Induction of murine AA amyloidosis by various homogeneous amyloid fibrils and amyloid-like synthetic peptides

We investigated amyloid-enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid-laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1-15 of mouse SAA (mSAA), 2-15 of cow SAA (bSAA), 1-15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33-45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid-like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid-like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species-specific and appears to be related to the primary amino acid sequence, especially the N-terminal segment of the amyloid protein.     

Spontaneous rupture of the liver following a normal pregnancy and delivery

Liver haematoma and rupture is a rare but potentially devastating complication of pregnancy. The majority of cases are associated with severe preeclampsia. We report a case of spontaneous hepatic rupture in a 39-year old woman following a normal pregnancy and delivery. Shoulder tip pain and serum haemoglobin of 6.8 prompted immediate imaging with computed tomography. A large subcapsular haematoma involving the right lobe of the liver was identified and managed conservatively. This diagnosis should be considered in the patient who presents with pain in the upper part of the abdomen and signs of haemorrhagic shock, even in the case of an uncomplicated pregnancy.     

Isolation and characterization of amyloid protein AA in the Abyssinian cat

Amyloid fibrils were isolated by extraction in deionized water from the kidneys of an Abyssinian cat with familial renal amyloidosis. The fibrils were suspended in a buffer containing 6 M guanidine hydrochloride and reduced and alkylated using dithiothreitol and iodoacetid acid. The resulting amyloid fibril subunit protein was isolated by chromatography on a column of Sepharose CL6B. It was fragmented using cyanogen bromide, and the resultant peptides were separated by reverse phase high performance liquid chromatography. The protein was characterized by determination of the amino acid sequence of the cyanogen bromide fragments using a Beckman 890C sequencer. The primary structure of this amyloid fibril subunit protein showed strong homology with amyloid protein AA found in man and animals with spontaneous and experimentally induced reactive systemic amyloidosis. This study confirms the reactive nature of familial renal amyloidosis in the Abyssinian cat and suggests that this disease may be a valuable spontaneous animal model for the study of familial Mediterranean fever in man.     

Evaluation of ectopic pregnancy by magnetic resonance imaging.

Patients (n = 37) suspected of ectopic pregnancy were prospectively evaluated with magnetic resonance (MR) imaging to assess the capability of MR imaging in the diagnosis of ectopic pregnancy. Five levels of confidence were defined: diagnostic, suspicious, equivocal, questionable, and negative. Tubal wall enhancement and presence of tubal haematoma or gestational sac-like structure were considered diagnostic findings. There were 21 diagnostic, two suspicious, eight equivocal, and six negative findings. MR findings were compared with the surgical findings in 18 patients. Surgical confirmation was obtained in 12 diagnostic, two suspicious, and four equivocal studies. Using the MR diagnostic criteria for tubal pregnancy, MR had 12 true positive, three true negative, three false negative, and no false positive results for the diagnosis of tubal pregnancy. Retrospective analysis of the signal intensity of haematoma and ascites was performed for these 18 surgically confirmed cases. The predominant signal intensity of tubal haematoma was an intermediate signal on T1-weighted image (WI) and a low signal on T2WI. Ascites showed signal intensity higher than that of urine on T1WI in 100% of 13 cases. In conclusion, MR imaging with use of intravenous contrast material allows a specific diagnosis of tubal pregnancy, recognizing tubal wall enhancement and fresh tubal haematoma.     

Polycystic disease of the kidney and liver in an adult Persian cat

Polycystic disease of the kidney and liver was diagnosed in a 10-year-old spayed female Persian cat. Previous reports of renal and hepatic polycystic disease in dogs and cats have described only juvenile forms, with death at an early age. The cat of this report had large, fluid-filled kidney and liver cysts, compared with the relatively small, spongiform cysts of puppies and kittens. The clinicopathological findings in this adult Persian cat are comparable to the adult form of renal and hepatic polycystic disease of man.     

