Studies in vivo and in vitro of serum amyloid P component in normals and in a patient with AA amyloidosis.

Pure serum amyloid P component (SAP) was isolated from a normal donor pool, from individuals with the different genotypes of an MspI restriction fragment length polymorphism (RFLP) linked to the SAP gene, and from a patient with AA amyloidosis. The SAP preparations were all identical and all behaved as a single homogeneous species in polyacrylamide gel electrophoresis, isoelectric focussing, reverse-phase chromatography, binding in vitro to phosphoethanolamine-Sepharose (binding constant 2.4 x 10(7) l/mol) and AL amyloid fibrils (1.6 x 10(8) l/mol), and binding to amyloid deposits in vivo in mice with casein-induced amyloidosis. The in vivo metabolism of 125I-SAP from a single donor was normal and identical in three healthy individuals representing the three different MspI RFLP genotypes. There is thus no frequent polymorphism of SAP in normal subjects, and SAP altered with respect to the characteristics studied here is not a necessary condition for pathogenesis of systemic AA amyloidosis.     

Gastrointestinal amyloid deposition in AL (primary or myeloma-associated) and AA (secondary) amyloidosis: Diagnostic value of gastric biopsy

Gastrointestinal amyloid deposition was investigated in 21 autopsy cases of nonhereditary systemic amyloidosis, 18 of the AL (primary or myeloma-associated) type and three of the AA (secondary) type. Vascular deposition of amyloid, most apparent in the submucosa, was found in all cases. Parenchymal deposition was observed mainly in the muscularis mucosae and muscularis externa in the AL type, and in the lamina propria mucosae in the AA type. Comparison of amyloid deposition in the stomach and rectum revealed no differences for the AA type. In the AL type, however, deposition in the lamina propria mucosae and muscularis mucosae was more frequent and marked in the wall of the stomach than in the rectum. Thus, gastric biopsy would be more valuable than rectal biopsy in the diagnosis of AL amyloidosis.     

Hepatic amyloidosis. A histopathologic analysis of primary (AL) and secondary (AA) forms.

The liver is a major site of amyloid deposition. The spectrum of histopathologic changes in the liver was studied in 38 patients with systemic amyloidosis (25 with primary or myeloma-associated amyloidosis [AL] and 13 with secondary, reactive [AA] amyloidosis). Overall architectural distortion, alterations of portal triads, as well as predilection for topographic deposition in the parenchyma and/or blood vessel walls were noted. Significant histopathologic differences in AL or AA amyloid liver involvement included 1) portal fibrosis, seen in 7 of 25 (28%) AL patients and 8 of 13 (62%) AA patients (P = 0.05), 2) parenchymal amyloid deposition in 25 of 25 (100%) AL amyloid and 10 of 13 (77%) AA amyloid patients (P = 0.04), and 3) vascular amyloid deposition found in 17 of 25 (68%) with AL amyloid and 13 of 13 (100%) patients with AA amyloid (P = 0.02). These data vary from the widely held concept that deposition of amyloid is predominantly vascular in the AL form and parenchymal in amyloid AA. Clearly, however, in individual cases significant overlap occurred, and characterization of amyloid types based on morphologic distribution of amyloid deposits may be possible in only a minority of cases. In most cases, differentiation of amyloid AL and amyloid AA forms requires clinical, histochemical, immunochemical, and sometimes more elaborate laboratory amino acid sequence studies for accurate identification.     

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.     

Histopathological diagnosis of amyloidosis

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.     

Splenic amyloidosis: correlations between chemical types of amyloid protein and morphological features

Sixty-one autopsy cases of splenic amyloidosis were reviewed to assess the relationship between the morphological patterns and chemical types of amyloid protein. On the basis of immunohistochemical reactions of amyloid protein, the cases were classified into 34 cases of AA and 27 of AL amyloidosis. Amyloid deposition in the spleen was divided into three major sites: the red pulp, the white pulp, and blood vessels. Red pulp involvement by amyloid was noted in 52% of the AL cases but in none of the AA cases. White pulp amyloid deposition was found in 70% of the AL and 35% of the AA cases. This difference was statistically significant (P less than 0.001). On the other hand, vascular deposition of amyloid was invariably noted in all cases with AA or AL amyloidosis, affecting the AA cases rather severely. These results strongly suggest that the widely held concept of deposition of amyloid as predominantly vascular in AL amyloidosis and parenchymal in AA amyloidosis requires revision. Our findings indicate that parenchymal, especially the red pulp, involvement is a consistent feature of AL amyloidosis, whereas vascular involvement is a finding common to both types of systemic amyloidosis.     

