Immunohistochemical analysis of constituents of senile plaques and cerebro-vascular amyloid in aged dogs.

Immunohistochemical analysis of constituents of senile plaques and cerebro-vascular amyloid in the brain of aged dogs was performed using antisera against beta protein, cystatin C, ubiquitin, tau, and neurofilament (NF). All types of senile plaques and cerebro-vascular amyloid in aged dogs were labeled by anti-beta protein serum. Cystatin C immunoreactivity was detected in neuronal cell bodies, primitive or classical plaques, and amyloid deposited around cerebral capillaries, but not in diffuse plaques and amyloid deposited in the media tunica of cerebro-meningeal arterioles. Ubiquitin-positive granules distributed widely in both gray and white matter of aged dogs, while they were very small in number in young dogs. Swollen neurites-like materials in primitive plaques or classical plaques were immunoreactive for anti-ubiquitin serum. Tau immunostaining labeled commonly axons and several neuronal or glial cells after hydrate autoclave pretreatment. Tau-positive components were observed very rarely in the corona of classical plaques. Most of swollen neurites-like structures of primitive or classical plaques were not reactive for anti-NF serum, and only a few plaques contained small numbers of NF-positive elements.     

Pathological studies on cerebral amyloid angiopathy, senile plaques and amyloid deposition in visceral organs in aged dogs.

The relationship between cerebral lesions such as amyloid angiopathy or senile plaques and amyloid deposition in the visceral organs were studied in 90 autopsy cases of dogs, 0 to 19-year-old. Cerebral amyloid angiopathy was detected in 28 aged dogs (mean age: 13.7-year-old) and was found mostly in or around the wall of cerebral meningeal arterioles and capillaries of the neocortex. That condition was often accompanied by cerebral hemorrhage in dogs more than 9 years of age. Senile plaques were detected in the neocortex of the brain of 12 dogs (mean age: 13.2-year-old) and classified into 3 subtypes, i.e., "diffuse plaque", "primitive plaque" and "classical plaque". Among those 3 subtypes of senile plaques, amyloid containing plaques were small in number. In the visceral organs of dogs with cerebral amyloid angiopathy, amyloid deposition was found in the vascular walls or connective tissues of small intestines at a high frequency and sometimes in the vascular walls of the heart, lung, liver and thyroid gland as well as in atrioventricular valves. Amyloid in both cerebral and visceral organs was congophilic and showed green birefringence under poralized light even after potassium permanganate oxidation.     

Amyloid angiopathy with cerebral hemorrhage and senile plaque in aged dogs

Amyloid angiopathy with cerebral hemorrhage and senile plaques was found in the brain of aged dogs. In all 9 cases examined, 13 to 19 years old, 6 males and 3 females, amyloid deposits were observed mostly in the wall of cerebral arterioles and capillaries showed hyaline degeneration. The accumulation of amyloid fibrils measuring about 10 nm in diameter was seen in the cerebral vessel wall by electron microscopy. The cerebral hemorrhage was observed in 6 of 9 dogs and 2 of them showed massive hemorrhage. The hemorrhagic foci were sometimes closely contact with the vessels involved in amyloid angiopathy. In addition, senile plaques being classified into 2 types were found in the cerebral cortex of 3 dogs. The first type was characterized by the accumulation of degenerative neurites and often contained granular argyrophilic material. The second type had a well-defined amyloid core with neuritic halo. Amyloid deposits were also found in or around intestinal vessel walls of 3 dogs. The amyloid deposited in the cerebral vessels, senile plaques and intestinal vessels showed characteristic green birefringence under the polarized light even after potassium permanganate treatment.     

Topographic relationship between senile plaques and cerebrovascular amyloidosis in the brain of aged dogs.

The distributions of senile plaques (SP) and cerebrovascular amyloidosis (CA) were studied by employing thioflavin S and modified Bielschowsky stains, and beta-protein immunohistochemistry on serial sections of the brains of aged dogs older than 10 years. Mature and perivascular plaques, both of which contained compact amyloid deposits, always showed a close topographic relationship to CA. In contrast, the majority of diffuse plaques showed no topographic relationship to CA. Cell bodies of neurons and/or glia were almost always involved in the diffuse plaques. In addition, beta-protein immunohistochemistry demonstrated amyloid deposits on the periphery of occasional neurons. These findings suggest that different mechanisms may be involved in the development of the different subtypes of SP in the brains of aged dogs.     