The lysosomal cysteine protease, cathepsin S, is increased in Alzheimer's disease and Down syndrome brain. An immunocytochemical study.

Expression of cathepsin (cat) S, a lysosomal cysteine protease, has recently been shown to cause an increase in production of amyloid beta-peptides in transfected human cells. In this study, we examined the presence and localization of cat S by immunocytochemistry in 21 control, 24 Alzheimer's disease (AD), and 10 Down syndrome (DS) postmortem brains. An antiserum to a human cat S fusion protein was affinity purified and its specificity confirmed by abolition of immunoreactivity after adsorption with cat S but not cat L fusion protein. A small minority of control cases showed light, focal staining of scattered cortical neurons. Many control cases, as well as most AD and DS cases, showed prominent staining of vascular smooth muscle cells, particularly in leptomeningeal vessels. Both AD and DS brain tissue showed increased immunoreactivity in a subset of neocortical and hippocampal neurons and glia. Cat S immunoreactivity occurred in a granular, cytoplasmic pattern in some neurons or in a more dense staining pattern in certain neurofibrillary tangle-bearing neurons. Cat S-positive neurons were also present in amygdala and basal forebrain in AD brains. A subset of astrocytes were immunoreactive with the cat S antibody in AD and DS but not in control brains. In rare AD cases, cat S immunostaining was observed in astrocytes in the periphery of amyloid-beta-containing plaques. These results suggest that cat S is up-regulated in AD and DS brain. The association of cat S immunoreactivity with tangle-bearing neurons, astrocytes, and rare senile plaques implies a role for altered cat S activity in the pathogenesis of AD.     

Simultaneous AL-type amyloid and light chain deposit disease in a liver biopsy: a case report

A 57-year-old male caucasian presented with a peripheral neuropathy which had an autonomic component. Clinical examination revealed hepatomegaly and laboratory tests showed derangement of liver function tests and IgG lambda myeloma. Biopsy of the liver was performed. Histological examination revealed AL-type amyloid in the hepatic arteries and a perisinusoidal deposit of diastase resistant, periodic acid-Schiff positive material which did not react in the same way as the arterial deposit, giving no apple green birefringence when stained with Congo red. Immunohistochemistry showed the material to consist of lambda light chains. Electron microscopy confirmed that the material did not have the ultrastructural characteristics of amyloid. A diagnosis of light chain deposit disease concurrent with vascular AL-type amyloid was made.     

Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques.

A novel putative polypeptide hormone identified as islet amyloid polypeptide (IAPP) was recently purified from islet amyloid (IA) of diabetic humans and cats, and also from amyloid of a human insulinoma. Although the function of IAPP is yet unknown, its occurrence in pancreatic endocrine tissue and its partial amino acid sequence identity with calcitonin gene-related peptide (CGRP) suggests an endocrine regulatory effect. In the present investigation, the authors utilized antisera to insulin, glucagon, somatostatin, pancreatic polypeptide, synthetic human CGRP, and a synthetic human IAPP (7-17) undecapeptide to immunohistochemically (PAP technique) document the presence of IAPP immunoreactive cells in the islets of the cat, dog, mouse, and rat, but not in the islets of the horse or calf. In serial sections of islets from these species it was shown that IAPP immunoreactivity occurred in insulin-reactive beta cells. This observation was confirmed immunocytochemically in cat islets by means of protein A-gold probes. With protein A-gold labeling techniques, IAPP immunoreactivity was localized to the outer lucent compartment of the beta cell secretory granule, whereas insulin immunoreactivity was associated with the electron-dense core. These findings provide strong evidence that IAPP or an IAPP precursor is synthesized by beta cells and is stored in beta cell granules for subsequent co-secretion with insulin. The conservation of IAPP in humans and multiple animal species and the localization of IAPP to pancreatic beta cells provide further evidence that IAPP has an important endocrine regulatory function. The propensity of IAPP to polymerize and form IA fibrils in diabetes associated with aging may indicate that IAPP is in some way also linked to the development of Type 2 diabetes.     

Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone.

Amyloid deposition is a very typical alteration in the islets of Langerhans in human Type 2 (non-insulin-dependent) diabetes mellitus and in feline diabetes mellitus. Amyloid infiltration is also commonly found in insulin-producing pancreatic tumors. It was shown recently that amyloid purified from an insulinoma was composed mainly of a novel polypeptide (insulinoma amyloid polypeptide, IAPP), which had partial identity with the neuropeptide calcitonin gene-related peptide (CGRP). Cat islet amyloid contained a similar polypeptide. This finding is verified in the present study, and it is shown that the cat IAPP differs from the human peptide only in two of the 16 elucidated amino acid residues. The authors now also show by N-terminal amino acid sequence analysis that human islet amyloid is of IAPP origin. Although the significance of IAPP is unknown, its occurrence in pancreatic endocrine tissue and partial identity with a known neuropeptide suggests an endocrine regulatory function.     

Cardiac amyloidosis mimicking hypertrophic cardiomyopathy

Amyloid infiltration of the heart may frequently masquerade as other cardiac disorders. The extended use of echocardiography may contribute to an erroneous diagnosis of hypertrophic cardiomyopathy, as both conditions show several features in common. This was the case with the patient reported below. A low QRS amplitude, an increased right ventricular wall thickness, thickened cardiac valves, and a pericardial effusion may, however, indicate amyloid infiltration. The diagnosis of systemic amyloidosis of immunocytic origin was subsequently established in our patient. A definitive diagnosis of amyloid heart disease requires endomyocardial biopsy, but it is suggested that typical noninvasive findings together with demonstration of amyloid in an organ other than the heart is sufficient for a reliable diagnosis. In addition, systemic manifestations may contribute to a correct diagnosis in generalized amyloidosis. Our patient had features consistent with the rare muscle pseudohypertrophy syndrome, which is associated with immunocytic amyloidosis.     

Histotopographic evidence that amyloid deposits in sclerocalcific heart valves and other chronic lesions of the cardiovascular system are related to old thrombotic material

Summary Deposition of amyloid in human sclero-calcific heart valves has been reported recently as a localized age-independant and dystrophic form of amyloidosis. Histochemical studies have shown that the deposits are permanganate resistant, contain tryptophan and P component and are immunologically unrelated to any known type of amyloid fibril protein. In this study histological observations from a series of four selected sclerotic heart valves show amyloid deposition in old thrombotic material covering fusing commissures or appositional collagen on the body of the leaflets. Similar cases from extravalvular sites have been added to the series: a partly hyalinized thrombus of the left atrium, a thrombotic aneurysm of the left ventricle, 2 thrombotic atherosclerotic aneurysms of the aorta and popliteal artery respectively, and an encapsulated haematoma of the scalp. The deposits are Congo red positive with typical green dichroism in polarized light, permanganate resistant and contain tryptophan. Electron microscopy of 3 cases displays small fibrils which are typical of amyloid.No patient showed evidence of systemic amyloidosis. The natural history of sclero-calcific valvulopathies and present observations favour the following pathogenesis: first, recurrent thrombotic deposition on thickened and fibrotic endocardium; second, degradation of a coagulation-related protein with potential during the aging of the clot with transformation into amyloid fibrils; finally, inclusion of the amyloid in sclerotic replacement tissue.     