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.     

A putative role for cathepsin K in degradation of AA and AL amyloidosis

The aims of this study were to investigate the role of cathepsin K in the pathology of amyloidosis by demonstrating its presence in multinucleated giant cells (MGCs) adjacent to amyloid deposits, and determining its ability to degrade amyloid fibril proteins in vitro. The study was performed using autopsy and biopsy specimens from patients with AA or AL amyloidosis. In six (55%) patients with AA amyloidosis and seven (58%) patients with AL amyloidosis, variable numbers of CD68-immunoreactive MGCs were found adjacent to amyloid deposits. In each case strong cytoplasmic immunostaining for cathepsin K was found in MGCs; immunostaining of amyloid deposits was present in five (45%) patients with AA amyloidosis and three (25%) patients with AL amyloidosis. In vitro degradation experiments showed that recombinant cathepsin K completely degraded AA amyloid fibril proteins at pH 5.5 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Less effective degradation took place at pH 7.4 and there was no degradation in the presence of a general cysteine protease inhibitor (E64) or in the absence of cathepsin K. This is the first study to show that cathepsin K is expressed in MGCs adjacent to amyloid deposits and to demonstrate its ability to degrade amyloid fibril proteins.     

The pattern of amyloid deposition in the lung

A review of routine histopathological samples and autopsies examined at the Department of Pathology, University of Malaya revealed 15 cases of amyloidosis of the lung. Two were localized depositions limited to the lung while in the remainder, lung involvement was part of the picture of systemic amyloidosis. Both cases of localized amyloidosis presented with symptomatic lung/bronchial masses and a clinical diagnosis of tumour. Histology revealed "amyloidomas" associated with heavy plasma cell and lymphocytic infiltration and the presence of multinucleated giant cells. In both cases, the amyloid deposits were immunopositive for lambda light chains and negative for kappa chains and AA protein. One was a known systemic lupus erythematosus patient with polyclonal hypergammaglobulinaemia. The other patient was found to have plasma cell dyscrasia with monoclonal IgG lambda gammopathy. Both patients did not develop systemic amyloidosis. In contrast, lung involvement in systemic AA amyloidosis was not obvious clinically or macroscopically but was histologically evident in 75% of cases subjected to autopsy. Amyloid was detected mainly in the walls of arterioles and small vessels, and along the alveolar septa. It was less frequently detected in the pleura, along the basement membrane of the bronchial epithelium and around bronchial glands. In one case of systemic AL amyloidosis associated with multiple myeloma, an "amyloidoma" occurred in the subpleural region reminiscent of localized amyloidosis. These cases pose questions on (1) whether localized "tumour-like" amyloidosis is a forme fruste of systemic AL amyloidosis and (2) the differing pattern of tissue deposition of different chemical types of amyloid fibrils, with the suggestion that light chain amyloid has a greater tendency to nodular deposition than AA amyloid.     

The pattern of amyloidosis in a Malaysian patient population

Congo red screening of 27,052 routine biopsy specimens from 22,827 patients over a 5 1/2-year period in the Department of Pathology, University of Malaya detected 186 cases of amyloidosis. The categories of amyloidosis encountered and their prevalences in relation to each other were: systemic AL (5.9%); systemic AA (3.2%); isolated atrial (14%); primary localized cutaneous (7.5%); other primary localized deposits (3.2%); localized intratumour (58%); and dystrophic (8.6%). A third of patients with systemic AL amyloidosis had coexistent immunocyte abnormality. The commonest underlying pathology for systemic AA amyloidosis was leprosy. Notable among the types of localized amyloidosis revealed by this study were isolated atrial amyloidosis, which appeared to complicate chronic rheumatic heart disease, and intratumour amyloidosis complicating nasopharyngeal carcinoma. Other tumours in which amyloid deposits were observed included basal cell carcinoma, islet cell tumour and medullary carcinoma of the thyroid. Dystrophic amyloidosis was observed in fibrotic tissues, such as damaged cardiac valves and osteoarthritic joints. Heredofamilial amyloidosis, senile systemic amyloidosis and degenerative cerebral amyloidosis were notably absent from this study.     

