Amyloid precursor protein, lipofuscin accumulation and expression of autophagy markers in aged bovine brain.

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.

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 ß-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 ß-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.

Muscular dystrophy in a dog resembling human becker muscular dystrophy.

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 ß-sarcoglycan, γ-sarcoglycan and ß-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.

Histiocytic sarcoma of the nasal cavity in a horse.

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.

Histopathological, histochemical and immunohistochemical findings of the small intestine in goats naturally infected by Trichostrongylus colubriformis.

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.

A multicancer-like syndrome in a dog characterized by p53 and cell cycle-checkpoint kinase 2 (CHK2) mutations and sirtuin gene (SIRT1) down-regulation.

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.

Type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog.

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.

A review of bovine urothelial tumours and tumour-like lesions of the urinary bladder.

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.

Morphological characterisation of malignant histiocytosis in a cat.

Malignant histiocytosis (MH) is a progressive systemic neoplastic proliferation of morphologically atypical histiocytes, well characterised in humans and dogs but only recently identified in the cat. In all species, liver, lung, lymph nodes, spleen and bone marrow are infiltrated by atypical histiocytes, and the disease is rapidly fatal. The purpose of this study was to describe the clinical, histological, immunohistochemical and ultrastructural findings of MH in a cat, together with the diagnostic work-up and a list of differential diagnoses. Clinical evaluation included a complete blood-cell count, serum biochemistry, urinalysis, serology and ultrasound examination. The cat had clinical signs of depression, thinness, dehydration, pale mucous membranes and tachycardia. Abdominal ultrasonography revealed generalised splenomegaly and hepatomegaly. Necroscopy showed whitish nodules, randomly scattered throughout the parenchyma in the spleen and liver. The periportal lymph nodes were greatly enlarged and the cut surface was uniformly greyish-white and translucent. Histological examination revealed pleomorphic proliferation of large round tumour cells, with numerous phagocytic vacuoles containing erytrocytes, leukocytes and haemosiderin. By immunohistochemistry, positivity for lysozyme and alpha1-antitrypsin and a scattered positivity for Mac 387 were observed. Ultrastructural features of tumour cells included cytoplasmic lipid droplets, lysosomes and phagolysosomes. MH in the cat needs to be differentiated from diffuse granulomatous disease, non-Hodgkin's lymphoma and Hodgkin's-like disease. The morphological features of the tumour cells, combined with immunohistochemical and ultrastructural observation, are consistent with a diagnosis of MH in the cat.

Expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma) in canine nasal carcinomas.

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.

Myopathy with central cores in a foal.

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 <290 U/liter) activities, suggesting muscle disease. Muscle biopsy was performed. In cytochrome oxidase-, succinate dehydrogenase-, and reduced nicotinamide adenine dinucleotide tetrazolium reductase-reacted sections, the dominant morphologic feature was the absence of oxidative enzyme activity in the cores. By use of immunohistochemical technique with a monoclonal antibody against desmin, the cores were clearly delineated and a desmin network was present within the cores. Ultrastructurally, the core areas were characterized by preserved sarcomeres with irregular Z-lines, with some streaming or zigzag appearance and abnormal sarcoplasmic reticulum profiles and T-tubules. Lack of mitochrondria within central cores was observed. Diagnosis of myopathy with central cores was made.

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.

[Histology in diagnosis of parasitic diseases]. / La istologia nella diagnosi delle parassitosi.

Many pathogenic organisms cause inflammatory lesions and microscopic findings are a useful diagnostic tool for the aetiological diagnosis. However, the histological lesions are limited in respect to many biological agents that can damage the tissues. The histologic hallmark of parasitic diseases is mostly granulomatous inflammation. It is characterized by a focal infiltration of macrophages and epithelioid cells. Many giant cells, lymphocytes, plasma cells, fibroblasts and granulocytes can be found. Agents inducing granulomas include helminths and parasites that replicate intracellularly. Some special stains are utilized in histopathology, for example Giemsa's stain is useful to identify Leishmania. Using specific antibodies, immunohistochemical methods provide an aetiological diagnosis. Sometimes, tissue damage can be immuno-mediated depending on deposit of circulating immunocomplexes or T-lymphocytes involvement rather than by direct parasitic injury. Generally, the lesions which can be observed are respectively vasculitis and inflammatory reactions predominantly composed of mononuclear cells, as observed in many viral or bacterial diseases. In these cases, aetiological diagnosis is improved by in situ-PCR. For microscopic identification of parasites in tissues it is also important to be familiar with the kind of parasites most likely to be found in the examined tissue and in that particular host. Localization of parasites can induce hyperplastic-neoplastic lesions. Many parasites have been associated with the occurrence of specific types of neoplasms, but the mechanisms involved are still not well defined. Chronic inflammation and/or immune suppression seem to induce neoplastic proliferation.

Expression of cyclooxygenase-1 and -2 in canine nasal carcinomas.

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.

Expression of vascular endothelial growth factor receptor Flk-1 in canine mammary tumours.

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.

Expression of Major Histocompatibility Complex class I and II in a case of Neospora caninum myositis in a dog.

Neosporosis was diagnosed in a 2-month-old dog by indirect immunofluorescence antibody test (IFAT) and confirmed by means of histopathology and immunohistochemistry. The associated myositis was characterized by Major Histocompatibility Complex expression on some muscle fibres. This finding indicates an immunological activation, of the muscle cells that, acquiring Major Histocompatibility Complex expression, may, in some way, contribute to antigen presentation. A possible role of these glycoproteins in the pathogenesis of Neospora-associated myositis is discussed.

Gentamicin administration in Duchenne patients with premature stop codon. Preliminary results.

Aim of the study was to investigate whether the administration of gentamicin could restore dystrophin expression in striated muscles of patients with Duchenne muscular dystrophy caused by premature stop codon, as reported in mdx mice. Four Duchenne patients, still ambulant or in wheelchair stage for less than 4 months, selected among those with point mutations resulting in premature stop codons, received two 6-day cycles of gentamicin sulfate, at an interval of 7 weeks, according to the protocol approved by the Ethics Committee of the Second University of Naples. A muscle biopsy was performed after the second cycle of administration; the specimens were analysed by both immuno-histochemistry and Western blotting. Skeletal muscle changes were monitored by dynamic tests and Creatine Kinase values; at the beginning and end of treatment, cardiac and respiratory status was evaluated by electrocardiography, echocardiography, acoustic densitometry and vital capacity. Side-effects such as nephrotoxicity and ototoxicity were also monitored. Three out of four patients, who had the most permissive UGA as stop codon, showed positive results. In one patient, there was a dramatic re-expression of dystrophin by both immuno-histochemistry and Western blot; in two patients, dystrophin positive fibres were seen by the antibody to the rod domain with immuno-histochemistry; the fourth patient, with UAA as stop codon, showed no expression of dystrophin at all. These results suggest that gentamicin is able to recover dystrophin expression in a subset of Duchenne patients with nonsense mutations, raising the possibility of the first pharmacological treatment for muscular dystrophy.

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.

Evaluation of angiogenesis in canine seminomas by quantitative immunohistochemistry.

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.