Immunohistochemical expression of tenascin in melanocytic tumours of dogs.

The aim of this study was to investigate tenascin-C (TN) immunolabelling and labelling for endothelium by von Willebrand Factor (vWF) in melanocytic tumours of dogs as compared with normal tissues, to evaluate the TN distribution in these types of tumours and to investigate whether a relation could be established between TN and angiogenesis in different types of tumour. Samples of normal dog skin (n=8), benign skin melanocytomas (n=10), malignant oral melanomas (n=9) and malignant toe melanomas (n=5) were studied. The percentages of TN and vWF immunolabelling per total microscopical area were analysed by morphometric methods. In normal skin, TN was found at dermo-epidermal junctions, around hair follicles, in the smooth muscles of hair follicles, and in the walls of blood vessels. TN immunolabelling (distribution and intensity) in melanocytomas was comparable with that found in normal skin. In melanomas, TN expression was considerably increased, its intensity in toe melanomas being twice that observed in oral melanomas. The degree of TN immunolabelling was not related to the histological malignancy of the melanomas. In melanomas, TN was found in the connective tissue surrounding the tumour cell nests and in narrow stromal strands inside the tumour. Regions infiltrated with lymphocytes were devoid of TN. The presence of TN around capillaries in melanocytomas and melanomas was investigated by double-immunolabelling (for TN and vWF). The intensity of vWF and TN immunolabelling was higher in melanomas than in melanocytomas, and higher in toe melanomas than in oral melanomas; however, no clear relation between TN expression and immunolabelling for vWF was found.

Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease.

In geriatric dogs, Alzheimer-like behavior is frequently observed. This behavior has been classified by several authors using questionnaires and a correlation has been described between cognitive dysfunctions and Alzheimer-like pathology. In the present study, cognitive performance was correlated with brain pathology for 30 dogs of varying ages. Within these animals, two age-matched groups of old dogs with and without behavioral changes were compared. The behavioral changes were analyzed and scored with questionnaires and necropsy was performed to rule out any other cause for changed behavior. Measurements, (immuno)-histochemical staining and fluorescence microscopy were used to detect cortex atrophy, amyloid, rest-products of oxidative damage, demyelination and accumulations of macrophages in the brains of these dogs. Spearman rank correlation coefficients (r) were calculated and adjusted according to Bonferonni. In the whole group (young to very old dogs), the age of the animal showed a significant correlation with various behavioral changes (r = 0.7 to 0.9, P < 0.01). The dementia score correlated significantly (r = 0.6 to 0.8, P < 0.01) with all the brain lesions studied, except one, i.e. demyelination (r = -0.4, P > 0.05). These results suggest that a questionnaire can be used to diagnose Alzheimer-like changes in canine practice. Oxidative damage on a cellular and a nuclear level plays an important role in behavior changes.

Acute phase reactants, challenge in the near future of animal production and veterinary medicine.

The future of acute phase proteins (APPs) in science is discussed in this paper. Many functions and associated pathological processes of APPs are unknown. Extrahepatic formation in local tissues needs attention. Local serum amyloid A (SAA) formation may be involved in deposition of AA-amyloid induced by conformational change of SAA resulting in amyloid formation, having tremendous food safety implications. Amyloidogenesis is enhanced in mouse fed beta pleated sheet-rich proteins. The local amyloid in joints of chicken and mammary corpora amylacea is discussed. Differences in glycosylation of glycoproteins among the APPs, as has been shown for alpha1-acid glycoprotein, have to be considered. More knowledge on the reactivity patterns may lead to implication of APPs in the diagnostics and staging of a disease. Calculation of an index from values of several acute phase variables increases the power of APPs in monitoring unhealthy individuals in animal populations. Vaccinations, just as infections in eliciting acute phase response seem to limit the profitability of vaccines because acute phase reactions are contra-productive in view of muscle anabolism. Interest is focused on amino acid patterns and vitamins in view of dietary nutrition effect on sick and convalescing animals. When inexpensive methodology such as liquid phase methods (nephelometry, turbidimetry) or protein array technology for rapid APP measurement is available, APPs have a future in routine diagnostics. Specific groups of patients may be screened or populations monitored by using APP.

Lymphosarcoma in a boar.

A 14-month-old Yorkshire boar was examined. In the abdomen, two big tumour masses were found in the intestines. In addition, pale nodules occurred in the liver, kidneys, in and upon the spleen, in the wall of the heart and in the testicles. By immunohistochemistry tumour cells were positive for CD3, but negative for B-lymphocyte antigen and CD79. From these findings it was concluded that the boar was suffering from multicentric lymphosarcoma probably of T-cell origin.

