S. Papparella

Expression of Vascular Endothelial Growth Factor in Canine Mammary Tumors

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.

Correlation of Nuclear Morphometric Features with Animal and Human World Health Organization International Histological Classifications of Canine Spontaneous Seminomas

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.

Expression of major histocompatibility complex class I and class II antigens in canine masticatory muscle myositis

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.

Number and size of silver-stained nucleoli (Ag-NOR clusters) in canine seminomas: correlation with histological features and tumour behaviour.

Twenty spontaneous canine seminomas were examined. A statistically significant difference (P < 0.01) was detected in both the mean silver-stained nucleolar area/cell and the mean silver-stained nucleolar number/cell between (1) two intraductal seminomas with no signs of invasion, and (2) three intraductal ones with signs of invasion and 15 diffuse-type seminomas (two of which metastasized 3 and 12 months after surgical treatment). Furthermore, the metastasizing seminomas showed a mean silver-stained nucleolar area/cell which was significantly greater than that observed in all the other seminomas examined (P < 0.01). It is concluded that quantitative studies of nucleolar changes are useful in evaluating the biological behaviour of spontaneous canine seminomas.

Canine inflammatory myopathy associated with Leishmania Infantum infection

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.

Syrian hamster infected with Leishmania infantum: A new experimental model for inflammatory myopathies

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. Muscle Nerve, 2009

Mitochondrial Myopathy in a German Shepherd Dog

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.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF TYPE IV COLLAGENASE IN NORMAL, DYSPLASTIC AND NEOPLASTIC CANINE MAMMARY GLAND

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.

Structural basis of cardiomyopathy in Duchenne/Becker carriers. Endomyocardial biopsy evaluation.

Dystrophin gene mutations provoke at least three diseases: ( 1) Duchenne muscular dystrophy (DMD) caused by marked dystrophin deficiency in all striated muscles; (2) Becker muscular dystrophy (BMD) caused by qualitative and/or quantitative dystrophin abnormalities in all striated muscles; and (3) X-linked dilated cardiomyopathy (XLCM) caused by qualitative dystrophin abnormalities only in myocardium. This third type of cardiomyopathy seems not to be related to familial cases of DMD or BMD. However, many Duchenne and Becker carriers can show exactly the same clinical profiles, probably arising from a similar pathogenesis. Such an apparently isolated cardiomyopathy was described by Comi et al. * in a population of females with a close relationship with subjects suffering from Duchenne or Becker muscular dystrophy. In that population 125 (59.6%) Duchenne and 15 (65.2%) Becker consultands showed signs of myocardial involvement. Since its classical definition, the pathogenesis of dilated cardiomyopathy remains unknown. It is therefore natural to assume that detailed inspection of the myocardium could be rewarding. However, the value of endomyocardial biopsy is very high in clarifying the pathogenesis of the disease. Olsen reported that biopsy of the heart was helpful in 85% of cases; biopsy confirmed the suspected clinical diagnosis in 59% of cases and disclosed unsuspected conditions in 26%. Dilated cardiomyopathy is considered an indication €or endomyocardial biopsy. It has already been used in skeletal muscle disorder to better clarify the pathogenesis of cardiac muscle abnormalities in these patients, in order to:

Age-Related Changes in Skeletal Muscle of Cattle

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.