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PubMed1985

A review of light, polarization and fluorescence microscopic methods for amyloid.

Traditional technics for amyloid are not always dependable. Therefore, several reactions, which do not require differentiation, were developed in this laboratory. This review describes technics that proved suitable for diagnostic pathology, including polarization and fluorescence microscopy. Effects of fixation on the reactivity of amyloid are also considered. The chemical mechanism of the alkaline Congo red, Mesitol WLS-Congo red and Phorwhite BBU reaction, and of a modified thioflavine T stain are reviewed briefly. Problems encountered with other methods, which cannot be recommended for diagnostic pathology, are outlined.

Amyloid↗
PubMed1988

[Bullous amyloidosis].

The patient, a 75-year old man, was admitted in May, 1986 for separation of the epidermis and extensive ecchymotic patches. Physical examination showed numerous haemorrhagic erosions on the extensor aspect of the limbs, feet and hands, and wide patches of epidermal separation in the axillary and dorsal regions. Ecchymotic purpura was present on the limbs, abdominal wall, neck and right orbital region. Nikolsky's sign was positive at the periphery of the lesions. Epidermal cysts, 1 to 5 mm in diameter, were visible on the back of the hands and on the upper part of the neck. There was no macroglossia. Several biopsies were performed in both diseased and healthy skin. Light microscopy of the diseased skin showed, at the junction of the papillary and middle dermis, a band of eosinophilic deposit in which were true intradermal bullae containing red cells. Congo red and thioflavine T stainings were positive, forming a dermal band. At direct immunofluorescence IgG, IgA, IgM as well as the C3 and C9 components of complement were absent. At electron microscopy there was no bullous separation at the dermoepidermal junction; the dermal deposits had a dense amyloid-like fibrillar structure without ramifications. Laboratory examinations showed lambda-2 monoclonal gammopathy with normal levels of IgG and IgA and slightly decreased IgM. Bence-Jones protein was found in the ruin. Bone marrow examination showed 8 p. 100 plasmocytes. The diagnosis was: non myelomatous lambda-2 monoclonal dysglobulinaemia. Amyloid deposits were found in biopsies of the gums and rectum. Other investigations gave negative results. Bullous lesions have been reported in about 20 cases of primary amyloidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Bis azo dye liquid crystalline micelles as possible drug carriers in immunotargeting technique.

Lyotropic liquid crystal mesophases, represented in this work by Congo Red bis azo dye solution, were proposed as systems to carry drugs to immuno-selected targets. The possible use of Congo Red for this aim arises from its liquid crystalline features, enabling it to attach to immune complexes as polymolecular, ordered conglomerates and simultaneously to incorporate many organic compounds into its mesophase, largely independent of the water with a specific but adaptive molecular organization. Molecules with planar rigid structure, and/or large hydrophobic fragments, especially those with a positively charged group in the molecule, are found to be incorporated best. Rhodamine B, Rhodamine 6G and adriamycine, which have the assumed binding features, were tested as model compounds and were found to be readily engaged into Congo Red mesophase. The effect of hydrophobicity on ligand binding was evaluated following the incorporation of homologic 10, 12, 14, 16 carbon chain organic acids and the effect of charge using small mobile tandem molecules of pI differing by a few pH units (lysine-norleucine, tyrosine-tyramine). Positive charge seems to affect binding especially by influencing the organization of molecules and the shape of the micelle simultaneously. Congo Red immobilized to heat-aggregated immunoglobulins and antibodies in the immune complex was found to retain its binding ability, confirming its possible usefulness for drug transport.

Antibodies↗
PubMed2000

Two novel proteins, PopB, which has functional nuclear localization signals, and PopC, which has a large leucine-rich repeat domain, are secreted through the hrp-secretion apparatus of Ralstonia solanacearum.

