Microdeposits of amyloid in sclerocalcific heart valves: a histochemical and immunofluorescence study.

Amyloid associated with seven sclerotic and two normal aortic and mitral valves was studied. The sclerotic valve amyloid contained microfibrils with typical random orientation and a fibril width of 9.5-12.5 nm. The amyloid deposits demonstrated permanganate-resistant Congophilia and contained the amino acid tryptophan. Immunofluorescence studies showed P-component in amyloid deposits of 6 of 7 valves, but none of the sclerotic valves contained amyloid fibril proteins of the AL (primary), AA (secondary), AEt (medullary thyroid carcinoma) or ASc1 (senile cardiac) types. Two non-sclerotic valves, removed from a patient with systemic amyloidosis, showed permanganate-sensitive Congophilic amyloid deposits which contained amyloid fibril protein AA.     

Senile amyloid deposition

Amyloid deposition in the aged was studied in a wide selection of tissues from 194 necropsies of subjects aged over 65 years, there being no clinical manifestation of amyloidosis. We employed conventional Congo red staining with the aid of a polarising filter for the identification of amyloid. We analysed the deposits histochemically for tryptophan content and reaction to oxidation using the potassium permanganate method in 26 subjects aged over 85 years. Both the frequency of amyloid deposition and the number of organs involved tended to increase with advancing age, and both increased after the ninth decade with little sex difference. Amyloid deposition preferentially occurred in the aorta, heart, lung, prostate and pancreas among the organs examined. Visceral involvement tended to be confined to the vasculature except in the prostate and pancreas. In cases associated with predisposing conditions considered to evoke secondary amyloidosis such as tuberculosis, multiple myeloma, and rheumatoid arthritis more amyloid was found but the distribution of the deposits was similar by organ and intravisceral site to that of cases unassociated with predisposing disorders. In both groups of cases amyloid was found to contain tryptophan, indicative of immunoamyloid and, with a few exceptions, to be resistant to oxidation by potassium permanganate indicating an immunoglobulin AL component. It can be concluded that senile amyloid should be classified as a form of so-called primary amyloidosis because of its widespread distribution, its occurrence independent of predisposing disorders and its chemical composition. Subclinical chronic inflammation of the respiratory, gastrointestinal and urogenital tracts may provide the necessary prolonged immunological stimulus to lead to amyloid deposition in ageing tissues.     

Transmission of mouse senile amyloidosis

SUMMARY: In mouse senile amyloidosis, apolipoprotein A-II polymerizes into amyloid fibrils (AApoAII) and deposits systemically. Peripheral injection of AApoAII fibrils into young mice induces systemic amyloidosis (Higuchi et al, 1998). We isolated AApoAII amyloid fibrils from the livers of old R1.P1-Apoa2(c) mice and injected them with feeding needles into the stomachs of young R1.P1-Apoa2(c) mice for 5 consecutive days. After 2 months, all mice had AApoAII deposits in the lamina propria of the small intestine. Amyloid deposition extended to the tongue, stomach, heart, and liver at 3 and 4 months after feeding. AApoAII suspended in drinking water also induced amyloidosis. Amyloid deposition was induced in young mice reared in the same cage for 3 months with old mice who had severe amyloidosis. Detection of AApoAII in feces of old mice and induction of amyloidosis by the injection of an amyloid fraction of feces suggested the propagation of amyloidosis by eating feces. Here, we substantiate the transmissibility of AApoAII amyloidosis and present a possible pathogenesis of amyloidosis, ie, oral transmission of amyloid fibril conformation, where we assert that exogenous amyloid fibrils act as templates and change the conformation of endogenous amyloid protein to polymerize into amyloid fibrils.     

Intervertebral disc amyloidosis: histochemical, immunohistochemical and ultrastructural observations

