondria. In some neuromuscular disorders there could be only non-specific myopathological features. However, a number of proteins, including sarcolemmal, sarcomeric, and nuclear proteins as well as enzymes with defects responsible for neuromuscular disorders, have been identified during the past two decades, allowing a more specific and firm diagnosis of muscle diseases. Identification of protein defects relies predominantly on immunohistochemical preparations and on Western blot analysis. While immunohistochemistry is very useful in identifying abnormal expression of primary protein abnormalities in recessive conditions, it is less helpful in detecting primary defects in dominantly inherited disorders. Abnormal immunohistochemical expression patterns can be confirmed by Western blot analysis which may also be informative in dominant disorders. Besides identification of specific protein defects, immunohistochemistry is also helpful in the differentiation of inflammatory myopathies by subtyping cellular infiltrates and demonstrating up-regulation of subtle immunological parameters. This review will summarize and describe the impact that histochemistry and immunohistochemistry has had and the possibilities it has opened up in the diagnosis of neuromuscular disorders in human as well as in veterinary myology.

Objective: to describe a case of a Isolated Lepromatous Conjunctivo-corneal granuloma in a cat living in Southern Italy. Methods: A two-year-old female black European shorthair cat, living in a suburban environment, with an outdoor lifestyle, was presented for evaluation of a 2 weeks’ duration nodular pinkish vascularized mass arising in the dorsal bulbar conjunctiva at the temporal cantus of left eye and invading the lateral surface of sclera and cornea. The cat didn’t show any other abnormality on both eyes by slit-lamp biomicroscopic and indirect ophtalmoscopic examination. Serologically feline leukemia virus and feline immunodeficiency virus were excluded, and IgG title for toxoplasmosis was 1:640. Cytobrush samples were obtained and a biopsy of the mass was performed. Results: Neuthophils, lymphocytes and acid-fast bacilli packed in the cytoplasm of foamy macrophages and giant cells were seen on cytological examination. The histological examination confirmed the presence of a granulomatous lesion with acid-fast bacilli within macrophages. Bacteriological culture of the material from the lesion and polymerase chain reaction technique were negative for Mycobacterium spp.. A diagnosis of feline leprosy was supposed. The owners refused any treatment, and one year later the lesion was still present. Conclusions: Veterinary ophthalmologists should be aware of conjunctivo-corneal leproma as an unusual symptom of leprosy.

Inflammatory myopathy associated with several infectious diseases occurs in dogs including those caused by Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Hepatozoon canis. However, muscle disease due to Leishmania infection has been poorly documented. The aim of this study was to examine the distribution and types of cellular infiltrates and expression of MHC class I and II in muscle biopsies obtained from 15 male beagle dogs from a breeder group with an established diagnosis of leishmaniasis. Myopathic features were characterized by necrosis, regeneration, fibrosis and infiltration of mononuclear inflammatory cells consisting of lymphocytes, plasma cells and histiocytes. The predominant leukocyte populations were CD3+, CD8+ and CD45RA+ with lesser numbers of CD4+ cells. Many muscle fibers had MHC class I and II positivity on the sarcolemma. There was a direct correlation between the severity of pathological changes, clinical signs, and the numbers of Leishmania amastigotes. Our studies provided evidence that: 1) Leishmania should be considered as a cause of IM in dogs; 2) Leishmania is not present within muscle fibers but in macrophages, and that 3) the muscle damage might be related to immunological alterations associated with Leishmania infection. Leishmania spp. should also be considered as a possible cause in the pathogenesis of human myositis.

