Incorporation of plasma proteins into amyloid

With the aid of pure antibodies labeled with fluorochrome and peroxidase, amyloid, occurring in various situations in patients with secondary amyloidosis, was found regularly to contain -globulin, fibrinogen, and albumin. The possible mechanisms of accumulation of proteins in amyloid are discussed.Laboratory of Age Pathology, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 5, pp. 117–119, May, 1975.     

Immunohistochemical characterization of amyloid proteins in sural nerves and clinical associations in amyloid neuropathy.

To test whether immunohistochemical characterization of proteins in amyloid deposits in biopsied sural nerves gives reliable and useful diagnostic information using commercially available reagents, biopsy specimens of sural nerves from 38 patients with amyloid neuropathy were studied. Transthyretin (TTR) was detected in the amyloid deposits of 11 nerves, lambda light chains (LC) in 8 nerves, kappa LC in 7 nerves, and both lambda and kappa LC in 3 nerves. In 9 nerves, the amyloid deposits were too small to allow adequate immunohistochemical characterization of amyloid proteins in serial sections. Evidence that immunohistochemical characterization was correct came from: 1) evaluation of kin, 2) search for monoclonal proteins in the plasma, and 3) sequencing of the gene abnormalities in TTR+ cases. In 9 of 11 TTR+ cases, in which DNA could be obtained, sequencing of the gene showed that each of the 9 cases was heterozygous for a gene mutation; 7 had previously described mutations and 2 undescribed mutations. Therefore, in the nine sporadic cases without plasma monoclonal light chains, the immunohistochemical characterization correctly identified the protein in amyloid as transthyretin. Likewise, there was a high concordance between immunoglobulin light chains in plasma and light chains in amyloid in primary amyloidosis. Evaluation of the type, distribution, and severity of the neurologic symptoms and deficits showed: 1) the sensorimotor and autonomic neuropathy of amyloidosis characteristically affects proximal as well as distal limbs, and 2) the type of amyloidosis probably cannot be determined from the characteristics or severity of the neuropathy alone or from the location or size of amyloid deposits in nerve.     

Zeroing in on amyloid proteins in Alzheimer disease therapy

The authors have provided us with a complete review of the approaches to the amyloid proteins of Alzheimer's disease in regards to targets for drug therapy. Sufficient information is now available concerning systemic amyloidogenesis, genes for familial Alzheimer's disease and the beta amyloid precursor protein, as well as the posttranslational processing requirements for amyloidogenesis and its prevention. Recent excitement concerning the roles for serine proteases and their inhibitors, both in the production and prevention of amyloidogenesis, make this review and its publication in the Neurobiology of Aging extremely timely.     

Histochemical analysis of senile plaque amyloid and amyloid angiopathy

Summary Histochemical methods were used to obtain information on the chemical constituents of brain amyloid in senile dementia of the Alzheimer type. The staining properties of brain amyloid (senile plaque and amyloid angiopathy) were compared with those of extraneural amyloidosis and endocrine amyloid. We found no histochemical differences between amyloid in senile plaques and in amyloid angiopathy. The content of aromatic amino acids was higher in amyloid of plaques and in amyloid angiopathy than in endocrine amyloid. Furthermore, we found persistent birefringence and affinity of brain amyloid for Congo red after exposure to potassium permanganate, suggesting that AA amyloid is not a major constituent of cerebral amyloid.     

Diagnosis of the type of amyloid in paraffin wax embedded tissue sections using antisera against human and animal amyloid proteins

Summary Different histochemical techniques were compared on paraffin wax embedded tissue sections for routine classification of amyloid; the following methods were used: potassium permanganate, the indirect immunoperoxidase method using polyclonal anti-human amyloid antisera (anti-AA, anti-A, anti-A and anti-AF) and the peroxidase-antiperoxidase (PAP) method using antisera against human, bovine, hamster and canine AA amyloid. Anti-human AA antiserum appeared to be a useful tool in this respect. Polyclonal anti-AL antisera may be helpful in diagnosing AL amyloid, but were of less value than anti-AA serum.Strong cross reactivity between anti-bovine AA antiserum and human AA amyloid deposits was found. This indicates that animal amyloid AA antisera can also be used for the diagnosis of AA amyloid in human tissues.This study was funded by the Division of Rheumatology, A.Z.U., the Netherlands League against Rheumatism and Sonderforschungsbereich 0207 (Project C6), München     

Involvement of amyloid proteins in the formation of biofilms in the pathogenic yeast Candida albicans

Candida species represent a major fungal threat for human health. Within the Candida genus, the yeast Candida albicans is the most frequently incriminated species during episodes of candidiasis or candidemia. Biofilm formation is used by C. albicans to produce a microbial community that is important in an infectious context. The cell wall, the most superficial cellular compartment, is of paramount importance regarding the establishment of biofilms. C. albicans cell wall contains proteins with amyloid properties that are necessary for biofilm formation due to their adhesion properties. This review focuses on these amyloid proteins during biofilm formation in the yeast C. albicans.     