Intervertebral discs selected at random from autopsies and surgical operations on herniated discs were examined for evidence of amyloid deposition. Amyloid deposits were detected in 53 (81.5%) of 65 autopsy cases. Discs from subjects over 50 years of age revealed a significantly high incidence of amyloid deposition. Among herniated discs, amyloid deposits were documented in 49 (75.4%) of 65 surgical specimens. The incidence of amyloid deposition in intervertebral discs increased with advancing age. Intervertebral disc amyloidosis consisted of amyloid deposits of three morphological types: linear amyloid deposits around the degenerating chondrocytes (perichondrocyte type), and nodular or band-like deposits in the annulus fibrosus (annulus fibrosus type) and nucleus pulposus (nucleus pulposus type). We suspect that the precursor protein of perichondrocyte type amyloid is derived from chondrocytes, and those of annulus fibrosus type and nucleus pulposus types are derived from the blood. The amyloid deposits were resistant to treatment with potassium permanganate. Immunohistochemically, the amyloid deposits reacted with antibody against amyloid P-component, but not with antibodies against AA, A k , Aλ, transthyretin or β₂-microglobulin. Ultrastructurally, the amyloid deposits were composed of 10 nm-wide nonbranching fibrils. The amyloid in herniated discs had the same biochemical and immunohistochemical properties as those found in autopsy cases. The immunohistochemical characteristics of the amyloid deposits suggest that it derives from an, as yet, unknown precursor protein.     

Senile systemic amyloidosis.

The senile amyloidoses comprise a heterogeneous group of disorders with deposition of amyloid in a variety of tissues. Most of these amyloidoses are localized to one tissue. It has been shown previously that the amyloid fibrils in one form of senile amyloidosis affecting the heart contains a prealbumin-related protein, ASc1. It is shown in this paper by immunohistochemical study using a specific anti-protein ASc1 antiserum that this type of amyloidosis, previously called senile cardiac amyloidosis, is a systemic disease with amyloid deposits in many organs. The designation senile systemic amyloidosis is proposed for this disease, which differs from other systemic amyloidoses in distribution of amyloid deposits.     

Senile Cardiac Amyloidosis: Evidence of Two Different Amyloid Substances in the Ageing Heart

In a material of seventy-two persons over 70 years of age, forty-seven cases of amyloidosis of the heart were found. In thirty-nine cases, deposits occurred only in the atria (isolated artrial amyloidosis, IAA), while in eight cases amyloid also was seen in the ventricles (senile cardiac amyloidosis, SCA). In some of the latter cases, small amyloid deposits occurred in other organs, especially the lungs. Some definite differences existed between the amyloid substance in SCA and IAA. Thus, tryptophan was demonstrated histochemically in SCA but not in IAA, and, furthermore, amyloid fibrils isolated from patients with IAA lacked protein Asca, the fibril subunit protein of senile cardiac amyloid. It is concluded that the ageing heart may be the target of two different forms of amyloid, one only affecting the atria, while the other is more widespread within the heart and sometimes also is found in other organs.     

Amyloidosis complicating idiopathic myelofibrosis

Three cases of amyloidosis occurring in the later course of idiopathic myelofibrosis were studied at autopsy. In these cases, the amyloid deposition was seen only in the renal glomerulus, which had caused proteinuria. Although their exact nature could not be determined by immunohistochemistry, the amyloid deposits in the three cases were permanganate resistant and exhibited the same organ distribution, indicating a similarity in their character. These cases suggest that amyloidosis might be a complication of idiopathic myelofibrosis, implying that idiopathic myelofibrosis could be a disorder underlying amyloidosis.     

Primary diffuse tracheobronchial amyloidosis in a dog

Primary diffuse tracheobronchial amyloidosis was diagnosed at necropsy in an intact male Akita dog aged 11 years, a non-productive chronic cough having been the only related clinical sign. Histologically, eosinophilic hyalinized deposits were found as a band in the lamina propria underneath the epithelium of the trachea and bronchi. When stained with Congo red, apple-green birefringence was observed in the deposits viewed with polarized light. The amyloid did not lose sensitivity to Congo red staining after incubation with potassium permanganate, indicating that it was of the AL (amyloid light chain) type. Ultrastructural features of the amyloid included a typical fibrillar meshwork with individual fibrils measuring 9.5 to 10.5 nm in diameter. This is the first report of primary diffuse tracheobronchial amyloidosis in the dog.     

