The serum amyloid A stimulating factor (SAASF) in the hamster.

The acute phase SAA response was studied in hamsters. An SAA-stimulating factor (SAASF) was detected in the early acute phase blood plasma of hamsters which were subcutaneously injected with casein-LPS. The latter is routinely used in our laboratory for amyloid induction in hamsters. Acute (4 h) inflammatory exudates (greater than 80 per cent polymorphonuclear leukocytes) were produced by intraperitoneal injection with either casein-LPS, latex or Freund's incomplete adjuvant. Chronic inflammatory exudate macrophages (greater than 98 per cent) were elicited by intraperitoneal injection with Bacillus Calmette Guérin (BCG). Cells were stimulated in vitro with latex. SAASF was detected in the supernates and lysates of the acute exudate cells but not in those of the chronic peritoneal exudate macrophages. Lymphocyte activating factor (LAF), however, was evidently present in the latter samples, indicating that SAASF and LAF (IL-1) are functionally different substances in hamsters.

Immunohistochemical identification and crossreactions of amyloid-A fibril protein in man and eleven other species.

Antisera were prepared in rabbits, sheep or chicken against purified amyloid fibril protein AA from man, mouse, stone marten, dog, cow and hamster. These antisera were tested by immunodiffusion against all purified antigens and applied to tissue sections containing amyloid from man, mouse, hamster, guinea pig, rabbit, cat, dog, mink, stone marten, pine marten, cow and horse. The binding of the antibodies to amyloid in tissue sections was assessed by the indirect immunoperoxidase method. The strongest reactions in the immunodiffusion and immunohistochemical methods were found between amyloid deposits of members of a given species and an antibody raised against protein AA from the same species. In contrast to the lack of cross-reactivity in immunodiffusion (except in the mouse-man relationship), extensive cross-reactions were observed immunohistochemically in phylogenetically related species, e.g. between stone marten, pine marten and mink, or between hamster and mouse. However, cross-reactions were also observed in combinations such as man-mouse, man-dog, man-cat, mouse-horse, and dog-cow. In addition, individual antisera showed variations in immunohistochemical reactivity with amyloid deposits of different members of one given species. Moreover, antisera prepared in rabbits reacted more restrictedly than those prepared in sheep, while rabbit antisera against any AA-protein did not react with rabbit amyloid. Finally, the widest degree of cross-reactivity including almost all mammalian species investigated was observed with a chicken antiserum to human amyloid AA protein.

Amyloid A proteins in different species.

Amyloid A (AA) proteins were purified from canine and bovine idiopathic amyloid and casein-induced hamster amyloid. The molecular weights of these proteins were in the same range (8,000-10,000) as those reported for AA proteins from man and other animal species. The amino acid compositions were comparable as well. Antisera against human, bovine, canine and hamster protein AA were tested with the indirect immunoperoxidase antiperoxidase (PAP) method and the indirect immunofluorescence technique on amyloid-containing human, bovine, canine and hamster tissue sections. Antihuman protein AA serum did not cross-react with hamster protein AA. In all the other combinations, cross-reactions were observed. Because of the high similarity of these different AA proteins, the amyloid diseases in the species mentioned can serve as interchangeable models to investigate the pathogenesis of AA amyloidosis.

Immunohistochemical identification of generalized AA-amyloidosis in a mountain gazelle (Gazella gazella).

Generalized amyloidosis was diagnosed post-mortem in a mountain gazelle (Gazella gazella). To test whether the amyloid deposits consisted of amyloid-A fibril protein a series of monoclonal and polyclonal antibodies directed against amyloid-A fibril protein of different species was applied to formalin-fixed paraffin sections using the indirect immunoperoxidase technique. The immunohistochemical results showed a moderate cross-reaction of gazelle amyloid with human, murine, hamster, and canine amyloid-A fibril protein. A strong cross-reaction, however, was found with one of two monoclonal anti-human amyloid-A antibodies and with an antiserum against bovine amyloid-A fibril protein, the amyloid fibril protein of another ungulate. These results demonstrate the presence of amyloid-A fibril protein in the gazelle amyloid and illustrate the diagnostic value of cross-reacting anti-amyloid-A antibodies for the identification of amyloid-A-amyloidosis in species and in individuals in which amyloid has not yet been examined.

Amyloid-enhancing factor (AEF) in the pathogenesis of AA-amyloidosis in the hamster.

