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Knowledge about the systemic amyloidoses has increased considerably during the last few years. This group of diseases is characterized by great biochemical variability, including at least 11 different amyloid fibril proteins and a remarkable range of clinical manifestations. With the understanding that the pathogenesis is different in the various forms of amyloidosis, it is now being increasingly accepted that an early and accurate diagnosis, including that of the underlying biochemical nature, is crucial for a successful treatment. The elucidation of the molecular mechanisms involved in amyloidogenesis is at the basis of the recent blossoming of new, innovative and more effective therapeutic approaches. [ABSTRACT FROM AUTHOR]

Merlini G, Westermark P (Amyloid Center, Biotechnology Research Laboratory, University Hospital IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy; and Rudbeck Laboratory, Uppsala University, Uppsala, Sweden). The systemic amyloidoses: clearer understanding of the molecular mechanisms offers hope for more effective therapies (Review). J Intern Med 2004; 255: 159–178. Knowledge about the systemic amyloidoses has increased considerably during the last few years. This group of diseases is characterized by great biochemical variability, including at least 11 different amyloid fibril proteins and a remarkable range of clinical manifestations. With the understanding that the pathogenesis is different in the various forms of amyloidosis, it is now being increasingly accepted that an early and accurate diagnosis, including that of the underlying biochemical nature, is crucial for a successful treatment. The elucidation of the molecular mechanisms involved in amyloidogenesis is at the basis of the recent blossoming of new, innovative and more effective therapeutic approaches. [ABSTRACT FROM AUTHOR]

Protein AA, the main fibril constituent in secondary systemic amyloidosis, was demonstrated by the peroxidase-anti-peroxidase method in kidney sections from five out of 14 cases of primary and myeloma associated amyloidosis, all having an immunoglobulin light chain derived protein as a major subunit. In three of these cases, protein AA was also demonstrated in double immuno diffusion of dissolved amyloid preparations. The protein had the characteristics of protein AA in elution position, immunodiffusion and isoelectric focussing pattern. The significance of protein AA in primary and myeloma associated amyloidosis is unknown. [ABSTRACT FROM AUTHOR]

Amyloid fibrils, purified from the spleen of four patients with amyloidosis associated with rheumatoid arthritis, had protein AA as a major protein. Besides this protein, all four amyloid fibril preparations contained a protein which in size, amino acid composition and N-terminal amino acid sequence was the same as the postulated serum precursor of protein AA, serum AA (SAA). The SAA-like amyloid fibril protein had a tendency to aggregate in neutral conditions, a phenomenon which is also seen in SAA but not in protein AA. [ABSTRACT FROM AUTHOR]

Antiserum specific for human prealbumin (HPA) was studied by indirect immunofluorescence on tissue sections of cardiac ventricles containing senile cardiac amyloid. The pattern of reactivity was identical to that previously reported for an antiserum specific for protein ASc1 (formerly designated AscA) present in these tissues. Anti-HPA failed to react with isolated atrial amyloid (IAA), primary amyloid (A,I, A;IV, A,VI), secondary amyloid (AA), amyloid asso- ciated with medullary carcinoma of the thyroid (AE1), pancreatic amyloid associated with adult onset diabetes, cerebral amyloid present in Alzheimer's disease or lichen amyloid. The reaction of anti-HPA was completely blocked by purified human prealbumin but was not influenced by absorption with purified human albumin or proteins extracted from any amyloid types tested. The anti-HPA reaction was also completely blocked by purified protein ASC1, and the reaction of Anti-Asc1 was similarly blocked by purified HPA. These studies suggest that senile cardiac amyloid of the ASc1 type contains prealbumin or a protein antigenically closely related to this molecule. [ABSTRACT FROM AUTHOR]

Amino-acid sequence analysis of an amyloid fibril protein from a patient with Swedish familial amyloidotic polyneuropathy showed homology with prealbumin hut with heterogeneous N-terminal deletions. One-third of the molecules had the same amino acid substitution, methionine for valine in position 30, as in familial amyloidosis of Portuguese. Japanese and Swedish-American type .A protein with the same antigenic properties and size was found in the fibrils of two other patients with Swedish FAP while the amyloid fibrils in two further patients predominantly contained a smaller prealbumin- derived protein. Cyanogen bromide cleavage of this protein revealed no evidence for a methionine residue in position 30. [ABSTRACT FROM AUTHOR]

