Disease-associated prion protein is not detectable in human systemic amyloid deposits.

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.

Organ transplantation in hereditary apolipoprotein AI amyloidosis.

Patients with hereditary apolipoprotein AI (apoAI) amyloidosis often have extensive visceral amyloid deposits, and many develop end-stage renal failure as young adults. Solid organ transplantation to replace failing organ function in systemic amyloidosis is controversial due to the multisystem and progressive nature of the disease and the risk of recurrence of amyloid in the graft. We report the outcome of solid organ transplantation, including dual transplants in 4 cases, among 10 patients with apoAI amyloidosis who were followed for a median (range) of 16 (4-28) and 9 (0.2-27) years from diagnosis of amyloidosis and transplantation, respectively. Eight of 10 patients were alive, seven with a functioning graft at censor. Two patients died, one of disseminated cytomegalovirus infection 2 months after renal transplantation and the other of multisystem failure following severe trauma more than 13 years after renal transplantation. The renal transplant of one patient failed due to recurrence of amyloid after 25 years. Amyloid disease progression was very slow and the natural history of the condition was favorably altered in both cases in which the liver was transplanted. Failing organs in hereditary apoAI amyloidosis should be replaced since graft survival is excellent and confers substantial survival benefit.

Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis.

The normal plasma protein serum amyloid P component (SAP) binds to fibrils in all types of amyloid deposits, and contributes to the pathogenesis of amyloidosis. In order to intervene in this process we have developed a drug, R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, that is a competitive inhibitor of SAP binding to amyloid fibrils. This palindromic compound also crosslinks and dimerizes SAP molecules, leading to their very rapid clearance by the liver, and thus produces a marked depletion of circulating human SAP. This mechanism of drug action potently removes SAP from human amyloid deposits in the tissues and may provide a new therapeutic approach to both systemic amyloidosis and diseases associated with local amyloid, including Alzheimer's disease and type 2 diabetes.

Structural diversity of ex vivo amyloid fibrils studied by cryo-electron microscopy.

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein.

Clinical and biochemical outcome of hepatorenal transplantation for hereditary systemic amyloidosis associated with apolipoprotein AI Gly26Arg.

BACKGROUND: Treatment of systemic amyloidosis comprises measures to support failing organ function coupled with attempts to reduce the supply of the respective amyloid fibril precursor protein. Orthotopic hepatic transplantation is effective in familial amyloid polyneuropathy associated with variant transthyretin, because this protein is produced almost exclusively in the liver. Hepatic transplantation has not been performed in hereditary apolipoprotein AI (apoAI) amyloidosis, and the liver's contribution to plasma apoAI levels has not been determined in vivo. METHODS: A 57-year-old Irish man with hereditary systemic amyloidosis associated with apoAI Gly26Arg, which had led to end-stage renal failure and progressive liver dysfunction, underwent hepatorenal transplantation. His outcome was followed clinically and his amyloid deposits were monitored with serum amyloid P component scintigraphy. The proportion of variant apoAI in the plasma was estimated by quantitative isoelectric focusing before and after liver transplantation. RESULTS: Plasma levels of variant apoAI decreased by 50% after liver transplantation, and the patient was asymptomatic 2 years after surgery. Subclinical amyloid deposits that were present in his spleen and heart preoperatively have regressed and stabilized respectively. CONCLUSIONS: Orthotopic liver transplantation substantially reduces the supply of the amyloid fibril precursor protein in hereditary apoAI amyloidosis, and the excellent outcome in this patient probably reflects the balance between deposition and turnover of amyloid having been altered in favor of the latter. These findings support the use of liver transplantation in patients with hereditary apoAI amyloidosis who develop hepatic dysfunction.

The protofilament substructure of amyloid fibrils.

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes the usually fatal, acquired and hereditary systemic amyloidoses and is associated with the pathology of Alzheimer's disease, type 2 diabetes and the transmissible spongiform encephalopathies. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. We have previously demonstrated that transthyretin amyloid fibrils contain four constituent protofilaments packed in a square array. Here, we have used cross-correlation techniques to average electron microscopy images of multiple cross-sections in order to reconstruct the sub-structure of ex vivo amyloid fibrils composed of amyloid A protein, monoclonal immunoglobulin lambda light chain, Leu60Arg variant apolipoprotein AI, and Asp67His variant lysozyme, as well as synthetic fibrils derived from a ten-residue peptide corresponding to the A-strand of transthyretin. All the fibrils had an electron-lucent core but the packing arrangement comprised five or six protofilaments rather than four. The structural similarity that defines amyloid fibres thus exists principally at the level of beta-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.