Pneumoperitoneum (PN) and the gas used to insufflate the abdominal cavity during laparoscopy seem to be responsible for local and systemic modifications. The aim of this study was to verify the effects of intra-abdominal carbon dioxide (CO(2)) and air insufflation on the peritoneum, as well as the cortico-surrenal response in prepuberal rats. Sixty prepuberal rats were divided into three groups: in the first (S, n = 36), PN was induced with CO(2), whereas in the second (A, n = 14), it was induced with filtered room air; in both conditions, insufflation lasted 30 min at a pressure of 10-12 mmHg. The third group (C, n = 10), underwent general anesthesia only. Two hours after inducing anaesthesia, 12 rats in group S, 6 in group A and 6 in group C were killed and the remaining, after 24 h; specimens of the visceral and the parietal peritoneum were obtained for histological examination, blood sample was taken for cortisol and DHEA-S assays at the different study periods. At the histological examination performed 2 h later, the groups S and A presented inflammatory cell infiltrate in the parietal and visceral peritoneum; this finding was even more marked in group A, which presented also congestion, hemorrhage and disruption of the cell line. Twenty-four hours after the experiment, the peritoneum of the two insufflated groups presented chronic infiltrate and reactive mesothelial cells with congestion, which was more evident in group A, but totally absent in group C. Cortisol levels were significantly higher in groups S and A (2.15:1 ratio) killed 2 h later compared to those killed 24 h later and to the control group. DHEA-S levels were not significantly different between the groups. Our results demonstrate that the chemical, physical and molecular impact of CO(2) on the peritoneum causes inflammation and tissue damage, this was even more evident 24 h after our experiment and in the air insufflated group. PN induced a significant variation in blood cortisol levels at 2 h. The CO(2) insufflation should be limited in patients with pre-existing peritoneal damage.

Background Telepathology is the process of diagnostic pathology performed on digital images viewed on a display screen rather than by conventional light microscopy with glass slides. At present, electronic mail (e-mail) attachment is the most common communication medium for telepathology, because it is relatively simple and incurs minimal cost. Objective Our objective was to test whether telediagnosis through Internet e-mail could be applied to veterinary diagnostic cytology. Methods Using 20 cytology cases, on which a consensus diagnosis was reached by 3 experienced pathologists, a total of 130 images were digitized and sent as e-mail attachments from the Unit of Anatomic Pathology of the Faculty of Veterinary Medicine of Naples to the Unit of Pathology of the Faculty of Veterinary Medicine of Messina, Italy. The images were reviewed at the Unit of Pathology of Messina, where the consulting pathologist formulated his diagnoses by visualizing the images on the monitor of a computer. The telediagnoses were compared with the consensus diagnoses and with the consulting pathologist's interpretation using conventional light microscopy at a later date. Results The median time to capture images was approximately 30 minutes for each case. The median time to make a telecytodiagnosis was a few seconds for each case. Overall, there was good agreement (85%) between the consensus diagnosis and the consultant's telediagnosis. In 100% of the cases there was agreement between the consulting pathologist's telediagnosis and conventional glass slide diagnosis. Conclusion Telepathology by e-mail provides acceptable efficacy and a faster turnaround time than post and can be applied to veterinary diagnostic cytology.

The aim of this study was to correlate nuclear morphometric features with animal and human World Health Organization International Histological Classifications in canine seminomas. Twenty-three canine seminomas were classified, according to Animal World Health Organization International Histological Classification as intratubular, intratubular with signs of invasion, or diffuse and according to Human World Health Organization International Histological Classification criteria as spermatocytic and typical. The morphonuclear characteristics of tumors were quantitatively evaluated by means of digital cell image analyses of hematoxylin and eosin-stained nuclei. In particular, the mean nuclear area, mean nuclear perimeter, mean nuclear form factor, and their respective standard deviations were calculated. The relationship between the different variables and the tumor histologic types was assessed. On the basis of animal and human classification systems, statistically significant differences were observed only between intratubular seminomas with signs of invasion and the other two types and between spermatocytic and typical seminomas, respectively. In humans, it is well known that typical seminomas are more common and aggressive than spermatocytic ones. In our study, the canine seminomas classified as typical showed significantly larger and more variable nuclear area and perimeter than spermatocytic seminomas. These results support the opinion that most canine seminomas correspond to human spermatocytic seminomas and could explain the benign behavior of canine seminomas, which derive from a more differentiated type of germ cell.