Globular hepatic amyloid: a diagnostic peculiarity that bears clinical significance

Hepatic amyloid deposition in the form of globular inclusions is a rare occurrence. Only 24 such cases have been reported to date. Its clinical and pathological significance are undefined. Unawareness of such a pattern can cause diagnostic confusion. We herein describe a case of globular hepatic amyloid in a patient with a B-cell lymphoma and chronic hepatitis C. The findings in our case support the literature data in that (1) it is often an incidental finding during workup for other coexisting conditions and (2) its morphology is peculiar but can be recognized with ease if one is aware of its existence. Our case also provides new insights into this condition. First, it represents the first nonautopsy case to demonstrate that globular hepatic amyloid is an indication of systemic amyloidosis, thus emphasizing the clinical importance of the recognition of this condition. Second, in our case, there was a coexisting B-cell lymphoma, the constituent cells of which showed immunoglobulin lambda light chain restriction, and patient's serum lambda light chain was elevated. However, light chain restriction was not demonstrated in the globular inclusions, and there was no evidence of monoclonal gammopathy by serum electrophoresis. Whether immunoglobulin light-chain abnormality played a causal role in this condition is to be determined.     

A feline model of experimentally induced islet amyloidosis

The study of the pathogenesis of islet amyloidosis and its relationship to the development and progression of type 2 diabetes mellitus has been hampered by the lack of an experimentally inducible animal model. The domestic cat, by virtue of the fact that it is one of the few species that spontaneously develop a form of diabetes mellitus that closely resembles human type 2 diabetes, including the formation of amyloid deposits derived from islet amyloid polypeptide (IAPP), was considered to be an excellent candidate species in which to attempt to develop a nontransgenic animal model for this disease process. To develop the model, 8 healthy domestic cats were given a 50% pancreatectomy, which was followed by treatment with growth hormone and dexamethasone. Once a stable diabetic state was established, cats were randomly assigned to groups treated with either glipizide or insulin at doses appropriate to control hyperglycemia. Cats were maintained on this treatment regimen for 18 months and then euthanized. Based on light microscopic examination of Congo red-stained sections of pancreas, all cats were negative for the presence of islet amyloid at the time of pancreatectomy. At the end of the study all 4 glipizide-treated cats had islet amyloid deposits, whereas only 1 of 4 insulin-treated cats had detectable amyloid. In addition, the glipizide treated cats had threefold higher basal and fivefold higher glucose-stimulated plasma IAPP concentrations than insulin-treated cats, suggesting an association between elevated IAPP secretion and islet amyloidosis. Blood-glycosylated hemoglobin concentrations were not significantly different between the two treatment groups. This study documents for the first time an inducible model of islet amyloidosis in a nontransgenic animal.     

Down-regulation of the major circulating precursors of proteins deposited in secondary amyloidosis by a recombinant mouse interleukin-1 receptor antagonist

An inflammatory response was induced in C57BL/6 mice using silver nitrate. Co-administration of a recombinant mouse interleukin-1 receptor antagonist (rmIL-1ra) significantly reduced the magnitude of hepatic induction of the mRNA specifying the serum amyloid A (A-SAA) isoforms A-SAA1 and A-SAA2 for up to 24 h. In relative terms, the amount by which the induction of serum A-SAA protein levels could be countered by the antagonist was less, probably reflecting extrahepatic A-SAA synthesis that is regulated independently of IL-1. Induction of hepatic serum amyloid P component (SAP) mRNA and other acute-phase reactant (APR) mRNA were all partially blocked by rmIL-1ra for up to 24 h, indicating that induction of these APR mRNA involves both IL-1 and additional factors acting independently of IL-1. Hepatic mRNA levels of the negative APR apolipoprotein A-I (apo A-I) and serum albumin were down-regulated by silver nitrate treatment; rmIL-1ra partially restored serum albumin mRNA levels but not those of apo A-I. The IL-1ra-mediated reduction in inflammation-induced hepatic mRNA and serum protein concentrations of A-SAA and SAP (the precursors of the main protein components of amyloid deposits in secondary amyloidosis) was, however, not sufficient to prevent or delay early amyloid deposition in the silver nitrate/amyloid enhancing factor model of accelerated amyloidosis. The rmIL-1ra may be a useful component in future therapies to control inflammation and secondary amyloidosis; in addition, it will be a useful tool for the detailed analysis of the IL-1-driven aspects of inflammation per se.     