Versican and hyaluronan expression in canine colonic adenomas and carcinomas: relation to malignancy and depth of tumour invasion.

Changes in the production and structure of glycosaminoglycans and proteoglycans have been reported in many neoplastic tissues, and versican and hyaluronan (extracellular matrix components) are frequently increased in tumours and promote tumour progression. The distribution of chondroitin sulphate, versican and hyaluronan in normal canine colonic wall (n=10), and normal colonic lymph nodes (n=10), colonic adenomas (n=22), colonic adenocarcinomas (n=28), colonic undifferentiated carcinomas (n=7), and colonic lymph node metastases (n=8), was examined, with antibodies against chondroitin sulphate and versican, and a specific biotinylated probe for hyaluronan. The epithelial cells of the normal colonic mucosa were negative for all three substances, whereas the stromal tissue and lamina propria were moderately positive for chondroitin sulphate and hyaluronan, and weakly positive for versican. Chondroitin sulphate expression was increased in adenomas and carcinomas. However, there was no significant correlation between grade of tumour and degree of chondroitin sulphate expression. Versican expression was increased in the peritumoral stroma of adenocarcinomas and reduced in adenomas. A significant correlation was observed between grade of tumour and degree of versican expression. In 13 adenocarcinomas and undifferentiated carcinomas with invasion into all layers of the colorectum, the intensity of stromal versican expression was significantly related to the depth of invasion; the intensity was increased in the stroma of tumour islands in deep layers of the colonic wall. Unlike versican expression, hyaluronan expression was increased in the stromal tissue of both adenomas and carcinomas. However, the degree of stromal hyaluronan expression was unrelated to tumour grade and depth of tumour invasion. Hyaluronan was also expressed in the membrane and in the cytoplasm of tumour cells in 3/22 (14%) adenomas, 18/28 (64%) adenocarcinomas and 2/7 (29%) undifferentiated carcinomas. These results suggest that altered levels of both versican and hyaluronan in canine colonic tumours affect tumour progression.

Canine counterpart of senile dementia of the Alzheimer type: amyloid plaques near capillaries but lack of spatial relationship with activated microglia and macrophages.

Senile plaques and cerebrovascular amyloidosis are major histopathological lesions in the brains of aged dogs. Different types of amyloid beta protein (A beta) positive plaques are known: diffuse ones and neuritic plaques. Diffuse plaques may contain membrane-bound A beta and/or small amounts of amyloid fibrils. Neuritic plaques are cored plaques with clusters of amyloid fibrils and degenerating neurities. In human amyloid plaques, a pathogenetic role for microglia cells has been described. The aim of this investigation was to study microglia cells in relationship to canine plaques and to investigate the localisation of amyloid plaques in relationship to vasculature. The lesions were studied by hematoxylin and eosin Congo red staining and immunohistochemistry with anti-A beta for plaques, with Mac 387, anti lysozyme and a series of lectins for mononuclear cells, with anti von Willebrand Factor and Lycopersicon esculentum (tomato) lectin for the endothelium of brain capillaries. Diffuse A beta-positive plaques were found in dogs of 10.8 years and older, and cored A beta-positive plaques with birefringent amyloid in Congo red-stained sections in subjects of 15 years and older. Accumulation of microglia cells in relationship to the plaques was not obvious. With anti A beta 8-17 the distribution of the plaques in the cortical layers varied. The younger dogs had primarily diffuse plaques in the deeper layers of the cortical grey matter. The older dogs showed more cored plaques than diffuse plaques which were found throughout all cortical grey matter layers. With anti A beta x-42 more plaques were found positive, especially diffuse ones, whereas staining results of anti A beta x-40 were more confined to amyloid plaques and vascular amyloid. A close spatial relationship was found between the cored plaques and capillaries.

Tenascin expression in relation to stromal tumour cells in canine gastrointestinal epithelial tumours.

