A transgenic mouse model reproduces human hereditary systemic amyloidosis.

Amyloidoses are rare life-threatening diseases caused by protein misfolding of normally soluble proteins. The fatal outcome is predominantly due to renal failure and/or cardiac dysfunction. Because amyloid fibrils formed by all amyloidogenic proteins share structural similarity, amyloidoses may be studied in transgenic models expressing any amyloidogenic protein. Here we generated transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII, a major protein of high density lipoprotein. According to amyloid nomenclature this variant was termed STOP78SERApoAII. STOP78SER-APOA2 expression at the physiological level spontaneously induced systemic amyloidosis in all mice with full-length mature STOP78SER-ApoAII identified as the amyloidogenic protein. Amyloid deposits stained with Congo red were extracellular, and consisted of fibrils of approximately 10 nm diameter. Renal glomerular amyloidosis was a major feature with onset of renal insufficiency occurring in mice older than six months of age. The liver, heart and spleen were also greatly affected. Expression of STOP78SER-APOA2 in the liver and intestine in mice of the K line but not in other amyloid-laden organs showed they present systemic amyloidosis. The amyloid burden was a function of STOP78SER-APOA2 expression and age of the mice with amyloid deposition starting in two-month-old high-expressing mice that died from six months onwards. Because STOP78SER-ApoAII conserved adequate lipid binding capacity as shown by high STOP78SER-ApoAII amounts in high density lipoprotein of young mice, its decrease in circulation with age suggests preferential deposition into preformed fibrils. Thus, our mouse model faithfully reproduces early-onset hereditary systemic amyloidosis and is ideally suited to devise and test novel therapies.

Carbamylation of the amino-terminal residue (Gly1) of mouse serum amyloid A promotes amyloid formation in a cell culture model.

Amyloid A (AA) amyloidosis is a fatal protein deposition disease afflicting a small percentage of patients with chronic inflammation. Factors other than inflammation that determine development of AA amyloidosis remain largely unknown. The subunit protein comprising AA amyloid fibrils is derived from serum amyloid A (SAA), specifically its amino-terminal portion. In this in vitro study, carbamylation of residues in this region (primarily Gly1 but also Lys24) was shown to markedly increase amyloid-forming propensity as judged by extensive accumulation of amyloid in cell cultures. Contrastingly, no amyloid deposition occurred in cultures given SAA having a noncarbamylated amino terminus. Carbamylation, known to occur during uremia or inflammation, merits investigation as a potential determinant of AA amyloid fibril formation.

Hereditary systemic immunoglobulin light-chain amyloidosis.

Several members of a family died from renal failure as a result of systemic amyloidosis. Extensive studies to detect previously documented gene mutations associated with amyloidosis failed to identify a causative factor. In search of the genetic basis for this syndrome, amyloid fibrils were isolated from renal tissue of a member of the kin who died while on renal dialysis. Amino acid sequencing of isolated amyloid protein identified sequences compatible with the constant region of the immunoglobulin κ light-chain. Isolation and characterization of κ light-chain protein from serum of an affected member of the kindred revealed mutation in the constant region of κ light-chain, with cysteine replacing serine at amino acid residue 131. This mutation (Ser131Cys) was confirmed by DNA analysis, which identified a single-base change of cytosine to guanine at the second position of codon 131 of the κ light-chain gene (TCT131TGT). DNA analysis of members of the extended family revealed transmission of the Ser131Cys mutation and association with systemic amyloidosis. This amyloid light-chain (AL) amyloidosis, which is a hereditary type of amyloidosis and not the result of a monoclonal plasma cell dyscrasia, may be misdiagnosed and lead to inappropriate chemotherapy.

Amyloid fibrils containing fragmented ATTR may be the standard fibril composition in ATTR amyloidosis.