Although hereditary transthyretin (h-ATTR) amyloidosis is a monogenetic disease, a large variation in its phenotype has been observed. The common hypothesis of amyloid fibril formation involves dissociation of the transthyretin (TTR) tetramer into monomers that after misfolding reassemble into amyloid fibrils. This notion is partly challenged by the finding of two distinct types of amyloid fibrils. One of these, type A, consists of C-terminal ATTR fragments and full-length TTR, whereas the other, type B, consists only of full-length TTR. All organs of an individual patient contain ATTR deposits of either type A or type B fibrils, and the composition in each individual remains unchanged over time. The finding of two distinct types of ATTR fibrils suggests that there are at least two different pathways in operation for ATTR fibril formation. For the most common European mutation, TTR Val30Met, ATTR fibril composition is related to the outcome of liver transplantation, which is the first successful treatment for the disease, and the penetrance of the trait. In addition, the presence of C-terminal ATTR fragments has an impact on the affinity for various tracers used for noninvasive imaging of amyloid depositions such as 99 m-technetium-diphosphono-propanodicarboxylic acid scintigraphy and positron emission tomography utilizing Pittsburgh component B, and even for the gold standard diagnostic procedure, tissue biopsy stained by Congo red and examined under polarized light. The importance of amyloid fibril composition needs to be taken into consideration when designing clinical trials of treatment modalities, and also in the evaluation of diagnostic methods such as imaging techniques. [ABSTRACT FROM AUTHOR]

The systemic amyloidoses constitute a group of life-threatening disorders at which one out of about 15 different proteins have polymerized into fibrils. Prognosis and treatment varies widely and depends on the biochemical type. Determination of this has usually to be performed by immunohistochemistry which is a challenge because of lack of monospecific antibodies that can be used on formaldehyde-fixed tissue sections. We have here used an old method to create immunogenic fragments of AL-amyloid fibrils by partial degradation and solubilization with sodium hydroxide. The mouse monoclonal antibody pwlam raised against this material, labelled AL-amyloid deposits of lambda origin strongly and specifically in sections of formaldehyde-fixed and paraffin-embedded tissues. [ABSTRACT FROM AUTHOR]

Abstract The amyloid fibril protein AL was isolated from the spleen of a patient with systemic amyloidosis. Size-exclusion chromatography of the solubilized amyloid fibrils revealed a distinct, retarded asymmetric peak. The symmetrical part of the peak showed on SDS-PAGE two positive periodic acid Schiff-staining bands at 14 and 16 kDa. Staining with Coomassie Brilliant Blue revealed in addition two proteins with masses of 13 and 20 kDa. The 14 and 16 kDa bands were the strongest ones. N-Terminal analyses of the four blotted bands showed that the N-termini were the same in all cases. Elucidation of the amino acid sequence established an AL-chain of 157 residues as well as a fragment covering positions 188–207 of the constant region. Two tryptic peptides derived from the same region, positions 25–46, showed an identical sequence, except for position 34 where both alanine and threonine residues occurred. Monosaccharide compositional analysis of the threonine-containing peptide revealed an oligosaccharide in the N-glycosylation site, position 32–34. Mass analysis of the glycopeptide verified the oligosaccharide. The AL-chains belong to the kappa 3a germline gene and verifies that the glycosylated chain is a mutated form. The AL-chains differ from that of the germline in 14 positions. The J-segment is of JκIII and is mutated in position 106. [ABSTRACT FROM AUTHOR]