The expression of tenascin, alpha-smooth muscle actin (alpha-SMA), desmin and vimentin was investigated immunohistochemically in the stroma of normal canine stomach, small intestine and colon, and in 30 epithelial tumours of the canine stomach, small intestine or colon. In addition, "co-localization" of tenascin and alpha-SMA was investigated by double immunohistochemistry. Tenascin was absent in the normal gastric mucosa but present in the normal intestine, with a gradual increase in immunolabelling intensity from the cryptal glands to the surface epithelium. Tenascin expression was greater in all adenomas and carcinomas than in normal tissues. Two different patterns of tenascin expression were observed in all carcinomas, irrespective of their site. In well-differentiated tumour regions of both gastric and intestinal tumours, a fibrillary sub-glandular expression was observed; in poorly differentiated tumour regions, however, the expression pattern was diffuse. Incomplete invasion of the muscularis mucosae was accompanied by thickening and increased tenascin expression. In normal stomach and intestines, alpha-SMA and desmin were demonstrated in pericryptal myofibroblasts and smooth muscle cells of the muscle layers. In colonic adenomas and gastric and intestinal carcinomas, alpha-SMA was demonstrated in all stromal cells surrounding tumour cells. In contrast to alpha-SMA labelling, desmin labelling was negative in tumour stromal cells (in both gastric and intestinal tumours), except in tumour regions close to the muscularis mucosae. This suggested that myofibroblasts in gastric and intestinal tumours originated from pre-existing fibroblasts, except in tumour regions close to the muscularis mucosae, where the myofibroblasts seemed to originate from smooth muscle cells of the muscularis mucosae. There was a strong co-localization of tenascin and alpha-SMA-expressing myofibroblasts, suggesting that myofibroblasts are responsible for tenascin secretion.

Detection of circovirus with a polymerase chain reaction in the ostrich (Struthio camelus) on a farm in The Netherlands.

This study describes for the first time the presence of circoviruses in ostrich tissue including embryos. A polymerase chain reaction (PCR) was used for the detection of the virus in liver samples. The use of a polymerase for low copy detection significantly increased the sensitivity of the test as well as a Southern blot. Viral DNA could be detected in chicks and eggs that did not hatch. For localisation of the virus in the liver in situ hybridisation was performed on a selection of positive liver tissues.

Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques.

In the aging dog brain lesions develop spontaneously. They share some morphological characteristics with those of Alzheimer 's disease in man. Diffuse and primitive plaques are well known, whereas neuritic plaques rarely develop. Neurofibrillary tangles have not been seen in the canine. The aim of the present investigation was to study major age-related changes of the dog's brain using paraffin sections with respect to cross-immunoreactivity of tau, A beta protein and other immunoreactive components including hydroxynonenal protein, which is a marker for oxidative damage. The occurrence of neurofibrillary tangles and of the protein tau therein was studied in serial brain sections of two dogs with the Gallyas stain and by immunohistochemistry with three different antibodies against tau. Senile plaques were stained with a monoclonal anti-A beta (residues 8-17), polyclonal anti-apolipoprotein E and a monoclonal antibody against 4-hydroxynonenal (HNE). Amyloid deposits and controls were screened by Congo red staining viewed in fluorescent light, followed by polarized light for green birefringence. With the Gallyas stain and one of the antisera against tau, neurofibrillary tangles were revealed in a similar dispersed pattern, whereas the other antitau antisera gave negative results. With the anti-HNE a positive reaction was found in cerebral amyloid deposits and in vascular wall areas where amyloid deposition was confirmed by Congo-red staining, and in perivascular cells and in some neurons. These results indicate that the canine with his tangles and plaques which show oxidative changes, forms a spontaneous modelfor understanding the early changes and their interrelationships in Alzheimer's disease.

Serum amyloid A and transferrin in chicken. A preliminary investigation of using acute-phase variables to assess diseases in chickens.

The concentrations of serum amyloid A protein (SAA) and transferrin in blood samples from broilers in various stages of natural Staphylococcus aureus infection, from healthy counterparts, and from turpentine- or saline-injected pullets were measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and densitometry. SAA was not detected in healthy chickens but was detected in turpentine-injected pullets and in S. aureus-infected broilers. Relative percentages of transferrin in turpentine- and saline-injected pullets were not different. Broilers with a S. aureus infection had a two-fold higher transferrin level than did their unaffected counterparts. There was also a difference between the transferrin levels of healthy broilers and healthy pullets, which indicates that other variables, such as breed or breeding conditions, may influence the transferrin level. This preliminary study showed that SAA is an acute-phase protein and a more reliable variable for diagnosing lesions in chickens than transferrin.

Influence of physical stress on the plasma concentration of serum amyloid-A (SAA) and haptoglobin (Hp) in calves.