Abstract The clinical phenotype of familial ATTR amyloidosis depends to some extent on the particular mutation, but differences exist also within mutations. We have previously described that two types of amyloid fibril compositions exist among Swedish ATTRV30M amyloidosis patients, one consisting of a mixture of intact and fragmented ATTR (type A) and one consisting of mainly intact ATTR (type B). The fibril types are correlated to phenotypic differences. Patients with ATTR fragments have a late onset and develop cardiomyopathy, while patients without fragments have an early onset and less myocardial involvement. The present study aimed to determine whether this correlation between fibril type and phenotype is valid for familial ATTR amyloidosis in general. Cardiac or adipose tissues from 63 patients carrying 29 different TTR non-V30M mutations as well as 13 Japanese ATTRV30M patients were examined. Fibril type was determined by western blotting and compared to the patients' age of onset and degree of cardiomyopathy. All ATTR non-V30M patients had a fibril composition with ATTR fragments, except two ATTRY114C patients. No clear conclusions could be drawn about a phenotype to fibril type correlation among ATTR non-V30M patients. In contrast, Japanese ATTRV30M patients showed a similar correlation as previously described for Swedish ATTRV30M patients. This study shows that a fibril composition with fragmented ATTR is very common in ATTR amyloidosis, and suggests that fibrils composed of only full-length ATTR is an exception found only in a subset of patients.

Human SAA1-derived amyloid deposition in cell culture: a consistent model utilizing human peripheral blood mononuclear cells and serum-free medium.

Amyloid A (AA) amyloidosis is a fatal disease caused by extracellular deposition of fibrils derived from serum AA (SAA). AA amyloid fibril formation has previously been modeled in macrophage cultures using highly amyloidogenic mouse SAA1.1, but attempts to do the same with human SAA invariably failed. Our objective was to define conditions that support human SAA-derived amyloid formation in peripheral blood mononuclear cell (PBMC) cultures. Two conditions were found to be critical - omission of fetal calf serum and use of StemPro34, a lipid-enriched medium formulated for hematopoietic progenitor cells. Cultures maintained in serum-free StemPro34 and provided with recombinant human SAA1 in the complete absence of amyloid-enhancing factor exhibited amyloid deposition within 7 d. Amyloid co-localized with cell clusters that characteristically included cells of fibrocytic/dendritic morphology as well as macrophages. These cells formed networks that appeared to serve as scaffolding within and upon which amyloid accumulated. Cells in amyloid-forming cultures demonstrated increased adherence, survival and expression of extracellular matrix components. Of the three human SAA1 isoforms, SAA1.3 showed the most extensive amyloid deposition, consistent with it being the most prevalent isoform in Japanese patients with AA amyloidosis. Attesting to the reproducibility and general applicability of this model, amyloid formation has been documented in cultures established from eight PBMC donors.

κ I light chain AL amyloidosis presenting with rapidly progressive renal and hepatic failure with unusual renal amyloid distribution.

A 65-year-old man suffering from generalized edema and jaundice was admitted to our hospital. Laboratory findings revealed marked renal dysfunction with heavy proteinuria as well as liver dysfunction with severe obstructive jaundice. On renal biopsy, the diagnosis of AL amyloidosis associated with κ I light chain was made. Interestingly, amyloid deposits were restricted to the glomeruli. Although hemodialysis was initiated, the patient died due to further deterioration of hepatic function. On autopsy, severe intrahepatic cholestasis was observed, and there was marked deposition of AL amyloid in the liver. Literature reviews showed that rapidly progressive renal failure is common in AL amyloidosis patients who presented with acute hepatic failure due to severe intrahepatic cholestasis. However, the detailed renal pathology in this condition has not been documented. The present case is very interesting because rapidly progressive renal and hepatic failure was simultaneously observed, and renal amyloid deposition was restricted to the glomeruli.

Progression of transthyretin amyloid neuropathy after liver transplantation.