Negative correlations between parenchymal amyloid and vascular amyloid in hippocampus.

Congo red was used to stain amyloid in 29 blocks of hippocampus from 17 unselected cases of Alzheimer's disease. Green birefringence under polarized light was used for evaluation of the average number of senile plaques and cross-sectional vessel profiles containing amyloid in five fields per slide, at a magnification of X100. Fields were selected that had large numbers of neurofibrillary tangles, also counted on the basis of green birefringence. The vascular involvement by amyloid was expressed as the ratio of amyloid positive to amyloid negative profiles. A negative correlation was found between Congophilic plaques or tangles on the one hand and vascular amyloid content on the other. In other words, cases with large numbers of Congophilic plaques had fewer Congophilic vessels, and vice versa: congophilic plaques = -3 (vessel amyloid) + 2.2, Spearman correlation coefficient, -0.61, P less than 0.01; tangles = -3.7 (vessel amyloid) + 15.6, Spearman correlation coefficient, -0.05, P greater than 0.05. When the slides were reexamined, using only fields with at least one Congophilic vessel, the negative correlation for plaque versus vessel amyloid remained highly significant, whereas that for tangles versus vessel amyloid became highly significant: Congophilic plaques = -1.2 (vessel amyloid) + 2.3, Spearman correlation coefficient, -0.48, P less than 0.01; tangles = -5 (vessel amyloid) + 19, Spearman correlation coefficient, -0.48, P less than 0.01. These data are most compatible with the hypothesis that amyloid is first produced in the parenchyma and is somehow cleared by the vessels. It is least compatible with the hypothesis that the amyloid precursor protein first enters the vessel wall to produce amyloid there, and then moves into the brain to produce amyloid in parenchymal sites.     

Pleuropulmonary pathology of vascular Ehlers–Danlos syndrome: spontaneous laceration,haematoma and fibrous nodules

Kawabata Y, Watanabe A, Yamaguchi S, Aoshima M, Shiraki A, Hatamochi A, Kawamura T, Uchiyama T, Watanabe A & Fukuda Y
(2010) Histopathology 56, 944–950
Pleuropulmonary pathology of vascular Ehlers–Danlos syndrome: spontaneous laceration, haematoma and fibrous nodules Aims: The aim was to clarify the pleuropulmonary pathological findings of vascular Ehlers–Danlos syndrome (vEDS). Methods and results: Nine patients with confirmed vEDS by means of cell culture and/or molecular biological studies who had undergone surgical lung biopsy (SLB), lobectomy or autopsy were studied. Six patients were male and three were female with a mean age of 23.2 years. Histological features were as follows: (i) the main pulmonary lesions related to fragility and spontaneous laceration, these being haematomas in seven, acute haemorrhage in nine, fibrous nodule in eight, with ossification or bone marrow formation in six; vascular disruption in five; intraluminal haemosiderosis in nine; interstitial haemosiderosis in seven, with iron deposition in the alveolar wall and/or vessel wall in five and foreign body reaction in two; emphysematous changes in eight; and bleb formation in two; (ii) secondary iatrogenic pleuropulmonary injuries during SLB or lobectomy comprised pleural laceration in seven of 10 and lung laceration in eight of 10 specimens. Conclusions: Spontaneous laceration of lung tissue is an essential feature and is followed by haematoma and possible fibrous nodule formation.     

Hepatic amyloidosis--two cases report.

Amyloidosis is classified according to the distribution pattern of amyloid deposition sites and associated diseases. Hepatic amyloidosis is not infrequent, although rarely causes clinical liver disease. We report two cases of amyloidosis diagnosed by liver biopsy. One presented with symptoms related almost to the liver disease, such as jaundice, hepatomegaly and indigestion. Echocardiogram revealed hypertrophic cardiomyopathy, suggesting cardiac involvement of the amyloidosis. The patient died of hepatic failure. The other case was found in a patient with an end stage renal disease. Features of congestive heart failure in this case may reflect cardiac involvement. The pattern of hepatic amyloid deposition in both of these cases was diffuse perisinusoidal. The predominant intralobular deposition suggests that these are amyloidosis of the secondary type.     