Isolation and characterization of amyloid fibrils from tissue.

Two simple protocols are described for the isolation of amyloid fibrils that consist of water extraction from homogenates of unfixed, frozen human amyloidotic tissues. Most of the contaminating plasma and fibril-associated proteins are removed to yield relatively pure amyloid fibrils, suitable for biochemical characterization, functional assays, and biophysical studies of their structure using many of the specialized techniques described elsewhere throughout this volume.

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Hereditary nephropathic systemic amyloidosis caused by a novel variant apolipoprotein A-I.

We report a family with autosomal-dominant hereditary systemic amyloidosis in three generations, presenting with renal involvement. Two members of the current generation received renal transplants for end-stage renal failure 16 and 18 years ago, and remain very well clinically despite massive visceral amyloidosis. Two other members of this generation, aged 32 and 47 years, have massive systemic amyloid but no clinical disability. Individuals known to be affected in previous generations died of renal failure in early adult life. Amyloid deposits in the proband, one of the transplanted individuals, were composed of apolipoprotein A-I (apoA-I), and among living family members there was complete concordance between amyloidosis and the presence of a novel 9 base pair in-frame deletion mutation in exon 4 of the apoA-I gene, causing a loss of residues Glu70Phe71Trp72. This predicts the acquisition of a single extra positive charge by mature apoA-I, and this variant was detected in the plasma of all carriers. All the previously reported amyloidogenic variants of apoA-I also carry an extra positive charge, indicating that this electrostatic change is likely to be relevant to the amyloidogenicity of apoA-I.

Transthyretin Ile73Val is associated with familial amyloidotic polyneuropathy in a Bangladeshi family. Mutations in brief no. 158. Online.

Amyloidosis is characterised by the extraceullular deposition of certain different proteins in a distinctively abnormal fibrillar conformation. All types of amyloid fibril share remarkably similar structural and biophysical properties despite substantial chemical heterogeneity among their respective precursor proteins. Hereditary amyloidosis associated with genetically determined protein variants is rare, but is extremely important as a model for studying the pathogenesis of amyloidosis generally. We report a novel mutation of the transthyretin (TTR) coding for TTR Ile73Val which is associated with familial amylodotic polyneuropathy (FAP) in a Bangladeshi family. The mutation was detected by direct sequencing of the PCR-amplified TTR exons. It creates an additional Accl restriction exzyme site in exon 3, allowing confirmation of its presence by RFLP. Amyloid detected in sural nerve and colonic biopsies was shown to be composed of TTR by immunohistochemistry. The predominant clinical features were progressive autonomic and sensori-motor peripheral neuropathy, beginning at age 50 years. The proband's father and two siblings had similar illnesses. These findings indicate Val73 is an amyloidogenic variant of TTR.

Amyloid deposition is delayed in mice with targeted deletion of the serum amyloid P component gene.

The tissue amyloid deposits that characterize systemic amyloidosis, Alzheimer's disease and the transmissible spongiform encephalopathies always contain serum amyloid P component (SAP) bound to the amyloid fibrils. We have previously proposed that this normal plasma protein may contribute to amyloidogenesis by stabilizing the deposits. Here we show that the induction of reactive amyloidosis is retarded in mice with targeted deletion of the SAP gene. This first demonstration of the participation of SAP in pathogenesis of amyloidosis in vivo confirms that inhibition of SAP binding to amyloid fibrils is an attractive therapeutic target in a range of serious human diseases.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Molecular mechanisms of fibrillogenesis and the protective role of amyloid P component: two possible avenues for therapy.

Amyloid deposits regress when the supply of fibril precursor proteins is sufficiently reduced, indicating that amyloid fibrils are degradable in vivo. Serum amyloid P component (SAP), a universal constituent of amyloid deposits, efficiently protects amyloid fibrils from proteolysis in vitro, and may contribute to persistence of amyloid in vivo. Drugs that prevent binding of SAP to amyloid fibrils in vivo should therefore promote regression of amyloid and we are actively seeking such agents. A complementary strategy is identification of critical molecular processes in fibrillogenesis as targets for pharmacological intervention. All amyloidogenic variants of apolipoprotein AI contain an additional positive charge in the N-terminal fibrillogenic region of the protein. This is unlikely to be a coincidence and should be informative about amyloidogenesis by this protein. The two amyloidogenic variants of human lysozyme, caused by the first natural mutations found in its gene, provide a particularly powerful model system because both the crystal structure and folding pathways of wild-type lysozyme are so well characterized. The amyloidogenic variant lysozymes have similar 3D crystal structures to the wild type, but are notably less thermostable. They unfold on heating, lose enzymic activity, and aggregate to form amyloid fibrils in vitro.