Binding of curcumin to senile plaques and cerebral amyloid angiopathy in the aged brain of various animals and to neurofibrillary tangles in Alzheimer's brain

The binding of curcumin to senile plaques (SPs) and cerebral amyloid angiopathy (CAA) was examined in the aged brain of various animal species and a human patient with Alzheimer's disease (AD), together with its binding to neurofibrillary tangles (NFTs). Brain sections were immunostained with anti-amyloid β protein 1-42 (Aβ42) and anti-amyloid β protein 1-40 (Aβ40) antibodies. These sections were also stained with alkaline Congo red, periodic acid-methenamine silver (PAM), and curcumin (0.009% curcumin solution) with or without formic acid pretreatment. The sections from the AD brain were also immunostained for anti-paired helical filament-tau (PHF-tau), and were stained with Gallyas silver for NFTs. Some SPs in the AD, monkey, dog, bear, and amyloid precursor protein transgenic mouse (APP Tg-mouse) brains contained congophilic materials, and were intensely positive for curcumin. In addition, curcumin labeled some diffuse SPs negative for Congo red in the AD, monkey, bear, and APP Tg-mouse brains. In all animals, CAA was intensely positive for both Congo red and curcumin. The specific curcumin staining activity was lost by formic acid pretreatment. In the AD brain, NFTs positive for PHF-tau and Gallyas silver were moderately stained with curcumin. These findings indicate that curcumin specifically binds to the aggregated Aβ molecules in various animals, and further to phosphorylated tau protein, probably according to its conformational nature.     

Transthyretin amyloidosis and two other aging-related amyloidoses in an aged vervet monkey

An aged male vervet monkey showed severe cardiac arrhythmia for more than 3 years. A multifocal amyloid consisting of transthyretin was deposited in all areas of the heart wall, especially in the extracellular stroma among muscle fibers and external tunica of arterioles. Moreover, the amyloid was deposited in the stroma and arterioles of other systemic organs except the liver and spleen. These characteristics are consistent with senile systemic amyloidosis in humans. A second amyloid consisting of amyloid beta protein was in senile plaques and cerebral amyloid angiopathy in the cerebral cortex. A third amyloid consisting of islet amyloid polypeptide was deposited in islets of the pancreas. Apolipoprotein E and amyloid P component colocalized with the 3 amyloids. Thus, 3 different aging-related amyloids were found in an aged vervet monkey. In particular, to our knowledge, this is the first report on spontaneous transthyretin amyloidosis in animals.     

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity

OBJECTIVE: To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. ANIMALS: 8 na?ve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). PROCEDURES: 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. RESULTS: In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.     

Detection of an autoantibody from Pug dogs with necrotizing encephalitis (Pug dog encephalitis).

An autoantibody against canine brain tissue was detected in the cerebrospinal fluid (CSF) and serum of two Pug dogs (Nos. 1 and 2) by indirect immunofluorescence assay (IFA). Dog No. 1, a 2-year-old male, exhibited severe depression, ataxia, and generalized seizures and died 2 months after the onset of symptoms. Dog No. 2, a 9-month-old male, exhibited severe generalized seizures and died 17 months after the onset of symptoms. Histopathologic examination revealed a moderate to severe multifocal accumulation of lymphocytes, plasma cells, and a few neutrophils in both the gray and white matter of the cerebrum in dog No. 1. In dog No. 2, the cellular infiltrates were mild, but there was a severe, diffuse, and multifocal necrosis in the cerebral cortex with prominent astrocytosis. With the aid of IFA using fluorescein isothiocyanate-labeled antidog IgG goat serum and a confocal imaging system, specific reactions for glial cells were detected in the CSF of these Pug dogs but not in six canine control CSF samples. Double-labeling IFA using CSF from these Pug dogs and a rabbit antiserum against glial fibrillary acidic protein (GFAP) revealed that the autoantibody recognized GFAP-positive astrocytes and their cytoplasmic projections. By immunoblot analysis, the autoantibody from CSF of these Pug dogs recognized two common positive bands at 58 and 54 kd, which corresponded to the molecular mass of human GFAP. The role of this autoantibody for astrocytes is not yet clear. However, if the presence of the autoantibody is a specific feature of Pug dog encephalitis, it will be a useful clinical diagnostic marker and a key to the pathogenesis of this unique canine neurologic disease.     