The Ralstonia solanacearum hrp gene cluster codes for components of a type III secretion pathway necessary for the secretion of PopA1, a hypersensitive response-like elicitor protein. In the present study, we show that several other Hrp-secreted proteins can be detected by growing wild-type bacteria in minimal medium in the presence of Congo red. Two of these proteins, PopB and PopC, are encoded by genes located downstream of popA and constitute an operon with popA. popABC mutants retain the wild-type ability to cause disease in hosts and to elicit the hypersensitive response on non-hosts. Expression of the popABC operon is controlled by the hrpB regulatory gene and is induced upon co-culture with Arabidopsis cell suspensions. This plant cell-specific induction depends on PrhA, a putative receptor for plant specific signal(s). The transcription of the popABC operon is not modified by the addition of Congo red to the growth medium and the intracellular pools of PopB and PopC are very similar in the absence or presence of Congo red. Preliminary data suggest that Congo red stabilizes secreted proteins in the extracellular medium. PopB is a 173-amino-acid-basic protein that contains a functional bipartite nuclear localization signal. PopC is a 1024-amino-acid protein that carries 22 tandem leucine-rich repeats (LRR). The LRR domain of this protein forms a consensus that perfectly matches the predicted eukaryotic cytoplasmic LRR consensus. We propose that PopB and PopC may be translocated into plant cells via the Hrp pathway.

Amino Acid Motifs↗
PubMed1998

New chelate-forming polymer microspheres carrying dyes as chelators for iron overload.

Dye-incorporated [poly(EGDMA-HEMA)] microspheres were investigated as a new chelate-forming polymer for iron overload. Poly(EGDMA-HEMA) microspheres, in the size range of 150-200 microm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dye-ligands (i.e. Cibacron Blue F3GA, Alkali Blue 6B and Congo Red) were covalently incorporated to the microspheres. The maximum dye incorporations were 16.5 micromol Cibacron Blue F3GA g(-1), 23.7 micromol Alkali Blue 6B g(-1), and 14.5 micromol Congo Red g(-1). The maximum Fe(III) adsorptions on the dye-incorporated microspheres from aqueous solutions containing different amounts of Fe(III) ions were 51.0, 37.3, and 25.1 mg g(-1) for the Cibacron Blue F3GA, Alkali Blue 6B, and Congo Red carrying microspheres, respectively. The maximum Fe(III) adsorptions were observed at pH 4.0 in all cases. Fe(III) removal from human plasma was also investigated. The maximum adsorption capacities of Fe(III) ions from human plasma for Cibacron Blue F3GA, Alkali Blue 6B, and Congo Red, were of 12.0, 7.5, and 3.8 mg g(-1) polymer, respectively. It was observed that Fe(III) could be repeatedly adsorbed and desorbed without significant loss in adsorption capacity.

Adsorption↗
PubMed2006

Comparative study on reaction selectivity of azo dye decolorization by Pseudomonas luteola.

This study is to inspect how the variation of molecular structures and functional groups present in our model azo dyes (i.e., Congo red, Eriochrome black T (EBT), methyl orange, and methyl red) affects biodecolorization capability of Pseudomonas luteola. The most viable decolorization was found at pH 7-9 and the optimal cellular age for the most effective decolorization was 7 days after static incubation in dye-free cultures. In decolorization, the maximal absorption wavelength in UV-vis spectra for the different dye-containing cultures shifted from visible light range towards the ultraviolet visible range. Methyl red was not decolorized in contrast to methyl orange, Congo red, and Eriochrome black T. The sulfonic group para to azo bond (-N=N-) in methyl orange was a strong electron-withdrawing group through resonance to cause an enhancement of color removal to be easily biodecolorized. As a charged carboxyl group on methyl red is at ortho position (i.e., in the proximity) to azo bond, this led to a complete inhibition to decolorization. However, decolorization of Congo red and EBT in the absence of charged group (e.g., hydroxy or amino group) near azo bond was not completely repressed like methyl red. Thus, the presence of electron-withdrawing groups as the substituents on azo dyes enhanced decolorization capability for biodegradability. In addition, Monod kinetic model provided better predictions to all dye decolorization at initial short periods of time due to negligible intermediate formed at initial short time duration, but significant intermediate accumulation took place at longer period of time. In contrast, the decolorization performances of methyl orange at 400ppm and EBT at 230ppm were significantly less than those predicted from the Monod kinetic model likely due to accumulated intermediates exceeding the threshold levels for feedback inhibition.

