Isolation and identification by sequence analysis of experimentally induced guinea pig amyloid fibrils.

Amyloidosis was produced experimentally in guinea pigs by multiple casein injections. Amyloid fibrils were isolated and fractionated and a protein obtained that had an amino acid composition comparable with A protein, a unique nonimmunoglobulin constituent of secondary amyloid deposits. N-terminal sequence analysis demonstrated a sequence homologous with that of A proteins from human and monkey preparations but preceded by a 5-residue peptide which had an N-terminal histidine. A definite species specificity in A protein from human and guinea pig was identified on immunologic analysis.

Linkage of protection against amyloid fibril formation in the mouse to a single, autosomal dominant gene.

Inbred strains of mice provide a model for studies of the pathogenesis of amyloid A (AA) amyloidosis. All susceptible strains of mice described to date codominantly express two serum amyloid A (apoSAA) isoforms, apoSAA1 and apoSAA2, of which only apoSAA2 serves as a precursor for amyloid fibrils. In previous studies, we have shown that the CE/J strain, which produces a single, novel apoSAA isoform, apoSAACE/J, is amyloid resistant. In the present study amyloid-resistant CE/J females were mated with amyloid-susceptible CBA/J males to produce F1 hybrid offspring which were then backcrossed to the parental CBA/J mouse strain. Amyloid susceptibility was determined in 30 backcrossed mice 72 h after injection of murine amyloid enhancing factor and silver nitrate. ApoSAA isoforms in plasma were separated by isoelectric focusing gel electrophoresis and visualized after immunoblotting with anti-AA antiserum. Amyloid A fibrils in spleen homogenates were denatured by formic acid and AA protein was quantified by ELISA using anti-mouse apoSAA antibodies. Values < 5 apoSAA equivalent units were considered negative. 13 mice expressed an apoSAA1 and apoSAA2 doublet characteristic of CBA/J mice, whereas 17 mice, expressed the apoSAACE/J isoform codominantly with apoSAA1 and apoSAA2. The correlation of amyloid resistance to expression of the apoSAACE/J isoform was absolute (17/17 were negative; mean score 2.6 +/- 0.17 [standard error of the mean] apoSAA equivalent units) and the correlation between amyloid susceptibility and the expression of apoSAA2/apoSAA1 was also striking (12/13 were amyloid positive; mean score 47.9 +/- 9.0 [standard error of the mean] apoSAA equivalent units (P < 0.001). This is not significantly different from the 50% segregation of apoSAA phenotypes expected for linkage to a single gene. These results indicate that a single gene governs apoSAACE/J expression and thus confers protection against amyloid deposition even in the presence of apoSAA1 and apoSAA2 isoforms and show for the first time that resistance to AA amyloidosis is a dominant trait governed by a single gene.

Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice.

B10.RIII and B10.G mice were transferred from a diet of laboratory rodent chow to a standard diet in which all the fat (5% by weight) was supplied as either fish oil (17% eicosapentaenoic acid [EPA], 12% docosahexaenoic acid [DHA], 0% arachidonic acid [AA], and 2% linoleic acid) or corn oil (0% EPA, 0% DHA, 0% AA, and 65% linoleic acid). The fatty acid composition of the macrophage phospholipids from mice on the chow diet was similar to that of mice on a corn oil diet. Mice fed the fish oil diet for only 1 wk showed substantial increases in macrophage phospholipid levels of the omega-3 fatty acids (of total fatty acid 4% was EPA, 10% docosapentaenoic acid [DPA], and 10% DHA), and decreases in omega-6 fatty acids (12% was AA, 2% docosatetraenoic acid [DTA], and 4% linoleic acid) compared to corn oil-fed mice (0% EPA, 0% DPA, 6% DHA, 20% AA, 9% DTA, and 8% linoleic acid). After 5 wk this difference between the fish oil-fed and corn oil-fed mice was even more pronounced. Further small changes occurred at 5-9 wk. We studied the prostaglandin (PG) and thromboxane (TX) profile of macrophages prepared from mice fed the two diets just before being immunized with collagen. Irrespective of diet, macrophages prepared from female mice and incubated for 24 h had significantly more PG and TX in the medium than similarly prepared macrophages from male mice. The increased percentage of EPA and decreased percentage of AA in the phospholipids of the macrophages prepared from the fish oil-fed mice was reflected in a reduction in the amount of PGE2 and PGI2 in the medium relative to identically incubated macrophages prepared from corn oil-fed mice. When this same fish oil diet was fed to B10.RIII mice for 26 d before immunization with type II collagen, the time of onset of arthritis was increased, and the incidence and severity of arthritis was reduced compared to arthritis induced in corn oil-fed mice. The females, especially those on the fish oil diet, tended to have less arthritis than the males. These alterations in the fatty acid pool available for PG and leukotriene synthesis suggest a pivotal role for the macrophage and PG in the immune and/or inflammatory response to type II collagen.