A 9-month-old male German Shepherd dog was referred for evaluation of progressive exercise intolerance. Clinical examination revealed a stiff, stilted gait and marked atrophy and hypotonia of skeletal muscle. The dog had raised creatine kinase (181 U/liter), lactate dehydrogenase (510 U/liter), and aspartate aminotransferase (123.6 U/liter) levels, suggesting a muscle disease. Histochemical evaluation of muscle biopsies revealed the presence of subsarcolemmal oxidative activity, reduced nicotinamide adenine dinucleotide, and succinate dehydrogenase, and the absence of cytochrome oxidase activity. Ragged red fibers were demonstrated with Gomori trichrome stain. Ultrastructural examination of the muscle confirmed the presence of subsarcolemmal accumulations of mitochondria and morphologically atypical mitochondria.

Cyclooxygenase-1 (COX-1) and cyclooxygenase -2 (COX-2) are known to play a role in the carcinogenesis of many human and animal primary epithelial tumours. However, expression of COX-1 and -2 has not been investigated in canine nasal epithelial carcinoma, a rare form of neoplasia. COX-1 immunolabelling was demonstrated in normal canine nasal mucosa and in a minority of neoplastic specimens. Cytoplasmic COX-2, however, was strongly expressed in the majority of canine nasal carcinomas. In addition, COX-2 expression was demonstrated in dysplastic epithelium and in a proportion of stromal cells. Co-expression of both enzyme isoforms was revealed by confocal laser scanning microscopy. The results indicate that COX-2 is overexpressed in a proportion of naturally occurring canine nasal carcinomas, suggesting its possible role in canine nasal tumorigenesis.

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, and the beginning of angiogenesis, by interacting with specific endothelial receptors termed VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). In this study, Flk-1 expression was evaluated immunohistochemically in 10 benign and 40 malignant canine mammary tumours. There was immunolabelling of endothelial cells located within the neoplastic proliferation and at the infiltrating periphery, and also of neoplastic cells. The number of positive endothelial and neoplastic cells, was higher in malignant than in benign tumours. Moreover, in the malignant tumours, expression of Flk-1 increased from well to less differentiated phenotypes (grade 1–3). The presence of VEGF receptor on neoplastic cells suggests that VEGF has an autocrine function in which neoplastic cells act as both VEGF producers and target cells. Thus, in malignant tumours, VEGF may contribute to neoplastic growth by inducing angiogenesis and by stimulating the proliferation of neoplastic cells.

Angiogenesis, which assists in supplying the nutritional and respiratory needs of proliferating cells, is essential for tumour growth. Angiogenic control is complex, involving a network of cytokines, in particular vascular endothelial growth factor ( VEGF ), a potent endothelial cell mitogen which also stimulates neoplastic cell proliferation. The purpose of this study was to evaluate VEGF expression and microvessel density (number of microvessels per mm 2 ), in canine seminomas. VEGF expression and microvessel density were higher in seminomas than in normal testicular tissue; both parameters were higher in diffuse tumours than in intratubular tumours. These data demonstrate an increase in angiogenesis in the more malignant histological types of seminoma and suggest that both VEGF and microvessel density are useful criteria for evaluating the intrinsic malignancy and growth potential of canine testicular tumours.

An immunohistochemical approach was used to examine the expression of MMP-2 and the activator MT1-MMP in a series of 50 canine mammary carcinomas of different histotype and stage. MMP-2 and MT1-MMP were mainly expressed in the cytoplasm of carcinoma cells and in fibroblasts. Immunolabelling for both MMP-2 and MT1-MMP was also seen on the tumour cell membranes. This labelling pattern showed no significant association with either the histological sub-type or stage of the carcinomas. Therefore, although distinct patterns of expression of MMP-2 and MT-MMP1 were shown by these carcinomas, functional studies by means of zymography would be required to provide useful information on tumour behaviour.

To investigate the diagnostic and prognostic value of nuclear morphometry in squamous cell carcinomas (SCCs) of canine skin, histological samples from 15 dogs were retrospectively analysed by computerized nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), standard deviation of MNA (SDa), mean nuclear perimeter (MNP), standard deviation of MNP (SDp), nuclear form factor (FF), and standard deviation of FF (SDf) were calculated. The morphometric data were examined in relation to tumour histological grade. No statistical differences were found between the different grades of SCC in terms of the mean values of SDa, SDp, FF or SDf. The MNA values, however, differed significantly between grades: I and III; I and IV; II and III; II and IV. The MNP values differed significantly between grades: I and III; I and IV; II and IV. The results indicate that nuclear morphometry analysis is a simple and reproducible method that could be used to provide objective diagnostic criteria for cutaneous SCCs in the dog.