Azo Compounds↗
PubMed1975

[The influence of dyes with special affinity to amyloid on the resorption of casein-induced amyloidosis in mice (author's transl)].

1. The influence of dyes with special affinity to amyloid on the resorption of experimental amyloidosis was studied in mice (A/J/Bom). 2. The amyloidosis was produced by injections of 5% casein over a period of seven weeks. After an interval of six weeks the animals received 0.5 ml of a 0.01% dye solution intraperitoneally (congo red, diamine green, Evans blue) or 0.9% NaCl (controls) every seventh day over a period of 22 weeks. 10, 17, and 22 weeks after the dye treatment was started 3-4 animals of each group were sacrificed and studied histologically. 3. In contrast to the in vitro findings of Shirahama et al. (1971), in vivo there is no acceleration of amyloid resorption by congo red. In the same way diamine green does not accelerate the resorption of amyloid. Evans blue produces a decrease in the amount of amyloid, but at the same time it causes wasting in the animals. Moreover, congo red and diamine green produce amyloidosis by themselves. 4. From our findings neither congo red nor the other amyloidaffine dyes studied, diamine green and Evans blue, are suitable for a therapy of amyloidosis.

Amyloid↗
PubMed1986

Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies.

Abdominal fat tissue aspirates from 12 patients with biopsy-proved amyloidosis were investigated by different morphologic techniques. By light microscopy, after staining of the fat tissue aspirates with Congo red and examination with a polarizing microscope, positive results were obtained in nine patients with amyloidosis, two of the three with primary (AL) amyloidosis and seven of the nine with secondary (AA) amyloidosis. By indirect immunofluorescence, using AA antiserum, positive results were obtained in five of the nine cases of AA amyloidosis (aspirates from these five patients were positive on Congo red staining). By electron microscopy, amyloid fibrils were observed in five cases of amyloidosis (two of the AL and three of the AA type, all positive on Congo red staining). Although amyloid was demonstrated less frequently by immunofluorescence and electron microscopy, perhaps because of the small numbers of fat particles examined, it seems that, with Congo red staining, abdominal fat tissue aspiration is a simple and sensitive method for the diagnosis of amyloidosis. Immunofluorescence studies allow discrimination between the different types of amyloidosis. The method could be used in patients in whom other types of tissue biopsy are not recommended because of risks of bleeding or other problems.

Adipose Tissue↗
PubMed2002

Egg yolk platelet proteins from Xenopus laevis are amyloidogenic.

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.

Amyloid↗
PubMed2002

AL amyloidosis is not present as an incidental finding in cutaneous biopsies of patients with multiple myeloma.

Multiple myeloma (MM) is a monoclonal B-cell neoplasm characterized by autonomous proliferation of immunoglobulin-secreting plasma cells that are capable of synthesizing amyloidogenic light chains resulting in AL amyloidosis. Clinically occult AL amyloid deposition may occur in up to 31% of patients with MM. The prognosis of combined amyloidosis and MM is improving with new therapeutic options. Thus it is imperative that patients with MM be screened for amyloidosis. Sixty-six consecutive skin biopsies from patients with MM and the diagnosis of graft vs. host disease (GVHD) were stained with Congo red and assessed for the presence of amyloid deposition. Twelve cases that had amyloid deposition in other tissue and had a cutaneous biopsy were also stained with Congo red and assessed for the presence of amyloid deposition. None of the 66 biopsies of GVHD, and none of the 12 cases that had documented amyloid deposition in other tissue showed evidence of amyloid deposition in the cutaneous biopsies. In the absence of specific cutaneous manifestations of amyloidosis, it is unlikely that amyloidogenic light chain deposition in the skin would be found. Type I collagen may appear similar to amyloid, both by light microscopy and fluorescence, after staining with Congo red. Thus care must be taken not to confuse type I collagen autofluorescence with positivity for amyloid when assessing skin biopsies stained with Congo red.