Histological and immunohistochemical characteristics of cerebral amyloid angiopathy in elderly dogs

Background: Cerebral amyloid angiopathy (CAA) is a disorder characterized by amyloid deposition in the wall of cerebral blood vessels. The deposits of amyloid occur frequently in the blood vessels of the frontal, parietal and occipital cortex.

Objective: To examine the characteristics of CAA classified according to the Vonsattel scale in elderly dogs histologically and immunohistochemically as well as the semi-quantitative evaluation of the amyloid deposits in the different segments of the brain.

Animals and methods: The brains of 36 dogs of different breeds and sexes, which had been routinely necropsied, were used and divided into two groups: dogs from 1 to 5 and 10 to 18 years old. The tissue sections were stained by hematoxylin–eosin, Congo red and immunohistochemically.

Results: Amyloid was accumulated in the wall of cerebral blood vessels in 70% of dogs over the age of 10 years predominantly in the frontal cortex. CAA was demonstrated in elderly dogs as follows: in the frontal cortex (n = 19 or 63%), the parietal cortex (n = 12 or 40%), the hippocampus (40%) and the cerebellum (n = 5 or 17%). The deposits of amyloid in the wall of blood vessels detected by Congo red staining were also Aβ1-14 and Aβ1-42 immunohistochemically positive. Most commonly, the amyloid deposits affected a moderate number of blood vessels. The accumulation of amyloid was immunohistochemically revealed in the blood vessel walls as well as in the senile plaques and neurons.

Conclusion: The amount of amyloid in the arterial walls increased with age in dogs, whereas the amyloid accumulated in plaques was Congo red negative.     

Age-related changes in the brain of the dog.

Although many age-related changes have been described in the nervous system of different species, few authors have specifically studied the topic. Knowledge of such changes is essential to veterinary pathologists, who must distinguish the lesions of specific pathologic processes from those arising as a result of normal aging. The brains of 20 old dogs, ranging in age from 8 to 18 years, were compared with those of 10 young dogs using routine staining techniques (hematoxilin and eosin, periodic acid-Schiff), special staining techniques (periodic acid-methenamine silver stain), and immunohistochemical techniques to detect glial fibrillary acid protein, neurofilaments, ubiquitin, and beta-amyloid. Changes affected meninges and choroid plexuses, meningeal and parenchymal vessels, neurons, and glial cells. Of special interest was the presence of polyglucosan bodies, cerebrovascular amyloid deposition, senile plaques, and ubiquitinated bodies. Some of the age-related changes found, particularly lipofuscin, polyglucosan bodies, and beta-amyloid protein deposition, may play a role in the pathogenesis of the canine cognitive dysfunction syndrome. The dog could be used as a natural animal model for the study of normal aging and human neurodegenerative diseases.     

Senile cardiac amyloidosis in the dog

Thirty-two cases of senile cardiac amyloidosis from a routine necropsy material of 594 dogs were studied. The age of the affected dogs ranged from 10 to 16 years. Amyloid was observed in the intramural arteries and arterioles, predominantly in the ventricular myocardium. The vessels were often obturated with amyloid, and myocardial necroses and fibroses were common consequences of the vascular lesions. In three cases amyloid deposits were also observed in the main subepicardial coronary arteries. In contrast to man, only slight interstitial amyloid degeneration was found in four of the dogs observed. Amyloid in the mitral valves, tricuspid valves and aortic cusps was observed in six, two and three cases respectively. It was concluded that the distribution of senile cardiac amyloidosis in the heart of the dog differs considerably from that in man.     

Ageing changes in cat brains demonstrated by beta-amyloid and AT8-immunoreactive phosphorylated tau deposits