Immunochemical microanalysis of amyloid proteins in fine-needle aspirates of abdominal fat.

Diagnosis of systemic amyloidosis by fine-needle aspiration biopsy of abdominal fat is well established. A complete diagnosis now must include determination of the chemical type of amyloid. Microextracts of amyloid proteins from 11 Congo red-positive aspirate samples were analyzed with immunochemical methods. There was correspondence of the results obtained by immunohistologic and Western blotting analyses in 3 of 4 specimens with kappa light chain amyloid, 5 of 6 with lambda amyloid, and 1 with amyloid A. The method provides rapid and reliable diagnostic information necessary for classification of the chemical type of amyloid required for initiation of specific modes of therapy, with little discomfort to the patient.     

Tissue transglutaminase colocalizes with extracellular matrix proteins in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a key histopathological hallmark of Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). CAA is characterized by amyloid-beta (Aβ) depositions and remodeling of the extracellular matrix (ECM) in brain vessels and plays an important role in the development and progression of both AD and HCHWA-D. Tissue transglutaminase (tTG) modulates the ECM by molecular cross-linking of ECM proteins. Here, we investigated the distribution pattern, cellular source, and activity of tTG in CAA in control, AD, and HCHWA-D cases. We observed increased tTG immunoreactivity and colocalization with Aβ in the vessel wall in early stage CAA, whereas in later CAA stages, tTG and its cross-links were present in halos enclosing the Aβ deposition. In CAA, tTG and its cross-links at the abluminal side of the vessel were demonstrated to be either of astrocytic origin in parenchymal vessels, of fibroblastic origin in leptomeningeal vessels, and of endothelial origin at the luminal side of the deposited Aβ. Furthermore, the ECM proteins fibronectin and laminin colocalized with the tTG-positive halos surrounding the deposited Aβ in CAA. However, we observed that in situ tTG activity was present throughout the vessel wall in late stage CAA. Together, our data suggest that tTG and its activity might play a differential role in the development and progression of CAA, possibly evolving from direct modulation of Aβ aggregation to cross-linking of ECM proteins resulting in ECM restructuring.     

Kinetic analysis of amyloid fibril polymerization in vitro

We investigated the polymerization kinetics of murine senile amyloid fibrils (fASSAM) in vitro. When sonicated murine senile amyloid fibrils was incubated with its constituent monomer protein, the extension of amyloid fibrils was observed in an electron microscopic analysis. Quantitative fluorometric analysis with thioflavine T (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989) revealed that (a) extension of amyloid fibrils occurred by a pseudo-first-order exponential increase in the fluorescence of thioflavine T; (b) the rate of extension was maximal around pH 7.5, and was inhibited with the increase in KCl or NaCl concentration in the reaction mixture; (c) the rate of polymerization was proportional to the product of the murine senile amyloid fibrils number concentration and the constituent monomer protein concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization with the equilibrium association constant K of 5 x 10(7) M-1. These results show that amyloid fibril formation can apparently be explained by a first-order kinetic model: that is, extension of amyloid fibrils proceeds by consecutive association of precursor proteins onto the ends of existing fibrils.     