Lmmunohistochemical classification of 140 autopsy cases with systemic amyloidosis

One hundred and forty autopsy cases of systemic amyloid-osis were examined using the potassium permanganate method for distinction of amyloid A protein from other amyloid proteins and an immunohistochemical technique. Of those cases, amyloid proteins were identified in 121 cases. There were 68 cases of amyloid A-related (AA) amyloidosis and these were the most common type among the cases (56.2%). There were 39 cases of immunoglobulin light chain-related (AL) amyloidosis (32.2%), six cases of β₂-microglobulin-related (Aβ2M) amyloidosis (5%), and five cases of transthyretin-related (ATTR) amyloidosis (4.1%). Minute areas of amyloid deposits in four cases with AA were resistant to potassium permanganate pretreatment. In Aβ2M amyloidosis amyloid deposits were either resistant or sensitive to potassium permanganate pretreatment, from case to case. The coexistence of two different amyloid proteins was seen in three cases: one case had ATTR and Ax types, and two cases had Aβ2M and AA types. Some discrepancies were seen between the immunohistochemical typing and clinical classification of amyloidosis referred to in the Annual of the Pathological Autopsy Cases in Japan, for example, one case of AA type in myeloma-associated amyloidosis and one case of AL type in secondary amyloidosis. From the present results, the importance of the immunohistochemical method in classifying amyloidosis is stressed.     

Senile aortic amyloid. A third distinctive type of age-related cardiovascular amyloid.

Aortic tissues from 22 elderly patients were analyzed by Congo red staining for amyloid deposits. All samples contained amyloid, which was resistant to the potassium permanganate reaction. Tryptophan was present in all amyloid deposits. The amyloid failed to react with antiserums to amyloid fibril protein ASc1 or human prealbumin, proteins previous demonstrated in generalized senile cardiac amyloid. It also differed from age-related isolated atrial amyloid, which has been shown to lack tryptophan. Deposits did not react with antiserums specific for amyloid fibril proteins of the A lambda IV, A lambda VI, AA, or AEt types. These results indicate that senile aortic amyloid is distinct from amyloid present in primary and secondary amyloidosis and appears to represent a third form of cardiovascular amyloid associated with the aging process.     

Localized Primary Amyloid Tumor of the Thyroid Developing in the Course of Hashimoto's Thyroiditis

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. lmmunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein. Acta Pathol Jpn 42: 210–216. 1992.     

Hepatic amyloidosis: morphologic differences between systemic AL and AA types

The liver is almost universally involved in systemic amyloidosis. Patterns of topographic distribution of amyloid within the liver lobule have been recognized, but the reliability of using these for classification of amyloid type is in question. We examined 286 livers from cases of systemic amyloidosis obtained from autopsies at Los Angeles County-University of Southern California Medical Center, classifying them as AL or AA type by means of the potassium permanganate Congo red-staining method along with a specific anti-AA antiserum. Prior publications have asserted that deposition of secondary (AA) amyloidosis is limited to the vessels in the portal tract, constituting a "vascular" pattern, and that in primary (AL) amyloidosis the deposits exhibit a "sinusoidal" pattern in that they are seen along hepatic sinusoids as well as in portal vessels. We confirmed that AL amyloid involves the portal vessels as frequently as AA amyloid and that deposition occurred significantly more frequently in the portal stroma, the central vein, and the "sinusoidal" areas. However, we also found a "sinusoidal" pattern in 29 of 78 cases of secondary (AA) amyloidosis; in 14 of these, more than half of the sinusoidal spaces were replaced by amyloid deposits. We also noted that in 23 of the 29 AA amyloidosis cases with "sinusoidal" involvement, a "sago" pattern of distribution of amyloid in the spleen was present. No consistent association of a specific chronic inflammatory disease with "sago" spleen and "sinusoidal" deposits could be documented. We conclude that topographic distribution of amyloid within the liver lobule is not a reliable method of distinguishing AA from AL amyloidosis and that specific staining methods must be used if the physician is to be able to attempt modern therapeutic modalities.     

Localized primary amyloid tumor of the thyroid developing in the course of Hashimoto's thyroiditis.

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. Immunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein.     