Vascular endothelial growth factor (VEGF) is a dimeric protein that stimulates angiogenesis in vitro and in vivo by inducing endothelial cell proliferation and migration. In this immunohistochemical study, VEGF-immunolabeled cells were counted in a series of 10 benign and 40 malignant canine mammary tumors. The morphologic pattern of VEGF positivity (intensity of immunolabeling and VEGF granule size and distribution) was also evaluated. A low number of cells weakly positive for VEGF with few and small granules polarized to the luminal pole was detected in benign neoplasms. In contrast, in malignancies a high number of VEGF-positive cells had strong immunolabeling, often with large granules found diffusely in the cytoplasm. This level of immunolabeling was more pronounced in the less differentiated, more malignant phenotypes (grade 3). Macrophages, which can synthesize VEGF, were strongly positive. Stromal and myoepithelial cells were negative. VEGF data were correlated statistically with intratumoral microvessel density (number of newly formed microvessels) and both measures were greater in less differentiated malignant neoplasms, demonstrating that angiogenesis and malignancy increase together. VEGF appears to be a powerful angiogenic factor in canine mammary tumors.

The distribution of type IV collagenase (cIVase) was evaluated immunohistochemically in a series of normal, dysplastic and neoplastic canine mammary glands, as well as in lymph node metastases. In normal and dysplastic mammary tissues, and benign mammary tumours, cIVase was observed in myoepithelial cells, but in malignant tumours it was mainly localized in the cytoplasm of carcinoma cells and fibroblasts close to infiltrating neoplastic cells. No significant difference was observed between carcinomas of different histological sub-types. These results suggest that the distribution of cIVase is a potentially useful indicator of malignancy in canine mammary tumours. (C) 1997 W.B. Saunders Company Limited.

Summary This study evaluates the mitotic phase distribution, mitotic activity and apoptosis in 17 basal cell tumours of canine skin. The number of mitotic and apoptotic cell/mm2 of neoplastic epithelium is counted and the volume corrected mitotic and apoptotic index, respectively, are obtained (M/V and A/V index). Transmission electron microscopy is performed on selected cases to confirm the typical features of apoptosis. The M/V index ranges from 9 to 45/mm2 of neoplastic epithelium, while the A/V index ranges from 5 to 111/mm2 of neoplastic epithelium. Even if the number of apoptotic cells is slightly higher than that of mitotic cells and vice versa, the mean values of the two parameters (mean M/V = 20.76 ± 10.86, mean A/V = 31.41 ± 24.53) tend to be equal. In fact, apoptotic and mitotic index are not statistically different (P = 0.11). Furthermore, in all the samples examined, an increased proportion of metaphases (46.82%) is observed. These findings suggest that the discrepancy between apparent mitotic activity and tumour growth may be mainly due to the increased duration of the entire cell cycle rather than to the high incidence of apoptosis.

Mutations in any of the genes encoding the alpha, beta or gamma-sarcoglycan components of dystrophin-associated glycoproteins result in both sporadic and familial cases of either limb-girdle muscular dystrophy or severe childhood autosomal recessive muscular dystrophy. The collective name 'sarcoglycanopathies' has been proposed for these forms. We report the identification of a fourth member of the human sarcoglycan family. We named this novel cDNA delta-sarcoglycan. Its mRNA expression is abundant in striated and smooth muscles, with a main 8 kb transcript, encoding a predicted basic transmembrane glycoprotein of 290 amino acids. Antibodies specifically raised against this protein recognized a single band at 35 kDa on western blots of human and mouse muscle. Immunohistochemical staining revealed a unique sarcolemmal localization. FISH, radiation hybrid and YAC mapping concordantly linked the delta-sarcoglycan gene to 5q33, close to D5S487 and D5S1439. The gene spans at least 100 kb and is composed of eight exons. The identification of a novel sarcoglycan component modifies the current model of the dystrophin-glycoprotein complex.