A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo

We synthesized (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) and used this compound to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial prion disease, and systemic amyloidosis, such as familial amyloidotic polyneuropathy, amyloid A (AA) amyloidosis, light chain (AL) amyloidosis, and dialysis-related amyloidosis. BSB showed reactions in all Congo red-positive and immunoreactive regions of the samples examined in the study, and some amyloid fibrils in the tissues could be detected more precisely with BSB than with the other methods. In the mouse model of AA amyloidosis, injected BSB reacted with amyloid in all regions in the serial sections in which Congo red staining was positive. A highly sensitive 27-MHz quartz crystal microbalance analysis revealed that BSB showed a significant affinity for amyloid fibrils purified from familial amyloidotic polyneuropathy and dialysis-related amyloidosis samples and suppressed formation of transthyretin amyloid in vitro. These results suggest that BSB may become a valuable tool for detection of amyloid deposits in amyloidosis and of the mechanism of amyloid formation.     

Amyloid localized to tenosynovium at carpal tunnel release. Natural history of 124 cases

One hundred fifty-two patients with amyloid in the tenosynovium who had carpal tunnel release were identified. Twenty-eight patients were excluded because of systemic amyloidosis: primary systemic amyloidosis (AL) in 24, secondary amyloidosis (AA) in 3, and familial amyloidosis (AF) in 1. The remaining 124 patients (82%) had carpal tunnel syndrome with local deposition of amyloid and no evidence of systemic amyloidosis. Median survival of the 124 patients from diagnosis of amyloidosis was 12 years. Only two patients had systemic amyloidosis develop--9 and 10 years after recognition of tenosynovial amyloid. Of particular interest were 12 patients who had an M-protein in the serum or urine. None of the 12 patients have had evidence of systemic amyloidosis or multiple myeloma during the median follow-up of 14 years. The authors conclude that amyloid may be localized to the tenosynovium and that systemic amyloidosis rarely develops during long-term follow-up.     

Diagnosis and treatment in systemic amyloidosis

Systemic amyloidosis is characterized by the involvement of multiple organs and the presence of an amyloid precursor protein in serum. This disorder is classified into four major forms: immunoglobulin light chain-derived (AL), reactive AA, dialysis-related (beta2M) and hereditary transthyretin (ATTR) type. Heart, kidney, gastrointestinal tract and peripheral nerves are commonly affected by amyloid deposition in systemic amyloidosis and histopathological demonstration of amyloid deposits on any of affected organs is the first step leading to the diagnosis of this disease. Immunohistochemical analysis of amyloid protein on tissue amyloid deposits is necessary to make classification of the disease and DNA testing is also useful in a hereditary form. Amyloidosis had been considered to be an incurable disease but during the past one decade several therapeutic approaches have been employed for the amyloidosis patients with diverse pathogenetic backgrounds: intravenous large dose of melphalan accompanied by autologous peripheral blood stem cell transplantation for AL amyloidosis and liver transplantation for hereditary ATTR type amyloidosis. As a result some amyloidosis patients have been rescued and are now enjoying their own social lives. It is likely that recent advance on the research of amyloidosis has changed the concept of this disease.     

Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis

The cerebrum, cerebellum, and choroid plexuses from 16 patients with systemic amyloidosis, and the pituitary glands from 14 of these patients, were investigated histologically and immunohistochemically. Cerebrovascular amyloid (CVA) was found in the leptomeninges and cortices of six patients with systemic amyloidosis, including two patients with amyloid A protein (AA) amyloidosis related to serum amyloid A protein, one with AL amyloidosis related to immunoglobulin light chain (AL), two with familial type I amyloidotic polyneuropathy (FAP), and one with senile systemic amyloidosis (SSA). CVA protein from two patients with FAP reacted with anti-human prealbumin antibody similar to that of the visceral organs of these two patients. CVA in SSA reacted with anti-human prealbumin antibody and anti-beta protein antibody. Vascular amyloid was frequently noted in the pituitary glands and choroid plexuses of patients with systemic amyloidosis, and was found to be identical to that in the visceral organs (heart, kidney, and intestine) of these patients. CVA in the leptomeninges and cortices from two patients with AA amyloidosis and one with AL amyloidosis reacted with anti-beta protein monoclonal antibody but not with anti-human AA monoclonal antibody, anti-human A lambda antisera, and anti-human A kappa antisera. We suggest that amyloid proteins of AA and AL amyloidosis do not readily accumulate in the vessels in the leptomeninges and cortices even though the proteins circulate, and that beta protein is not derived from a serum precursor.     