The life expectancy of domestic pet cats is increasing, along with the occurrence of geriatric-onset behavioural problems, such as cognitive dysfunction syndrome (CDS). While the cause of CDS is unclear, it has been suggested that it may result from age-related neurodegeneration. In aged and in particular senile human beings, histopathological changes may include the extracellular accumulation of plaque-like deposits of beta-amyloid (Abeta) protein and the intracellular accumulation of an abnormally hyperphosphorylated form of the microtubule-associated protein, tau. In severe cases, the latter may form into neurofibrillary tangles. Brain material was assessed from 19 cats, aged from 16 weeks to 14 years; 17 of which had clinical signs of neurological dysfunction. Immunohistochemical methods were used to detect Abeta and its intracellular precursor protein (amyloid precursor protein (APP)) and hyperphosphorylated-tau. APP was constitutively expressed, with diffuse staining of neurons and blood vessels being detected in all cats. More intense staining and diffuse extracellular Abeta staining deposits were found within the deep cortical areas of the anterior- and occasionally mid-cerebrum of seven cats, all of which were over 10 years of age. Neurons staining intensely positive for AT8-immunoreactivity were seen in two cats, aged 11 and 13 years. However, no mature neurofibrillary tangles were detected. This study demonstrated that extracellular Abeta accumulation and AT8-immunoreactivity within neurons are age-related phenomena in cats, and that they can occur concurrently. There are similarities between these changes and those observed in the brains of aged people and other old mammals.     

Familial renal amyloidosis in Chinese Shar Pei dogs

Renal amyloidosis was diagnosed in 14 young Chinese Shar Pei dogs, all of which were related. Clinical signs were those of renal failure and included vomiting, anorexia, lethargy, polydipsia, polyuria, weight loss, and dehydration. Some dogs had a history of intermittent fever and joint swelling. Laboratory findings also were compatible with renal failure and included azotemia, hyperphosphatemia, low total CO2 content in serum, isosthenuria, proteinuria, and hypercholesterolemia. All dogs had medullary deposition of amyloid, and 9 of 14 (64%) had glomerular involvement. The remaining renal lesions were typical of end-stage renal disease. In some dogs, amyloid deposits were found in other tissues (eg, liver, spleen, stomach, small intestine, myocardium, lymph node, prostate gland, thyroid gland, and pancreas). Amyloid deposits were sensitive to potassium permanganate oxidation, suggesting the presence of amyloid protein AA.     

Serum immunoglobulin E against storage mite allergens in dogs with atopic dermatitis

OBJECTIVE: To determine the prevalence of serum IgE against the storage mites Acarus siro, Blomia tropicalis, and Tyrophagus putrescentiae in a population of dogs with atopic dermatitis. SAMPLE POPULATION: Sera from 84 dogs with atopic dermatitis residing in various regions of the United States and Europe. PROCEDURE: Immunoblotting of sera from atopic dogs was used to identify proteins in mite extracts that bound IgE. RESULTS: 94% of the dogs had serum IgE against proteins in extracts of 1 or more of the storage mite species. Ninety-five, 92, and 89% of the storage mite-sensitive dogs had serum IgE against proteins in extracts of A siro, B tropicalis, and T putrescentiae, respectively. Eighty-two percent had serum IgE against at least 1 protein in all 3 species. Most of the major allergens had molecular weights > 80 kd. A greater percentage of the dog sera had IgE against storage mite proteins, compared with proteins of the house dust mites Dermatophagoides farinae and D pteronyssinus. CONCLUSION AND CLINICAL RELEVANCE: Many dogs with atopic dermatitis have serum IgE against many allergens of storage mites. Most of these allergens, like allergens of dust mites, had molecular weights > 80 kd. Storage mite sensitivity in dogs may be as important, if not more important, than dust mite sensitivity.     

Immunohistochemical identification of generalized AA-amyloidosis in a mountain gazelle (Gazella gazella)

Generalized amyloidosis was diagnosed post-mortem in a mountain gazelle (Gazella gazella). To test whether the amyloid deposits consisted of amyloid-A fibril protein a series of monoclonal and polyclonal antibodies directed against amyloid-A fibril protein of different species was applied to formalin-fixed paraffin sections using the indirect immunoperoxidase technique. The immunohistochemical results showed a moderate cross-reaction of gazelle amyloid with human, murine, hamster, and canine amyloid-A fibril protein. A strong cross-reaction, however, was found with one of two monoclonal anti-human amyloid-A antibodies and with an antiserum against bovine amyloid-A fibril protein, the amyloid fibril protein of another ungulate. These results demonstrate the presence of amyloid-A fibril protein in the gazelle amyloid and illustrate the diagnostic value of cross-reacting anti-amyloid-A antibodies for the identification of amyloid-A-amyloidosis in species and in individuals in which amyloid has not yet been examined.     