Transmission of amyloidosis in offspring of mice with AApoAII amyloidosis

Pre-existing amyloid fibrils can induce further polymerization of endogenous precursor proteins in vivo. Thus, transmission of amyloid fibrils (AApoAII) may induce a conformational change in endogenous apolipoprotein A-II and accelerate amyloid deposition in mouse senile amyloidosis. To characterize transmissibility, we examined amyloidosis in the offspring of AApoAII-injected mother mice that possessed the amyloidogenic Apoa2(c) allele of the apolipoprotein A-II gene. At 4 months of age, amyloid deposits were detected in the intestines of offspring born from and nursed by amyloid fibril-injected mothers, with intensity of deposition increasing thereafter. No amyloid deposits were detected in the offspring of noninjected control mothers. Accelerated amyloidosis was also observed in offspring born from mothers without injection but nursed by amyloid fibril-injected mothers. However, this was not observed in offspring born from amyloid fibril-injected mothers but nursed by control mothers. This fostering excluded vertical transmission through the placenta, suggesting the presence of factors that accelerate amyloidosis during the nursing period. In addition, milk obtained from amyloid fibril-injected mothers induced AApoAII amyloidosis in young mice, and transmission electron microscopy detected noodle-like amyloid fibrils in milk of amyloid fibril-injected mothers. These results provide important insight into the etiology and pathogenesis of amyloid diseases.     

Relationship between amyloid deposition and intracellular structural changes in familial amyloidotic polyneuropathy

Transthyretin-related familial amyloidotic polyneuropathy is a systemic amyloidosis caused by mutations in the transthyretin gene. Extracellular deposition of amyloid is the common pathologic hallmark of amyloidoses including Alzheimer disease, AL amyloidosis, AA amyloidosis, and familial amyloidotic polyneuropathy. However, the exact relationship between amyloid deposition and cell death has not yet been clarified. To elucidate this relationship, we studied the effect of transthyretin amyloid fibrils and prefibrillar aggregates on cells by using autopsy tissues obtained from 8 patients with familial amyloidotic polyneuropathy, as well as cultured cell lines. Ultrastructural studies of amyloid-laden cardiomyocytes showed that intracellular structural changes correlated with the degree of amyloid deposition and may reflect metabolic disturbances caused by physical limitations imposed by the amyloid deposits. Amyloid-laden vascular endothelial cells, mesangial cells, smooth muscle cells, Schwann cells, and cardiomyocytes, however, had well-preserved cell nuclei and showed no apoptotic changes, even when cells were completely surrounded by prefibrillar transthyretin aggregates and amyloid fibrils. Synthesized prefibrillar transthyretin aggregates, transthyretin fibrils, and amyloid fibrils obtained from patients with familial amyloidotic polyneuropathy evidenced no cytotoxicity in cell culture experiments. Our data thus indicate that neither transthyretin amyloid fibrils nor prefibrillar transthyretin aggregates directly induced apoptosis. However, cellular metabolic disturbances caused by cells' being physically confined by amyloid deposits may induce cell degeneration.     

Pathological study on amyloidosis in <Emphasis Type="Italic">Cygnus olor</Emphasis> (mute swan) and other waterfowl

Between 2004 and 2007, we examined a total of 70 waterfowl. Forty of 51 (78.4%) mute swans (Cygnus olor) had amyloidosis. Amyloid deposits were detected in the spleen of 39 of 49 birds (79.6%), liver of 37 of 47 birds (78.7%), intestine of 38 of 50 birds (76.0%), pancreas of 30 of 42 birds (71.4%), kidney of 32 of 47 birds (68.1%), thyroid gland of 20 of 30 birds (66.7%), heart of 26 of 49 birds (53.1%), and lung of 5 of 45 birds (11.1%). In some birds, there was a globular pattern of amyloid deposition or infiltration of foreign-body giant cells around amyloid nodules in the spleen. Immunostaining with anti-AA antibody and Western blotting revealed that all were cases of AA amyloidosis. In sections treated with potassium permanganate, which removes Congo red stain, the green refringence under polarized light had mostly vanished. However, staining was not completely eliminated in some areas. Electron microscopy confirmed that the star-shaped amyloid fibrils were 10 nm in diameter and lacked branching. We also demonstrated amyloid bundles and star-shaped amyloid fibrils. A high percentage (96.3%) of mute swans had an inflammatory condition known as bumblefoot. Swans are useful model for studies of animals that have high amounts of amyloid. This research may help in the elucidation of the mechanism of amyloidogenesis in humans, and more research regarding amyloidosis in birds that are consumed as food is necessary.     