OBJECTIVE: To biochemically characterize peripheral nerve amyloid in subjects with transthyretin (TTR) amyloidosis and assess effect of orthotopic liver transplantation (OLT) on progression of neuropathy. METHODS: Amyloid fibrils were isolated from peripheral nerve tissues of 6 patients with TTR amyloidosis who were heterozygous for an amyloid-associated TTR mutation. Ratio of variant to wild-type TTR in the fibrils was determined by amino acid sequencing of tryptic peptides containing either the variant amino acid residue or the corresponding normal amino acid. RESULTS: Amyloid fibrils from 3 subjects who died without having received a liver transplant were composed of 60%-65% variant TTR and 35%-40% wild-type. Amyloid fibrils from a subject who died 5 years after liver transplantation contained 25% variant and 75% wild-type TTR. CONCLUSION: Ratios of variant to wild-type TTR in amyloid patients heterozygous for an amyloid-associated TTR mutation are similar to published ratios for amyloid fibrils in cardiac tissue. Survival after liver transplantation for TTR amyloidosis may be associated with progression of neuropathy due to continued deposition of amyloid derived from wild-type TTR.

Hereditary fibrinogen A alpha-chain amyloidosis: phenotypic characterization of a systemic disease and the role of liver transplantation.

Variants of fibrinogen A alpha-chain (AFib) cause the most common type of hereditary renal amyloidosis in Europe and, possibly, the United States as well. Variant fibrinogen is produced in the liver, and solitary renal allografts fail within 1 to 7 years with recurrent amyloidosis. We assessed 22 AFib patients for combined liver and kidney transplantation (LKT) and report the clinical features and outcome. Twenty-one had E526V and 1, the R554L variant. Coronary atherosclerosis was identified in 68% and systemic atheromatosis in 55%. Vascular atheroma excised at endarterectomy and endomyocardial biopsies contained purely variant fibrinogen amyloid. Half had autonomic neuropathy. Six of 9 patients who underwent LKT are alive (67%), with good allograft function and no amyloidosis at median 67 months (range, 33-155 months) of follow-up. Serial technetium-99m-labeled dimercaptosuccinic acid ((99m)Tc-DMSA) renal scintigraphy in 2 cases of preemptive LKT demonstrated preserved native kidney residual function at 5 years. Four explanted livers were used successfully for domino transplantation. Fibrinogen amyloidosis is a systemic amyloid disease with visceral, vascular, cardiac, and neurologic involvement. LKT is curative; however, cardiovascular amyloidosis may preclude this option. Our data encourage evaluation of preemptive solitary liver transplantation early in the course of amyloid nephropathy to prevent hemodialysis and kidney transplantation.

Biochemical characterisation of amyloid by endomyocardial biopsy.

Cardiomyopathy is a major cause of death in patients with systemic amyloidosis. There are several forms of systemic amyloidosis which cause cardiomyopathy and determination of the exact type of amyloid in each affected patient is essential for treatment and determination of prognosis. In this study, we tested the feasibility of determining the type of amyloidosis by biochemical analysis of endomyocardial biopsies. Right ventricular endomyocardial biopsies were obtained from 10 patients with restrictive cardiomyopathy. Three patients had monoclonal protein demonstrated in serum or urine and all three had bone marrow findings consistent with monoclonal gammopathy. Seven patients had isolated cardiomyopathy without evidence of monoclonal gammopathy. A portion of each myocardial biopsy was submitted for histologic evaluation and all demonstrated amyloid by Congo red staining. Each biopsy was analysed biochemically by isolation of amyloid fibrils and the protein characterised by amino acid sequence analysis. Four amyloid isolates were characterised as immunoglobulin light chain proteins. Two specimens obtained from patients with transthyretin (TTR) DNA mutations contained TTR peptides proving the hereditary nature of the disease. Biopsies from four patients without a TTR mutation contained TTR and were consistent with the diagnosis of senile cardiac amyloidosis (SCA). All endomyocardial biopsy specimens that were analysed had sufficient amyloid fibril subunit protein to allow characterisation by amino acid sequence analysis. This methodology is particularly useful in differentiating SCA with TTR amyloid fibrils from immunoglobulin light chain amyloidosis which also occurs in the elderly age group.

Leukocyte chemotactic factor 2: A novel renal amyloid protein.