Kinetics of serum amyloid protein A in casein-induced murine amyloidosis.

Serum amyloid protein A (SAA), the precursor of secondary amyloid protein, is elevated in chronic diseases which are associated with an increased incidence of amyloid. However, SAA is also elevated in acute bacterial and viral infections and somes forms of cancer. The murine model of casein-induced amyloidosis was studied to determine the relationship between SAA production and amyloid deposition. SAA levels measured by radioimmunoassay were found to be as high as 200 times the normal level in CBA/J mice receiving daily parenteral casein. After a single injection of casein the SAA level was elevated by 3h and peaked by 12-18 h. Similar levels were found in casein-treated A/J mice, a strain less susceptible to the induction of amyloid. Parenterally administered bovine serum albumin, which has low potential for amyloid induction, gave SAA levels in CBA/J and A/J mice comparable to casein treatment. These data show that, while SAA levels are elevated during chronic antigenic stimulation, there are other factors involved in amyloid formation. These factors may include alterations in the degradation of SAA by the reticuloendothelial system caused by substances such as casein. Nude (athymic) mice were shown to attain high levels of SAA after receiving casein parenterally. Therefore, thymus-derived lymphocytes are not necessary for the synthesis of SAA.

A unique amyloidogenic apolipoprotein serum amyloid A (apoSAA) isoform expressed by the amyloid resistant CE/J mouse strain exhibits higher affinity for macrophages than apoSAA1 and apoSAA2 expressed by amyloid susceptible CBA/J mice.

CBA/J and other inbred strains of mice that express the amyloidogenic apolipoprotein serum amyloid A (apoSAA) apoSAA2, together with apoSAA1, are susceptible to amyloid A (AA) amyloidosis, whereas CE/J mice that express a single unique isoform, apoSAACEJ, are resistant. Studies indicate that CBA/JxCE/J hybrid mice that express apoSAA2 in the presence of apoSAACEJ are protected from amyloidogenesis. To define a mechanism by which expression of apoSAACEJ may protect from AA formation in the presence of apoSAA2, binding of recombinant apoSAA (r-apoSAA) isoforms, validated by N-terminal sequencing, to a murine macrophage cell line was investigated. Maximal specific binding occurred after incubation of radiolabeled apoSAA with IC-21 macrophages (1x105 cells/ml) for 30 min at 4 degreesC. The binding of 125I-r-apoSAA1, 125I-r-apoSAA2 and 125I-r-apoSAACEJ was specific and saturable, with an affinity (Kd) of about 2.8, 3.2 and 1.3 nM, respectively, and approximately 2-4x106 sites per cell. Competitive binding experiments indicate apoSAACEJ binds with higher affinity to macrophages than does either apoSAA1 or apoSAA2. We suggest that greater cellular affinity of apoSAACEJ compared to apoSAA2 may contribute to protection from AA amyloid in certain CBA/JxCE/J hybrid mice by interfering with interaction of apoSAA2 by macrophages and hence either membrane associated or intracellular degradation.

The acute phase response in apolipoprotein A-1 knockout mice: apolipoprotein serum amyloid A and lipid distribution in plasma high density lipoproteins.