Morphologic differences in the pattern of liver infiltration between systemic AL and AA amyloidosis

Biopsy and necropsy tissue from 31 unselected patients with systemic amyloidosis, in which there was histologic evidence of liver involvement, were reviewed with reference to the location and pattern of amyloid deposition in the liver. Amyloidosis was classified into AA and AL types on the basis of immunohistochemistry and permanganate reaction of the amyloid deposits. Nineteen were categorized as AA (secondary) and 12 as AL (primary) amyloidosis. Deposition of AA amyloid was limited to the walls of vessels in the portal tract, constituting a "vascular" pattern. In AL amyloidosis, the deposits exhibited a "sinusoidal" pattern in that they were seen along hepatic sinusoids as well as in vessel walls. This difference was statistically significant (P less than .001). The histologic pattern of liver infiltration offers a valuable clue in the classification of systemic amyloidosis and provides information that may be useful in the selection of patients for therapy.     

Atrial amyloid deposits in the failing human heart display both atrial and brain natriuretic peptide-like immunoreactivity

Atrial amyloid deposits are common in the ageing human heart and contain alpha-atrial natriuretic peptide (proANP99-126) immunoreactivity. However, atrial myocytes secrete both amino and carboxy terminal fragments of the ANP prohormone (proANP1-126) and also express an homologous, but separate brain natriuretic peptide (BNP). Characteristic amyloid deposits were identified in the atria of 9/22 patients (26-63 years of age) with end-stage heart failure. Amyloid fibrils displayed immunoreactivity for both amino and carboxy terminal fragments of proANP1-126 and for the distinct BNP sequence. As in other endocrine organs, both mature and precursor peptide sequences appear to be constituents of amyloid fibrils. Whilst immunoreactivity for cardiac peptide hormones is co-localized in atrial amyloid deposits, it is uncertain whether the increase in natriuretic peptide expression which accompanies cardiac failure contributes to the incidence of isolated atrial amyloidosis.     

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Microglia are a key component of the inflammatory response in the brain and are associated with senile plaques in Alzheimer's disease (AD). Although there is evidence that microglial activation is important for the pathogenesis of AD, the role of microglia in cerebral amyloidosis remains obscure. The present study was undertaken to investigate the relationship between beta-amyloid deposition and microglia activation in APP23 transgenic mice which express human mutated amyloid-beta precursor protein (betaPP) under the control of a neuron-specific promoter element. Light microscopic analysis revealed that the majority of the amyloid plaques in neocortex and hippocampus of 14- to 18- month-old APP23 mice are congophilic and associated with clusters of hypertrophic microglia with intensely stained Mac-1- and phosphotyrosine-positive processes. No association of such activated microglia was observed with diffuse plaques. In young APP23 mice, early amyloid deposits were already of dense core nature and were associated with a strong microglial response. Ultrastructurally, bundles of amyloid fibrils, sometimes surrounded by an incomplete membrane, were observed within the microglial cytoplasm. However, microglia with the typical characteristics of phagocytosis were associated more frequently with dystrophic neurites than with amyloid fibrils. Although the present observations cannot unequivocally determine whether microglia are causal, contributory, or consequential to cerebral amyloidosis, our results suggest that microglia are involved in cerebral amyloidosis either by participating in the processing of neuron-derived betaPP into amyloid fibrils and/or by ingesting amyloid fibrils via an uncommon phagocytotic mechanism. In any case, our observations demonstrate that neuron-derived betaPP is sufficient to induce not only amyloid plaque formation but also amyloid-associated microglial activation similar to that reported in AD. Moreover, our results are consistent with the idea that microglia activation may be important for the amyloid-associated neuron loss previously reported in these mice.     

Morphologic and chemical variation of the kidney lesions in amyloidosis secondary to rheumatoid arthritis

The kidneys of 20 patients who died of secondary systemic amyloidosis due to rheumatoid arthritis were studied histologically, and four of these were shown to have an uncommon pattern of deposition with almost no glomerular involvement but heavy deposits in the outer zone of the medulla. In three of the four patients frozen tissue was available. Immunochemical characterization of amyloid fibrils from these three cases showed that the major subunit amyloid fibril protein was protein AA, typical of secondary amyloidosis. Gel chromatography of fibrils revealed an uncommon elution pattern with two retarded major protein peaks. Both these proteins showed immunologic identity with protein AA and had N-terminal amino acid sequences identical with that protein but differed in size obviously due to a shortening of the C-terminal in one of the proteins. The reason for the correlation between the pattern of deposition of amyloid and alterations in protein AA is unclear but might be due to variations in enzymes responsible for the cleavage of the amyloid fibril subunit precursor protein SAA.