Renal amyloid deposits can often be seen in primary amyloidosis (immunoglobulin light chain disease) or in secondary forms such as reactive amyloidosis as well as in several hereditary forms where a variety of mutant proteins 'precipitate' as amyloid plaques. However, in rare cases, amyloidosis may be identified by renal biopsy, but no definitive diagnosis could be made. We have isolated amyloid fibrils from such a case in which the patient presented with nephrotic syndrome and subsequent azotemia requiring hemodialysis. Evaluation for amyloid deposition in other organ systems was negative and immunohistochemical analysis of the kidney deposits for known contributing proteins was unrevealing. Biochemical analysis of the fibrils identified a new amyloid subunit protein, leukocyte chemotactic factor 2, originally identified as a possible chemotactic and growth factor. A monoclonal antibody to this protein reacted specifically with the amyloid deposits in the glomeruli and interstitium by immunohistochemistry. This study emphasizes the importance of biochemical characterization of amyloid present in renal biopsies.

Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation.

It has been hypothesized that transthyretin (TTR) amyloidosis may progress after orthotopic liver transplantation (OLT) as a result of continued amyloid fibril synthesis and deposition from normal TTR. To test this hypothesis amyloid fibrils were isolated from cardiac tissues of three patients who died 1(1/2) to 5(1/2) years after OLT: two with Val30Met and one with Thr60Ala TTR. The ratio of variant to normal TTR in each case was determined and compared with the ratio of variant to normal in cardiac tissues from seven patients who died with TTR amyloidosis but who had not had liver transplantation. Tissues from patients with TTR amyloidosis without OLT included three with Val30Met, two with Thr60Ala, one with deltaVal122, and one with Val122Ile. All tissues from patients without OLT had greater amounts of variant TTR than normal TTR except for the Val122Ile in which the ratio was 50:50. The overall median variant to normal ratio was 60:40 with a range of 50-70% variant. In contrast, the mean percentage of variant TTR in the three tissues from patients after OLT was 25% (range 20-35). These data are consistent with the continued deposition of normal TTR in cardiac tissue after liver transplantation.

Lambda II immunoglobulin light chain protein in primary localized rectal amyloidosis.

Rectal involvement is usually part of a systemic amyloidosis, whereas, localized rectal amyloidosis is a rare entity. We present a case of asymptomatic localized rectal amyloidoma. Amyloid fibrils were isolated from rectal biopsy tissue and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which showed bands at 17 kDa, 21 kDa and 28 kDa, a broad doublet band at 7-8 kDa and weaker bands at 15 kDa and 24 kDa. Edman sequence analysis of the isolated protein and its tryptic peptides showed that the amyloid protein was derived from an immunoglobulin lambdaII-light chain. To our knowledge, this is the first reported case to isolate and chemically characterize amyloid fibrils from a localized rectal amyloidoma. The development of specific therapies for patients with amyloid-associated disorders emphasizes the need to characterize the biochemical nature of the amyloid fibril protein.

Structural analyses of fibrinogen amyloid fibrils.

Hereditary fibrinogen amyloidosis is characterized by deposition of amyloid fibrils in renal glomeruli. The subunit protein of the amyloid fibrils is a proteolytic fragment of the fibrinogen Aalpha-chain. To investigate the structure of fibrinogen amyloid, fibrils were extracted from the tissues of a patient and studied by X-ray fiber diffraction and electron microscopy. We have carried out a full structural characterization of amyloid fibrils taken from disease tissue. These studies revealed that ex vivo fibrinogen amyloid fibrils have a cross-beta structure similar to other chemical types of amyloid fibrils.

Cellular events associated with the initial phase of AA amyloidogenesis: insights from a human monocyte model.