Redistribution of amyloid deposits.

After 21 daily subcutaneous injections of 0.5 ml 10% casein, CBA/J mice were left untreated and evaluated periodically for 6 months for the development of amyloid in spleens, livers, and kidneys. At the end of the amyloid-inducing regimen, the mice developed moderate to heavy splenic amyloid, trace to light hepatic amyloid, and virtually no renal amyloid. Renal amyloid appeared about 2 months after cessation of the casein and then increased steadily, while splenic and hepatic amyloid gradually diminished. Six months after the cessation of casein, moderate amyloid deposits were observed in the kidneys whereas no, or only traces of, amyloid remained in spleens and livers. This renal amyloid was localized predominantly in the peritubular area and differed from the renal amyloid seen in rapidly induced disease, when it localizes dominantly in glomeruli. This phenomenon is interpreted in the light of possible redistribution of amyloid deposits from organ to organ, and the clinical and investigative significances of this possibility and others are discussed.     

A very severe case of feline amyloidosis with spontaneous hepatic rupture and chronic renal failure

A 3.5-year-old neutered male domestic shorthaired cat presenting with inappetence, depression, polydisia, and severe icteric mucous membranes was diagnosed as having systemic amyloidosis with spontaneous hemorrhage from the liver and chronic renal failure. Laboratory findings were remarkable for anemia, thrombocytopenia, mature neutrophilia, hyperbilirubinemia, azotemia, and hyperphosphatemia. Little treatment was possible and the cat was euthanased later. At necropsy, the entire abdominal cavity was filled with intra-abdominal hemorrhage. Histopathological examination revealed extensive deposition of eosinophilic homogenous material in the parenchyma of the liver and sinusoids. Hepatic amyloid was detected primarily in periacinar regions associated with atrophy of adjacent hepatocytes. In the kidney, marked eosinophilic homogenous material was found in numerous glomeruli and outer medullary area. Amyloid deposits were demonstrated by the Congo red stain. Whether underlying diseases present as risk factors for the severity of clinical and biochemical picture of generalized amyloidosis in this case is unclear.     

Avian amyloidosis.

Although amyloid deposits have been described for more than a century and a half, its proteinaceous and fibrillar nature was not revealed until after 1950. Biochemical characterization of amyloids has brought to light that several non-related proteins can re-organize into amyloid fibrils. In some domestic and caged wild birds, and especially waterfowl, amyloidosis is a well recognized pathological disorder and is an important cause of death in Anseriformes. Its regular occurrence in Galliformes has been recognized more recently, where amyloid deposits occur mainly in the joints in contrast to other species studied so far. Avian amyloidosis is systemic in nature, being classified by amino acid sequencing and, monoclonal and polyclonal antibodies as of the AA-type amyloid, also named reactive or secondary amyloid. The pathogenesis of both AA and other types of amyloidosis is a complex phenomenon that is not well understood. It has been shown that the occurrence of certain predisposing conditions and chronic infections, inflammations or tumours increase strongly the serum levels of the hepatic acute phase reactant serum amyloid A (SAA), the precursor protein of amyloid protein A (AA). Although an increased pool of precursor protein is necessary for amyloid to develop and while certain amino acid substitutions may favour amyloidogenicity giving rise to unstable intermediate protein conformations that easily re-organize into fibrils, the action of other factors which are discussed in this review, seems of vital importance at the initiation of fibrillogenesis. As the clinical symptoms of amyloidosis generally are non-specific, diagnosis requires histopathology following biopsy or necropsy. AA-amyloidosis is a fatal progressive disease in birds and other species. Currently no curative treatment is available, therefore special attention should be paid to prevention focusing on hygiene and avoidance of stress.