Amyloidosis↗
PubMed1998

Generalized AA-amyloidosis in Siberian tigers (Panthera tigris altaica) with predominant renal medullary amyloid deposition.

Generalized amyloidosis with predominant renal medullary amyloid deposition was found in four closely related Siberian tigers (Panthera tigris altaica) suffering from end stage kidney diseases. Only minimal to mild amounts of amyloid were deposited in various organs outside the kidneys with individually variable organ involvement. The Congo red staining affinity of amyloid deposits was sensitive to potassium permanganate oxidation. The deposits were further characterized as being of the amyloid-A (AA) type by immunohistochemistry using the mouse monoclonal antibody mc4 directed against a conserved region of the human AA-protein. A combination of immunohistochemistry and Congo red staining was much more sensitive for the diagnosis of amyloid deposits than Congo red staining alone. With this combination, even minimal amyloid deposits were detected that had been missed in the first reading using Congo-red-stained slides alone. Since no common primary cause was identified, the amyloidosis was classified as idiopathic generalized AA-amyloidosis with a potential familial predisposition.

Amyloidosis↗
PubMed1993

Bis azo dyes--studies on the mechanism of complex formation with IgG modulated by heating or antigen binding.

The mechanism of binding of azo dyes (bis azo) to immunoglobulin G of altered conformation, induced by heating or interaction with antigen was analysed in this work. Azo dyes: Congo Red, Evans Blue and Trypan Blue were selected for these studies. The molecules of Congo Red and Evans Blue associate readily in water and exist as polymolecular micellar species of liquid crystalline organization. Such organization of molecules appeared necessary for these dyes to interact with antibodies and to affect the formation of immune complex. It was proved by studying the properties of isomeric dyes Evans Blue and Trypan Blue, whose ability to form polymolecular conglomerates in water differs, being high for Evans Blue and low for Trypan Blue. The dyes seem to influence the formation of the immune complex generally by interacting with individual immunoglobulin molecules. The rate of exchange of dye molecules in conglomerate bound to protein varies and is the lowest for the portion of molecules which are engaged directly in the complex with protein. Electron microscopic studies also confirmed the polymolecular form of the dye in the dye-protein complex. The computational simulation of dye-dye and dye-protein interaction was performed in a model system. The micelle in the system was represented by three molecules of Congo Red. The peptide loop composed of amino acids 68-88 originating from VL IgG domain, was selected to represent protein. Amino acid side chains of this fragment were reduced to C beta. The best fitness was found for peptide chains of twisted beta conformation and independently optimized conformation of the dye in a form of "twisted ribbon" micelle. It was concluded that the IgG domains become accessible for penetration of the dye after being relaxed in the result of heating or interaction with the antigen.

Agglutination Tests↗
PubMed2002

Amyloid-like features of polyglutamine aggregates and their assembly kinetics.

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.

Amyloid beta-Peptides↗
PubMed2006

Albumin binds self-assembling dyes as specific polymolecular ligands.

Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

Animals↗
PubMed2005

Immunohistochemical identification of amyloid, using an anti-human serum amyloid P component (SAP) antibody, is possible in ruminants but not in dogs and cats.