In plasma, the bulk of apoSAA, a positive acute phase reactant protein, is transported in high density lipoproteins (HDL), especially HDLH (apoA1-rich HDL). In this study we tested whether apoA1 deficiency would adversely affect apoSAA concentration and lipid distribution in mouse plasma lipoproteins. Acute phase response (APR) was induced in C57BL/6J (apoA1+/+) and apoA1-knockout mice (apoA1-/-) by a subcutaneous injection of silver nitrate. The APR increased cholesterol concentrations in LDL of apoA1-/- mice and apoA1+/+ mice in a like manner. In contrast to apoA1+/+ mice, concentrations of cholesterol, phospholipids and proteins in both HDLL (1.063

Diet, amyloid enhancing factor (AEF) and amyloidogenesis: an hypothesis.

At least two forms of amyloidosis, amyloid A (AA) and prion protein (PrP), can be transmitted by dietary ingestion of an agent(s) present in crude mammalian tissues. Although the incubation time for PrP or scrapie-induced diseases to develop in experimental animals extends over months or years, AA or secondary amyloidosis in mice is inducible within a week. In response to inflammatory stimuli we hypothesize that dietary factor(s) modulate the rate at which beta-pleated sheet fibrils accumulate in most forms of amyloidosis. The critical importance of precursor protein polymorphism, cell surface proteoglycans (PG), lipids and apolipoprotein metabolism has also been addressed in this hypothesis.

Fish oil fatty acids and experimental arthritis.

Maintenance of mice on dietary regimens containing fish oil decreases severity of collagen-induced arthritis. Macrophages from fish oil fed animals had decreased omega-6 and significant amounts of omega-3 polyunsaturated fatty acids in membrane phospholipids and produced significantly less prostaglandins than macrophages from corn oil fed animals. Gender differences in both prostaglandin production and susceptibility to arthritis were noted.

Current concepts in management of lupus nephritis.

Since prognosis seems to vary according to which of several possible types of disease is present, the first step is renal biopsy and histologic classification. Management options thereafter-both conventional (steroid therapy) and investigational (cytotoxic agents plus steroids, thymic hormone replacement, steroid "pulse" therapy)-are discussed in terms of recent clincal results and theoretical mechanisms of action.

New concepts in the pathogenesis of primary and secondary amyloid disease.

Despite the marked progress obtained in the structural and amino acid sequencing data of amyloid proteins our understanding of the cellular mechanisms causing the deposition of amyloid fibrils is still poor. Some of the questions about the cellular events leading to the synthesis of amyloid fibrils can be approached by evaluating the immune reactivity of animals that develop amyloid after repeated daily casein injections. Recent studies carried out in a mouse model indicate that macrophage activation associated with T-cell suppression and followed by B-cell proliferation appear to be responsible for the immunopathological abnormalities in both primary and secondary amyloid disease.

Amyloid resistance in A/J mice. Studies with a transfer model.

Amyloidosis was studied in CBA/J and A/J mice using a classic method of amyloid induction and a transfer model. A/J mice required over 3 times as many injections of azocasein to develop splenic amyloidosis as the CBA/J strain. Perifollicular cellular proliferation occurred after fewer injections in CBA/J mice. In vitro azocasein-stimulated DNA synthetic activity, assayed by incorporation of tritiated thymidine in spleen cell cultures, was greater in CBA/J than A/J mice. Both strains developed amyloidosis after three or four azocasein injections, following sublethal irradiation and intravenous administration of spleen homogenates from azocasein-treated CBA donors. Amyloidosis was accelerated by A/J spleen homogenates only when the donors were given a prolonged course of azocasein. Transfer amyloidosis could be induced in both strains when the irradiation step was eliminated, although the amount of perifollicular amyloid was less. These results demonstrate that the mechanism of amyloid resistance in A/J mice lies in the response to the inducing agent in the preamyloid phase of amyloidogenesis.

Amyloidosis in (CBA/J X A/J)F2 mice: correlation of amyloid resistance and low mitogenic response to concanavalin A.

A/J mice are highly resistant to induction of amyloidosis with serial injections of azocasein, compared to the CBA/J strain. (CBA X A) F2 hybrid mice were treated with azocasein, after spleen biopsy for assay of 3 immunologic functions differing in the parent strains. The proportions of amyloid susceptibility and resistance in the hybrid population conformed to the expectations for a single gene. Low responses to the T cell mitogen concanavalin A correlated with resistance to amyloid induction, whereas the response to lipopolysaccharide and the level of natural killer activity were independent of susceptibility to amyloidosis.