Reactive amyloidosis is a systemic protein deposition disease that develops in association with chronic inflammation. The deposits are composed of extracellular, fibrillar masses of amyloid A (AA) protein, an N-terminal fragment of the acute-phase serum protein serum amyloid A (SAA). The pathogenic conversion of SAA into amyloid has been studied in two human cell culture models, peritoneal cells and peripheral blood monocytes. Human monocyte cultures proved more robust than either mouse or human peritoneal cells at initiating amyloid formation in the absence of a preformed nidus such as amyloid-enhancing factor and particularly well suited for examination of individual cells undergoing amyloid formation. Amyloid-producing monocyte cultures were stained with Congo red and Alcian blue for detection of amyloid and glycosaminglycans, respectively; immunocytochemistry was performed to identify SAA/AA, CD68, CD14, lysosomal protein Lamp-1, and early endosomal protein EEA1. SAA interaction with monocytes was also visualized directly via fluorescence confocal microscopy. Amyloid was initially detected only in intracellular vesicles, but with time was seen extracellularly. Morphologic changes in lysosomes were noted during the early phase of amyloid formation, suggesting that exocytosis of fibrils may occur via lysosome-derived vesicles. Cultures engaged in amyloid formation remained metabolically active; no cytotoxic effects were observed. Mimicking in vivo phenomena, amyloid formation was accompanied by increased glycosaminoglycan content and C-terminal processing of SAA. The ability of human monocytes to endocytose and intracellularly transform SAA into amyloid via a mechanism that requires and maintains, rather than compromises, metabolic activity distinguishes them as a useful model for probing earliest events in the disease process.

Biochemical characterization of vitreous and cardiac amyloid in Ile84Ser transthyretin amyloidosis.

Plasma transthyretin (TTR) is synthesized in the liver and is the source for visceral amyloid deposits in TTR amyloidosis. However, TTR is also synthesized in the retinal pigment epithelium of the eye and choroid plexus of the brain. It has been postulated that vitreous amyloid, which is associated with approximately 20% of the known amyloidogenic TTR mutations, results from local synthesis of TTR in the eye. In order to elucidate if differences in amyloid between organs exists, we have analyzed vitreous and cardiac amyloid fibrils in Ile84Ser TTR patients for comparison. Analysis of guanidine hydrochloride solubilized protein from isolated vitreous and cardiac amyloid fibrils indicated that the amyloid TTR in both organs is highly proteolyzed with minor amounts of intact TTR present. While vitreous protein was amenable to direct Edman sequence analysis, cardiac protein gave low yields indicating it was mostly N-terminally blocked or inaccessible to Edman degradation. While vitreous contained major 11 kDa and minor 9 kDa fragments, cardiac contained at least three major fragments of 7-11 kDa. Vitreous protein was cleaved between Lys48-Thr49, while cardiac protein was cleaved at multiple sites in the residue 46-52 region. While deposits in both tissues were enriched in variant TTR, vitreous fibrils contained more variant protein than cardiac fibrils (80-89% vs. 60-65% Ser84TTR). These differences suggest that the mechanism or pathway of fibril formation may differ in various tissues.

Aging in heterozygous Dnmt1-deficient mice: effects on survival, the DNA methylation genes, and the development of amyloidosis.

We previously reported that heterozygous DNA methyltransferase 1-deficient (Dnmt1(+/-)) mice maintain T-cell immune function and DNA methylation levels with aging, whereas controls develop autoimmunity, immune senescence, and DNA hypomethylation. We therefore compared survival, cause of death, and T-cell DNA methylation gene expression during aging in Dnmt1(+/-) mice and controls. No difference in longevity was observed, but greater numbers of Dnmt1(+/-) mice developed jejunal apolipoprotein AII amyloidosis. Both groups showed decreased Dnmt1 expression with aging. However, expression of the de novo methyltransferases Dnmt3a and Dnmt3b increased with aging in stimulated T cells from control mice. MeCP2, a methylcytosine binding protein that participates in maintenance DNA methylation, increased with age in Dnmt1(+/-) mice, suggesting a mechanism for the sustained DNA methylation levels. This model thus provides potential mechanisms for DNA methylation changes of aging, and suggests that changes in DNA methylation may contribute to some forms of amyloidosis that develop with aging.