A new apolipoprotein Al variant, Trp50Arg, causes hereditary amyloidosis.

A man with hereditary non-neuropathic systemic amyloidosis had amyloid fibril protein subunits consisting of N-terminal fragments (residues 1-86, 1-92 and 1-93) of a previously unknown variant of apolipoprotein Al, Trp50Arg, encoded by a thymine-cytosine transition. This is the third reported amyloidogenic apoAl variant. All involve substitutions of single neutral amino acids by the cationic residue arginine, suggesting a common mechanism of amyloidogenesis. However, the phenotypic expression of these mutations varies both within and between the seven known families with hereditary apoAl amyloidosis. These findings should facilitate analysis of the molecular basis of fibrillogenesis and of factors that modulate amyloid deposition and its consequences in vivo.

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Binding of pentraxins to different nuclear structures: C-reactive protein binds to small nuclear ribonucleoprotein particles, serum amyloid P component binds to chromatin and nucleoli.

Binding of the human pentraxin plasma proteins, C-reactive protein (CRP) and serum amyloid P component (SAP), to the nuclei of human cells was studied using whole acute phase serum as the source of the proteins and confocal immunofluorescence microscopy. CRP and SAP clearly bound to distinct, different structures. Double staining with MoAbs to the Sm D and Sm B/B' components of small nuclear ribonucleoproteins confirmed that CRP bound exclusively to these particles. As expected, SAP bound to chromatin and, in addition, binding to the nucleolus was observed for the first time. These interactions demonstrated under relatively physiological conditions, with native pentraxins unseparated from serum and with nuclear constituents in situ, are likely to be of functional importance in vivo.

Rapid automated enzyme immunoassay of serum amyloid A.

Serum amyloid A (SAA), a sensitive acute-phase protein, is the precursor of AA fibrils in reactive amyloidosis. However, SAA is poorly immunogenic, and development and standardization of immunoassays of this protein have been difficult. We established an automated polyclonal/monoclonal microparticle capture enzyme immunoassay, standardized with the World Health Organization prospective reference standard for SAA. A stabilized concentrate of SAA was used for controls and calibrators. The assay range was 1-750 mg/L with CVs < 7% throughout. Plasma and serum gave identical results and no interferences were observed. Linear regression against radial immunodiffusion assay gave a slope of 1.04 (95% confidence interval 0.99-1.10), intercept of -9 mg/L (95% confidence interval -14-3), and residual SD (SEE) of 20 for samples containing < or = 200 mg/L (n = 173). In 105 apparently healthy adults the mean (SD) SAA concentration was 3.7 (3.6) mg/L, the median was 3.0 mg/L, and the range, 0.7-26.4 mg/L. In clinical acute-phase sera, values up to 2200 mg/L were seen. This method will facilitate measurement and investigation of SAA in clinical practice generally and in AA amyloidosis.

Studies of the structure and binding properties of hamster female protein.

We report here the characterization of hamster female protein (FP), a member of the pentraxin family of plasma proteins, as a molecule composed of glycosylated subunits of 25,655 MW containing a single intrachain disulphide bridge. In the presence of EDTA the subunits are non-covalently associated as pentamers of mass approximately 128,000 MW, and in the presence of calcium they aggregate further, probably to form decamers. This pentamer-decamer transition at physiological ionic strength has not been described in other pentraxins. As previously reported, FP shares the capacity of C-reactive protein (CRP) in other species to bind phosphocholine and we show here that it also resembles human CRP in binding only weakly to agarose, to human AA amyloid fibrils in vitro, and to mouse AA amyloid deposits in vivo. It thus differs markedly from human and mouse serum amyloid P component (SAP) but it is nevertheless deposited in hamster AA amyloid in vivo and clearly is the hamster counterpart of SAP in other species. These results illustrate the subtle diversity among members of the otherwise conserved pentraxin family of vertebrate plasma proteins.