Amyloidosis represents a heterogenous group of diseases that have in common the deposition of fibrils composed of proteins of beta-pleated sheet structure, a structure which can be specifically identified by histochemistry using the Congo red or similar stains. Amyloid consists primarily of the amyloid fibrils but also of the amyloid P component (AP). This component, which is identical with the serum counterpart (SAP), is found in all types of human amyloid, and immunohistochemical identification of AP has been proposed as an adjunct to the universal, type-independent diagnosis of human amyloidosis. In the present study of animal amyloidosis, we compared the amyloid-specific Congo red stain with an immunohistochemical protocol using an anti-human SAP antibody for the identification of amyloid in formalin fixed tissue samples. The species and types of amyloidoses investigated were: (i) seven cows, one yak (Bos grunniens), and one sheep affected with amyloidosis of presumed AA type, (ii) one dog with a pancreatic endocrine tumour producing amyloid of presumed AIAPP type, (iii) two cats with presumed AIAPP-amyloidosis of the islets of Langerhans, one cat with presumed AA-amyloidosis, and one cat with an amyloid-producing odontogenic tumour. Intense immunostaining co-localized with amyloid, identified by its congophilia and green birefringence, using a protocol without any antigen retrieval in each of the seven cows, the yak and the sheep. The method seemed more sensitive in the ruminants than the Congo red stain, but was unable to detect amyloid in the dog and the cats regardless of the application of various antigen retrieval protocols. However, specific identification of amyloid still rests on the Congo red method or similar histochemical techniques.

Amyloid↗
PubMed1987

Synovial fluid analysis for diagnosis of amyloid arthropathy.

To detect the presence of amyloid, we studied synovial fluid samples from 7 patients who had biopsy-proven amyloidosis and clinical arthropathy and from 98 patients who had other forms of arthritis. Centrifuged sediments from synovial fluids of all 7 patients with amyloid and clinical arthropathy were positive on staining with Congo red, whereas the 98 others were negative. Under polarized microscopy, the amyloid appeared as an amorphous material with strong Congo red uptake and typical apple-green birefringence. These findings indicate that analysis of synovial fluid sediments with Congo red may represent a simple, yet sensitive, test for the diagnosis of amyloid arthropathy.

Aged↗
PubMed1998

Primary localized amyloid tumor of the breast with osseous metaplasia.

A case of primary localized amyloid tumor of the breast is described. It is an extremely rare condition and has not been seen in literature in Japan. A 76-year-old woman visited a hospital because of a painless, hard mass of the right breast. A relatively well demarcated, calcified mass was excised under clinical diagnosis of fibroadenoma. Histologically, massive eosinophilic amorphous material was deposited in breast stroma. It was stained red-orange by Congo red and displayed apple-green birefringence under polarized light. The staining persisted after incubation with KMnO4 and immunolabeling by immunoglobulin chi-light chain antiserum, consistent with the amyloid of AL (Achi) type. Osseous metaplasia with bone marrow cavity, foreign body type giant cells in response to amyloid, and scattered plasma cell infiltration were also recognized. Osseous metaplasia in the breast amyloid tumor has been reported in only one case before. To date, the patient has not developed any clinical or laboratory evidence of systemic amyloidosis or multiple myeloma.

Aged↗
PubMed1990

[Renal involvement in amyloidosis: analysis of 14 cases].

UNLABELLED: 14 cases were investigated between 1963 to 1988. Age ranging from 25-66 (mean 48.2). 9 cases (64%) were over age 50. Diagnosis was established by renal biopsy or autopsy. 11 cases (79%) were primary. 3 cases (21%) were secondary with chronic infection. Besides renal involvement, primary amyloidosis principally affected the heart, gastrointestinal tract and tongue. Hepatosplenomegaly and anemia were found in secondary cases. Proteinuria was found in 14 cases. Nephrotic Syndrome (NS) found in 8 cases, both renal insufficiency and hypertension were 21% respectively. In 3 cases performed renal venography, 2 cases showed renal vein thrombosis. 9 cases (64%) showed kidneys were enlarged. Congo red test showed positive reaction were 100% in secondary and 50% in primary. By light microscopy stained with congo red there was a specific protein deposited in glomeruli and mesangium, by electron microscopy. There was fine amyloid fibrils. These findings are characteristic and to have diagnostic value. CONCLUSION: (1) Renal amyloidosis is not very rare in China. (2) Proteinuria and NS were major clinical manifestations. (3) Renal biopsy and autopsy have diagnostic value, congo red test does not prove the absence of primary amyloidosis.

Adult↗