Cleavage of AL amyloid proteins and AL amyloid deposits by cathepsins B, K, and L

Cathepsin (Cath) B, CathK and CathL are cysteine proteases that participate in the lysosomal protein degradation system and are expressed in macrophages, epithelioid cells, and multinucleated histiocytic giant cells (MGCs). Both macrophages and MGCs are commonly found adjacent to immunoglobulin light chain-associated (AL) amyloid deposits, which raised the question of whether cysteine proteases are able to cleave AL amyloid proteins and AL amyloid deposits. The present study has investigated whether recombinant human CathB, CathK, and CathL are able to degrade AL(VlambdaVI) amyloid proteins and AL amyloid deposits. Using immunohistochemistry, CathB, CathK, and CathL were found adjacent to AL amyloid deposits. In vitro degradation experiments using purified AL amyloid proteins showed that CathB, CathK, and CathL degrade AL(VlambdaVI) amyloid proteins. Furthermore, using unfixed tissue sections from an amyloidotic spleen as an in vitro model for extracellular proteolysis of intact amyloid deposits, it was demonstrated that all three cysteine proteases are also capable of degrading AL amyloid in situ. This is the first study to show that cysteine proteases are able to cleave AL amyloid proteins. However, the efficiency with which proteolysis occurs depends on the concentration of active protease recruited at the sites of amyloid deposition, and possibly on the structure of the AL amyloid proteins.     

Gastrointestinal beta2microglobulin amyloidosis in hemodialysis patients: biochemical analysis of amyloid proteins in small formalin-fixed paraffin-embedded tissue specimens.

We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. A new microtechnique was applied for extraction and immunochemical/chemical characterization of amyloid proteins in small amounts of fixed tissue, thus allowing precise identification of beta2microglobulin amyloid (Abeta2M) in both cases studied. The molecular mass of the identified amyloid beta2M was close to that of intact beta2M (12 kDa), with no evidence of the products of proteolytic fragmentation of these molecules. The isoelectrofocusing of the purified Abeta2M demonstrated a shift to more acidic pI as compared to the normal beta2M analyzed under the same experimental conditions. The obtained data suggest that the intestinal amyloid deposits in dialysis-related amyloidosis contain disease-specific beta2M isoforms, which could play a role in the pathogenesis of amyloid disease. The new methodology used might be useful in obtaining precise diagnosis of amyloidosis that is necessary for appropriate therapy, and also provide new important information on the chemical structure of amyloid proteins.     

Histochemical classification of systemic amyloid fibril proteins. Alkaline guanidine method

The alkaline guanidine method facilitates differentiation between different types of amyloid fibril proteins in formaldehyde-fixed, paraffin-embedded tissue sections. Systemic AA-type amyloids lost Congophilia (affinity of Congo red) after incubation with alkaline guanidine for one minute. Systemic AL-type amyloids lost or markedly decreased Congophilia after two hours of treatment with alkaline guanidine. Systemic prealbumin-type amyloids were resistant to incubation for two hours. On the other hand, some cerebral amyloid plaques from patients with Creutzfeldt-Jakob disease and Gerstmann-Str?ussler syndrome markedly decreased Congophilia, while in other amyloid plaques, Congophilia was not decreased even with two hours of treatment. The senile plaques from those patients with Alzheimer's disease did not diminish Congophilia after alkaline guanidine treatment. Thus, while this method does not differentiate types of cerebral amyloid protein, it does clearly differentiate types of systemic amyloid fibril proteins.     

The formation of bioactive amyloid species by prion proteins in vitro and in cells

Amyloid proteins are a group of proteins that can polymerize into cross beta-sheeted amyloid species. We have found that enhancing cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis is a common property of bioactive amyloid species formed from all of the amyloid proteins tested to date. In this report, we show that the infectious amyloid species of the prion protein HET-s of the filamentous fungus Podospora anserina, like other amyloidogenic proteins, also enhances MTT formazan exocytosis. More strikingly, cellular MTT formazan exocytosis revealed the formation of bioactive amyloid species in prion-infected mouse N2a neuroblastoma cells. These findings suggest that cellular MTT formazan exocytosis can be useful for studying the roles of bioactive amyloid species in prion infectivity and prion-induced neurodegeneration.     

Temporal accrual of complement proteins in amyloid plaques in Down's syndrome with Alzheimer's disease

The complement system constitutes a series of enzymatic steps involved in the inflammatory response and is activated in Alzheimer's disease (AD). Using Down's syndrome (DS) brains as a temporal model for the progression of AD, we examined components of the complement cascade and their relationship to other principal events in AD pathology: Abeta42 deposition, neuritic changes, neurofibrillary tangles (NFTs), and gliosis (reactive astrocytes, activated microglia). Adjacent sections of frontal cortex from 24 DS subjects ranging in age from 12 to 73 years were immunohistochemically examined for immunoreactivity (IR) of classical complement proteins (Clq and C3), markers indicating activation of complement (C4d and C5b-9), the complement inhibitor apolipoprotein J (apo J), and markers of AD neuropathology. Abeta42-labeled diffuse plaques were first detected in a 12-year-old DS subject and were not labeled by any of the complement antibodies. Colocalization of Abeta42 with Clq, C3, C4d, and/or apo J was first detected in compacted plaques in the brain of a 15-year-old DS patient with features of mature AD pathology, such as reactive astrocytes, activated microglia, dystrophic neurites, and a few NFTs. IR for C4d and C5b-9 (membrane attack complex, MAC) was observed in small numbers of plaque-associated dystrophic neurites and in focal regions of pyramidal neurons in this 15-year-old. The only other young (相似文献   