AA-amyloidosis was induced in hamsters receiving amyloid-enhancing factor (AEF) by daily subcutaneous injection with either an aged casein solution or casein supplemented with lipopolysaccharide (LPS). Both amyloid inducers gave similar results with respect to amyloid development in spleen, liver and kidneys and to serum amyloid A (SAA) concentrations and plasma cathepsin D activities. AEF was isolated from amyloid-containing tissue by the method described by Hol et al. (1985), and amyloid-enhancing material was also extracted from isolated hamster amyloid fibrils by intensive sonification. This fibril-derived amyloid-enhancing material lacked typical green birefringence after staining with Congo red and appeared as amorphous material on electron microscopy. AEF shortened the pre-amyloid phase for splenic and hepatic amyloid development and also the subsequent interval before renal amyloid deposition. This indicates that endogenous AEF, unlike passively transferred preformed AEF, is not distributed throughout the body and is probably generated at the site of amyloid deposition. Moreover, these results suggest that amyloid deposition in the kidneys, like that in the spleen and liver, involves an AEF-dependent pathway. Thus redistribution of amyloid is probably not an important cause of renal amyloid involvement. In addition to the reduction in the lag phase for splenic and hepatic amyloid deposition, AEF also speeds the changes in SAA concentration and plasma cathepsin D activity. This indicates that AEF accelerates rather than eliminates the pre-amyloid phase.

Amyloid enhancing factor in hamster.

An amyloid enhancing factor (AEF) was extracted from spleens and livers of casein-treated hamsters. It shortened the induction time of experimental amyloidosis in recipient hamsters from 14 days to 4 days. Sepharose-4B gel filtration resolved the extract into 4 different fractions, with molecular weights of: higher than IO(7) (Vo-fraction), about 280 000, 59 000 and 12 000 respectively. In all 4 fractions AEF was present, indicating that AEF is probably a low molecular weight substance that easily aggregates, or associates with other compounds present in the spleen extract. AEF was precipitable at 50% ammonium sulphate saturation. On anion exchange chromatography in PBS (pH 7.2) of this precipitate, AEF was found in the fraction eluting at high NaCl concentration. This fraction did not show any relation to hamster protein AA, IgG or albumin with double immunodiffusion. Ultraviolet absorption spectrophotometry indicated nucleotide-like material, suggesting AEF to be related to nucleoproteins.

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis.

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.

A second component in bovine AA amyloid fibrils not identical with protein AA is essential for AA amyloid fibrillogenesis.

Amyloid fibrils were isolated from the renal papillae and glomeruli of cows with spontaneous AA amyloidosis. The fibrils were solubilized by treatment with guanidine hydrochloride (Gu HCl) and subjected to gel filtration on Sephacryl S-200. Two other fractions were obtained beside the void volume and the AA fractions. Reaggregation studies were performed by dialysing the fractions, separately or in combinations, against Gu-HCl-free solutions. Protein AA alone (about 10 kd) appeared not to precipitate. The other fractions alone and the combinations of fractions tested formed precipitates. The precipitates containing all fractions (including protein AA) or protein AA plus a fraction containing a 19- and a 23-kd protein revealed congophilic green birefringent fibrillar material. Dialysis against acidic and calcium-containing solutions gave the best results. Amyloid fibril-like material was visible on electron microscopic examination. The amino acid composition of the 19 + 23-kd material appeared to be slightly different from protein AA and evidently unlike SAP. On immunofluorescence-absorbance studies the 19 + 23-kd material appeared evidently unlike protein AA and SAP. From these findings it is concluded that for spontaneous formation of AA amyloid fibrils other non-AA proteins are necessary.

Enhancement of amyloid induction by amyloid fibril fragments in hamster.

An extract (fibril amyloid-enhancing factor) prepared by sonication of a suspension of water-purified hamster AA-amyloid fibrils showed an amyloid-enhancing effect upon intraperitoneal injection into hamsters. The fibril amyloid-enhancing factor was identical to the original fibrils according to the results of infrared spectroscopy, gel filtration, gel electrophoresis, and Western blotting; although lyophilized samples of the extract did not show any green birefringence after staining with Congo red. From these results, it was concluded that fibril amyloid-enhancing factor represents small fragments of amyloid fibrils which enhance in vivo formation of amyloid fibrils.

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

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 lambda, anti-A kappa 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 less of 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.

Experimental amyloidosis in the hamster: correlation between hamster female protein levels and amyloid deposition.

Reactive systemic AA amyloidosis was induced in female, male and castrated male hamsters either by repeated injection of casein or by injection of amyloid enhancing factor (AEF) followed by casein. The circulating concentrations of serum amyloid A protein (SAA), the putative precursor of the AA amyloid fibril protein, and of female protein (FP), the pentraxin homologue of serum amyloid P component (SAP) of other species, were measured and correlated with the speed and extent of amyloid deposition. The SAA responses of the three groups of hamsters were indistinguishable in both experiments but, in confirmation of previous reports, castrated males had FP levels higher than those of control males though still lower than in females. No differences were seen between groups in amyloid induction by casein injection alone. However, in the accelerated model using AEF, amyloid deposition occurred sooner and was more extensive in both females and castrated males than in unoperated males. These results strengthen the association between SAP, of which FP is the hamster counterpart, and the pathogenesis of amyloidosis.