The influence of physical stress on the plasma concentration of the acute-phase proteins serum amyloid-A (SAA) and haptoglobin (Hp) was studied in 10 calves. Two different stress levels were created by housing two groups of five calves, each on different types of floor. The stress level was assessed by studying videotapes of the animals, and, subsequently, by quantifying the problems related with moving across the pens and the time the calves spent lying down and standing. Plasma concentrations of Hp, SAA, aldolase, and cortisol were measured in blood samples obtained by jugular venepuncture. Plasma SAA concentrations were significantly (p < 0.001) elevated in animals housed on the floor type associated with the highest level of physical stress, although the concentrations were within the normal range for healthy adult cattle. Hp concentrations were not elevated. The floor type did not alter the stress related biochemical variables aldolase and cortisol. It is concluded that plasma SAA concentrations rise upon physical stress, whereas Hp concentrations do not change. The absence of a significant difference in aldolase or cortisol concentrations indicates that the difference in the level of neuro-endocrine stress between the animals housed on the two floor types is only minimal. Consequently, SAA is suggested to be a sensitive variable to assess physical welfare in calves.

Concentrations of serum amyloid-A (SAA) and haptoglobin (HP) as parameters of inflammatory diseases in cattle.

The concentrations of the acute-phase proteins, serum amyloid-A (SAA) and haptoglobin (Hp), were determined in the plasma of healthy cows (n = 25) and cows with spontaneous acute (n = 6), subacute (n = 37), or chronic (n = 7) inflammatory diseases. The plasma concentration of SAA alone, Hp alone, and the Hp/SAA ratio, differed significantly (p < 0.001) between healthy animals and animals with inflammatory diseases. Plasma Hp concentrations in the group of cows with acute inflammatory diseases were significantly (p < 0.01) different from those in the group with chronic inflammatory diseases. Moreover, the Hp/SAA ratio in chronic inflammatory diseases was significantly different from this ratio in acute (p < 0.01) and subacute (p < 0.05) inflammatory diseases. It is therefore concluded that the plasma concentrations of SAA and Hp and the Hp/SAA ratio are useful parameters to distinguish healthy animals from animals with inflammation and can be helpful in distinguishing between acute and chronic of inflammatory diseases.

Experimental amyloidosis in the hamster: correlation between hamster female protein levels and amyloid deposition.

Reactive systemic AA amyloidosis was induced in female, male and castrated male hamsters either by repeated injection of casein or by injection of amyloid enhancing factor (AEF) followed by casein. The circulating concentrations of serum amyloid A protein (SAA), the putative precursor of the AA amyloid fibril protein, and of female protein (FP), the pentraxin homologue of serum amyloid P component (SAP) of other species, were measured and correlated with the speed and extent of amyloid deposition. The SAA responses of the three groups of hamsters were indistinguishable in both experiments but, in confirmation of previous reports, castrated males had FP levels higher than those of control males though still lower than in females. No differences were seen between groups in amyloid induction by casein injection alone. However, in the accelerated model using AEF, amyloid deposition occurred sooner and was more extensive in both females and castrated males than in unoperated males. These results strengthen the association between SAP, of which FP is the hamster counterpart, and the pathogenesis of amyloidosis.

Amyloid enhancing factor in hamster.

An amyloid enhancing factor (AEF) was extracted from spleens and livers of casein-treated hamsters. It shortened the induction time of experimental amyloidosis in recipient hamsters from 14 days to 4 days. Sepharose-4B gel filtration resolved the extract into 4 different fractions, with molecular weights of: higher than IO(7) (Vo-fraction), about 280 000, 59 000 and 12 000 respectively. In all 4 fractions AEF was present, indicating that AEF is probably a low molecular weight substance that easily aggregates, or associates with other compounds present in the spleen extract. AEF was precipitable at 50% ammonium sulphate saturation. On anion exchange chromatography in PBS (pH 7.2) of this precipitate, AEF was found in the fraction eluting at high NaCl concentration. This fraction did not show any relation to hamster protein AA, IgG or albumin with double immunodiffusion. Ultraviolet absorption spectrophotometry indicated nucleotide-like material, suggesting AEF to be related to nucleoproteins.

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis.

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.

Deposition of amyloid in the liver of hamsters: an enzyme-histochemical and electron-microscopical study.

In induced amyloidosis the amyloid was first deposited in the portal areas, then in the centre of the lobule and disseminated through it in the spaces of Disse. No intracellular amyloid was found in the predeposit phase. In the hepatic lobule both mononuclear phagocytes and hepatocytes showed a topographic relationship to the first deposits of amyloid. Some macrophages showed invaginations or vacuoles containing amyloid fibrils. Between the microvilli of hepatocytes, parallel amyloid fibrils occurred. Between the amyloid fibrils were 30-50 nm membrane-bounded spherical particles which may have been lipoprotein aggregates. There was no large-scale phagocytosis of amyloid during the induction period or after survival without casein treatment up to 18 weeks. Lysosomal enzyme activity was seen in the deposits of extracellular amyloid.