A synthetic amyloid lawn system for high-throughput analysis of amyloid toxicity and drug screening

Amyloid-beta (Abeta) is the major constituent of senile plaques in the brains of Alzheimer's disease patients. In order to develop an efficient in vitro system for studying the interaction of cells with Abeta aggregates, we have prepared a synthetic amyloid lawn by immobilizing Abeta peptides over a functionalized glass surface and subsequently incubating the template in a fresh Abeta solution. On the top of different types of amyloid lawns (e.g. monomeric, oligomeric, and fibrillar), we cultivated PC12 cells, creating physical contacts between the cells and the lawns. Results indicated that cell viability was differentially affected when grown atop different Abeta lawns while cells were well adhered onto the surface of these Abeta lawns. The mode of cell death by Abeta lawn was confirmed to be apoptotic rather than necrotic, showing that cells undergo suicide by just contact with Abeta lawn. While conventional 'solution-based' methods for testing amyloid toxicity suffer from problems such as lot-to-lot variations, continued fibrillation, and heterogeneous population of aggregates, our 'surface-based' lawn system is suitable for high-throughput analysis of amyloid toxicity, which may enable high-throughput screening of potential drug candidates for treating amyloid diseases with the goal of reducing the cell death on the lawn.     

Deposition of amyloid proteins in the epicardial coronary arteries of 58 patients with primary systemic amyloidosis.

INTRODUCTION: We sought to determine the distribution and the effect of amyloid on epicardial coronary arteries in patients with primary cardiac amyloidosis. METHODS: We reviewed pathologic specimens taken after autopsy or cardiac transplantation from 58 patients with primary cardiac amyloidosis. Patients were seen from 1981 to 2000. Multiple sections of epicardial coronary arteries (left anterior descending artery, left circumflex artery, and right coronary artery) were examined to determine the degree of amyloid deposition in the intima, media, adventitia, and vasa vasorum (vasa vasorum are nutrient arteries for the coronary arteries themselves). RESULTS: In 56 of 58 patients (97%), amyloid was present in epicardial coronary arteries. Amyloid was identified in all artery layers (intima, media, and adventitia), and more patients had amyloid in the adventitia. However, amyloid did not cause intraluminal obstruction of epicardial coronary arteries in any patient. The vasa vasorum had considerable deposits and, in many patients, were obstructed by amyloid. Patients with obstruction of the vasa vasorum were significantly more likely to have obstructive intramural coronary amyloidosis than patients without vasa vasorum obstruction (P=.002). CONCLUSIONS: The epicardial coronary arteries of patients with primary cardiac amyloidosis had extensive amyloid deposition. This deposition, however, did not lead to obstruction of epicardial coronary arteries and therefore did not contribute to ischemic syndromes observed in these patients. Obstruction of the vasa vasorum was associated with obstructive intramural coronary amyloidosis.     

Micropurification and two-dimensional polyacrylamide gel electrophoresis of immunoglobulins for studying the clonal diversity of antigen-specific antibodies.

Presented here is a simplified method for purifying antigen-specific antibodies and analysing them by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). For this procedure, specific immunoglobulins (Ig) are bound to resin beads coated with antigen and, in the presence of sodium dodecyl sulphate and dithioerythritol, loaded directly onto isoelectric focusing strips for 2D-PAGE analysis. This technique bypasses antigen-specific Ig elution and concentration and therefore minimises antibody loss and simplifies sample preparation. Because of its high sensitivity, this technique permits clonal analysis of samples containing small quantities of Ig. We studied Ig anti-tetanus toxoid (TT) and IgG in sera from several sources. The resulting oligoclonal Ig anti-TT patterns contrasted with the polyclonal patterns of total IgG visualised after 2D-PAGE analysis of the respective serum samples.