Distribution pattern of matrix metalloproteinases 1, 2, 3, and 9, tissue inhibitors of matrix metalloproteinases 1 and 2, and α2-macroglobulin in cases of generalized AA- and AL amyloidosis

Matrix metalloproteinases (MMPs) degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix which is disrupted by the deposition of amyloid. This immunohistochemical study investigated the distribution pattern of matrix metalloproteinases (MMP-1, -2, -3, and -9) and their inhibitors [alpha 2-macroglobulin (alpha 2-M), tissue inhibitors of MMPs (TIMP)-1, and TIMP-2] in human AA- and AL amyloid deposits. Specimens of liver, kidney, and spleen from 22 autopsy cases were investigated. Nine patients had suffered from generalized AA amyloidosis, eight from generalized AL amyloidosis, and five from rheumatoid arthritis or tuberculosis with no histological evidence of amyloid. In all amyloidotic and non-amyloidotic patients, each protease and protease inhibitor was detected in almost every organ investigated. In the amyloidotic cases, there was no indication that a specific protease or protease inhibitor was absent or expressed, but a difference was observed in their spatial distribution patterns. The most noticeable difference was found in immunostaining of amyloid. Only MMP-1, -2, and -3, and alpha 2-M were present in AA amyloid deposits, and only TIMP-1 and TIMP-2 were found in deposits of AL amyloid. This is the first study to show that MMP-1, -2, and -3 are present in AA amyloid deposits. They may be involved in tissue remodeling or in proteolysis of the precursor and fibril proteins.     

Diagnosis and immunohistochemical classification of systemic amyloidoses

 Fourty-three cases of systemic amyloidosis were identified in an unselected autopsy series from our institute (6305 autopsies between 1979 and 1993) and classified immunohistochemically by means of a panel of antisera directed against five major amyloid fibril proteins. Amyloid A (AA) amyloidosis was the most common type, being found in 21 cases (48.8%). Transthyretin-derived (ATTR) amyloidosis was present in 11 cases (25.6%), and immunoglobulin light chain-derived (AL) amyloidosis in 10 cases (23.3%). A single case (2.3%) contained deposits of more than one type of systemic amyloid. AA amyoloidosis was associated with chronic inflammatory or infectious diseases (81%), malignant tumours (19%) or both (9.5%). Immunoglobulin light chain-derived amyloidoses were associated with myeloma (50%) or primary (idiopathic; 50%). In AA and AL amyloidosis the kidney was the organ most frequently involved. ATTR amyloid affecting mostly the heart and lungs presented as senile systemic amyloidosis. Systemic amyloidosis was the cause of death in 5 cases (12%) and caused symptoms in 17 cases (39%). Our results suggest that most cases can be classified by using a panel of sensitive and specific antibodies against five major amyloid fibril proteins. This technique may make amyloid type-specific therapy possible for AL amyloid patients who do not have evidence of an underlying plasma cell dyscrasia. Received: 9 December 1997 / Accepted: 2 February 1998     

Circulating serum amyloid P component is the precursor of amyloid P component in tissue amyloid deposits.

Intravenous administration of 125I-labelled isolated mouse serum amyloid P component (SAP) to mice with systemic amyloidosis was followed by specific deposition of the labelled protein in amyloidotic organs. Although only a small proportion of the total injected dose became localized in this way, the amount correlated with the quantity of amyloid present in different organs and was greatest in the spleen. No such localization was detected in the organs of control, untreated mice or animals which had received inflammatory stimuli but did not have amyloidosis. The labelled SAP was found by autoradiography to be present in the same distribution within the tissues as the Congophilic amyloid deposits. These observations establish directly, for the first time, that circulating SAP is the precursor of the amyloid P component (AP) in systemic amyloidosis. They were confirmed by the further finding that intravenous injection into amyloidotic mice of human SAP, either in whole human serum or in isolated pure form, was followed by appearance of the human SAP in the mouse amyloid deposits. In addition to elucidating one aspect of the pathogenesis of amyloid deposition and strengthening the homology of functional behaviour between SAP of different species, the present results suggest a means for selective targeting of diagnostic tracers and/or effector agents to amyloid deposits in vivo.     

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.     

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.     

Fibronectin and basement membrane components in renal amyloid deposits in patients with primary and secondary amyloidosis.

Kidney biopsies from one patient with primary (AL) and three with secondary (AA) amyloidosis were used for an ultrastructural study of the collocalization of basement membrane proteins and the extracellular matrix protein fibronectin within amyloid deposits. Antibodies against amyloid P component, laminin, and heparan sulphate proteoglycan core protein all reacted with the basement membranes and the amyloid depositions in AA and AL amyloidosis. Monoclonal and polyclonal antibodies against collagen type IV reacted only with the basement membranes. Anti-fibronectin reaction was found in association with the basement membranes in all four cases, while labelling of amyloid depositions was found only in one of the AA amyloid cases and in the AL amyloid depositions. It is concluded that basement membrane components may be of importance for the formation of amyloid fibrils.