Background: Wild‐type transthyretin (ATTRwt) amyloidosis is the most common systemic amyloidosis in Western countries and manifests mainly as progressive restrictive cardiomyopathy. Objective: To study the prevalence of ATTR deposits in ligament tissue in patients undergoing surgery for lumbar spinal stenosis and to assess whether these deposits are associated with cardiac amyloidosis. Materials and methods: A total of 250 patients, aged 50–89 (57% women), none with known cardiovascular disease, were included. Ligaments were investigated microscopically for amyloid. ATTR type was determined by immunohistochemistry and fibril type by Western blot. The amount of amyloid was graded 0‐4. All patients with grade 3‐4 ATTR deposits were offered cardiac investigation including ECG, cardiac ultrasound, plasma NT‐proBNP and cardiac magnetic resonance (CMR), including modern tissue characterization. Results: Amyloid was identified in 221 of the samples (88.4%). ATTR appeared in 93 samples (37%) of whom 42 (17 women and 25 men) were graded 3‐4; all had fibril type A (mixture of full‐length TTR and fragmented TTR). Twenty‐nine of 42 patients with grade 3‐4 ATTR deposits accepted cardiovascular investigations; none of them had definite signs of cardiac amyloidosis, but five men had a history of carpal tunnel syndrome. Conclusions: The prevalence of ATTR deposits in ligamentum flavum in patients with lumbar spinal stenosis was high but not associated with manifest ATTR cardiac amyloidosis. However, the findings of fibril type A, the prevalence of previous carpal tunnel syndrome and ATTR amyloid in surrounding adipose and vascular tissue indicate that amyloid deposits in ligamentum flavum may be an early manifestation of systemic ATTR disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The distribution of proteoglycans in kidneys from patients with secondary (AA) systemic amyloidosis was investigated. Antisera reacting with the protein cores of chondroitin sulphate proteoglycan (CSPG), dermatan sulphate proteoglycan (DSPG) and heparan sulphate proteoglycan (HSPG) were used in conjunction with the peroxidase-antiperoxidase (PAP) method. HSPG was the only proteoglycan found to be specifically localized to the amyloid deposits. The staining was most intense on the endothelial side of the deposits in both the glomeruli and in the vessel walls. No staining was observed after absorption of the HSPG antiserum with a fraction of the amyloid preparations.corresponding in size to that reported for glomerular HSPG. The possible role of HSPG and endothelial cells in the pathogenesis of the amyloid deposits is discussed. [ABSTRACT FROM AUTHOR]

There are around 30 human diseases associated with protein misfolding and amyloid formation, each one caused by a certain protein or peptide. Many of these diseases are lethal and together they pose an enormous burden to society. The prion protein has attracted particular interest as being shown to be the pathogenic agent in transmissible diseases such as kuru, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Whether similar transmission could occur also in other amyloidoses such as Alzheimer's disease, Parkinson's disease and serum amyloid A amyloidosis is a matter of intense research and debate. Furthermore, it has been suggested that novel biomaterials such as artificial spider silk are potentially amyloidogenic. Here, we provide a brief introduction to amyloid, prions and other proteins involved in amyloid disease and review recent evidence for their potential transmission. We discuss the similarities and differences between amyloid and silk, as well as the potential hazards associated with protein-based biomaterials. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine the chemical nature of amyloid deposits found in knee joint menisci. METHODS: Amyloid was extracted from the menisci of 3 adults who underwent knee joint replacement surgery. The primary structural features of the purified proteins were determined by sequential Edman degradation and tandem mass spectrometry (MS/MS). Tissue specimens were also subjected to in situ hybridization analysis, as well as complementary DNA cloning by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally, specimens from these 3 patients, as well as other patients with amyloid in the knee joint menisci, were examined immunohistochemically. RESULTS: Amino acid sequence and MS/MS analyses of the extracts revealed the presence of 60-77-residue components identical to the N-terminal portion of apolipoprotein A-I (Apo A-I). The Apo A-I nature of the amyloid was confirmed by the demonstration that the green birefringent congophilic deposits in the 7 meniscus samples were recognized by an anti-human Apo A-I antibody. That the meniscus itself was the source of the amyloidogenic protein was evidenced through Southern blot analysis, in which an Apo A-I product was generated by RT-PCR from synovial tissue, and further, by the demonstration that the cytoplasm of chondrocytes reacted with the specific Apo A-I probe used for in situ hybridization and was immunostained by the anti-Apo A-I antiserum. CONCLUSION: Amyloid in the knee joint menisci is formed from Apo A-I that is produced by chondrocytes within the meniscal cartilage. This entity represents yet another localized form of amyloidosis associated with the aging process and may be of pathophysiologic import. [ABSTRACT FROM AUTHOR]

Primary localized amyloidosis has been described in many different organs in the body. Studies by immunohistochemical techniques have suggested an immunoglobulin light chain origin of the amyloid material. Only in a limited number of cases has the amyloid protein been characterized by amino acid sequence analysis as subtypes of immunoglobulin light chain or heavy chain. In this report, two cases of primary localized amyloidosis of the eyelid are presented. The amyloid substance has been extracted and a major fibril protein subjected to amino acid sequence analysis. Both amyloid proteins were part of the variable region of immunoglobulin light chains, subtype &lamda;V and subtype &lamda;VI, respectively. While &lamda;VI has been shown to be a common subtype in systemic immunoglobulin light chain-amyloidosis, it has never been demonstrated in localized amyloid. Very few &lamda;V immunoglobulin light chains have been characterized and the subgroup has never been found in amyloid before. [ABSTRACT FROM AUTHOR]