Detection of antigenic fractions from Brucella abortus S45/20 which bind to non-agglutinating antibodies using electroblotting and enzyme-linked antibody probes

Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.     

Amyloid in the horse: a report of nine cases

Out of approximately 16,000 horses referred for clinical examination, nine had amyloidosis. Six of these horses had localised amyloid deposits in the wall of the nasal meatus and ventral turbinates associated with epistaxis. Horse 1 also developed malignant histiolymphocytic lymphosarcomas. The amyloid deposits were potassium permanganate-resistant and tryptophan-positive. Gel filtration of solubilised amyloid fibrils from Horse 1 revealed a major retarded fraction with an apparent molecular weight of 20 kD. This protein had an amino acid composition similar to human AL-amyloid proteins and horse immunoglobulin light chains. On Western blot a strong cross-reaction was observed between horse 1gG2a light chains and the Horse 1 amyloid. Horses 7 to 9 had suppurative verminous aneurysm, tuberculosis and an adrenal cortical adenoma, respectively, and had generalised amyloid deposits in liver and spleen. These amyloid deposits were found to be potassium permanganate-sensitive and positive for tryptophan. Gel filtration of solubilised amyloid fibrils from Horse 8 revealed a major retarded fraction (protein AA) with an apparent molecular weight of 10 kD. Immunoperoxidase-antiperoxidase staining showed the localised deposits to be negative or only weakly positive with antisera against bovine, hamster, dog and human protein AA and to be positive with anti-horse-one amyloid protein. The generalised deposits were found to be positive with the antisera against allogenic protein AA. The results of the potassium permanganate incubation, biochemistry, immunoblotting and immunochemistry, indicate that the localised amyloid of Horse 1 and most likely the amyloid of Horses 2 to 6, is of the AL-type. The generalised amyloid deposits were found to be of the AA type.     

Feline insular amyloid: immunohistochemical and immunochemical evidence that the amyloid is insulin-related

Utilizing islet amyloid-laden pancreatic tissues from six diabetic cats, we demonstrated substantial immunoreactivity (peroxidase-antiperoxidase technique) of the islet amyloid with antiserum to a B chain-rich insulin fraction, but no reactivity with antisera to insulin, glucagon, or somatostatin. Islet amyloid was purified from two cats and a protein unique to the diabetic and islet amyloid-laden cats was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoreactivity of this protein with antiserum to the B chain-rich insulin fraction was also shown by immunoblotting. Attempts to obtain the amino acid composition of the purified unique protein (represented by a single 25,000 dalton band on gel electrophoresis) were not successful because the amount of protein was too small. These results provide important additional evidence that an insulin-related protein is involved in the formation of islet amyloid. Our study also shows that the diabetic cat provides several advantages for the continued study of the etiopathogenic relationship of islet amyloid and diabetes mellitus.     

Isolation and purification of a low molecular weight protein from bovine urine

A low molecular weight protein was separated from urine samples obtained from a heifer with spontaneous renal disease and from cows with CaNa2EDTA-induced renal dysfunction. The molecular weight and electrophoretic mobility of the separated protein were examined. The low molecular weight protein collected by gel filtration chromatography was further separated into two fractions by ion exchange chromatography using DEAE-cellulose. One of the two fractions, the lowest molecular weight protein showed a single band in SDS-PAGE, and its molecular weight was approximately 12,000. An antiserum against this protein formed a single precipitin line with the urine from cows with experimentally induced renal dysfunction and a heifer with spontaneous renal disease by the double immunodiffusion technique. However, the antiserum did not form any precipitin line with the concentrated urine of healthy cow and human beta 2-microglobulin. In cellulose acetate membrane electrophoresis, this protein migrated in the same position as that of serum gamma-globulin from healthy cow.     

Duration of antibodies against 24 kd protein of Rhipicephalus sanguineus extract in dogs infested with the adult ticks

A 24 kd protein from Rhipicephalus sanguineus (Rs24p) which was common to larvae, nymphs, male and female whole body and salivary gland extract of males and female was detected specifically in the serum from dogs after repeated infestation with adult R. sanguineus. The duration of antibodies against Rs24p in dogs infested with adults was examined by Western blotting analysis. Anti-Rs24p antibody was detected in two of 4 dogs during the period of 40 days in the first infestation. In the second infestation, all dogs showed positive reaction against Rs24p, but the duration of the antibodies varied greatly among the animals.