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We have previously reported the specific association of glycosaminoglycans (GAG) and proteoglycans (PG) with amyloid fibrils and characterized the polysaccharides directly extracted from amyloid-laden tissues. In the present study we further elucidate the association between purified amyloid fibrils and GAG/PG with special reference to those GAG/PG associated with amyloid P-component (AP) and the interactive role of Ca2+ ions. Amyloid fibrils were isolated from human hepatic AA amyloid employing water extraction with and without preceding removal of AP, an extrafibrillar protein component of all amyloids, using sodium citrate. GAG/PG co-isolated with the amyloid extracts, with and without AP, were isolated and characterized. Agarose-affinity chromatography of extracts containing AP was performed, and the GAG associated with this extrafibrillary protein were characterized as well. Several different GAG/PG populations were demonstrated in the various extracts. The abolition of calcium-dependent binding markedly influenced the amount of GAG/PG recovered in the fibril extracts, as well as the total amount of amyloid material obtained. Thus, it seems that calcium plays an important role in the association between the fibrils and the sugar moieties, and that a significant fraction of the GAG found in amyloid exhibits a Ca(2+)-dependent fibril-GAG interaction. No significant difference in the proportion between galactosaminoglycans and glucosamines was, however, disclosed when the two extraction protocols were compared, suggesting that no particular GAG species has a higher affinity for the fibrils themselves. Both dermatan/chondroitin sulphate and heparan sulphate identified in the present study exhibited a Ca(2+)-dependent interaction with AP, supporting previous findings. However, the amyloid-associated galactosaminoglycans found, especially the large PG appearing in small amounts, seemed to have a higher affinity for the extrafibrillar AP than the other GAG.

Serum IgE concentrations were elevated in thirty-seven out of sixty-seven patients (55%) with acute or chronic liver disease of widely differing aetiology. The mean IgE concentrations in these patients showed an eight-fold increase above that observed in control subjects. Increased IgE levels in patients with liver disease occurred in the absence of eosinophilia, clinical evidence of atopy or other known causes of IgE elevation. No IgE-containing plasma cells were detected in the liver biopsies from thirty-two of the sixty-seven patients tested. Peripheral blood T cells were significantly decreased from normal in the patients with liver disease, but no correlation emerged between serum IgE levels and absolute peripheral blood T-cell numbers. These findings emphasize the importance of liver disease as a significant cause of serum IgE elevation.

Amyloid fibrils obtained from a case (Hud) of localized, tumorous amyloidosis of the skin were investigated by immunological and biochemical methods including amino acid sequence studies. Evidence was obtained that a lambda immunoglobulin light chain protein, A lambda I, and differently sized N terminal fragments (mol. wt 14,000 and 10,000) of the same protein comprised a major component of the fibrils. The chemical nature of this protein suggested that this case of cutaneous amyloidosis belonged to the idiopathic form of amyloidosis, probably as a special type of systemic, idiopathic amyloid disease.

One-hundred and twenty-four patients with spondylarthropathies were studied for antibodies to the peptides from HLA-B27.1 and Klebsiella pneumoniae nitrogenase which share a QTDRED hexamer sequence. Of 60 male Norwegian ankylosing spondylitis (AS) patients 23.3% showed positive ELISA reactivity for B27.1 peptide compared with 4% of Norwegian male controls (P less than 0.10). This difference was not observed among patients and controls from New Mexico. All patients with anti-B27.1 antibody were HLA-B27+. Antibody to B27.1 peptide was present in 20% of normal female controls with at least one previous pregnancy. No female control without previous pregnancy showed positive anti-B27.1 peptide reactivity. Anti-Klebsiella peptide antibody was neither significantly elevated in AS nor correlated with anti-B27.1 peptide antibody. Significant migration inhibition by these peptides was not observed in AS or normal controls. The possible influence of epitope conformation, rather than sequence homology, in potentially cross-reacting determinants shared by bacterial antigens and human Class I molecules requires further study.

Cultures of non-elicited mouse peritoneal macrophages were used as a test system to study the effect of sera from patients with rheumatoid arthritis (RA) on intra-cellular cathepsin B activity of macrophages. Sequential sera obtained during and after pregnancy from six RA patients, and sera from six actively ill, non-pregnant RA patients were compared to six healthy female controls and 3rd trimester healthy women. Sera from actively ill RA patients (both pregnant and non-pregnant) caused macrophage cathepsin B levels significantly above normal controls, while intra-cellular activities of beta-glucuronidase and N-acetyl-glucosaminidase did not differ from the controls. A significant correlation between activity of RA and intracellular cathepsin B was found in pregnant patients. It is suggested that a factor (or factors) present in serum from patients with active RA causes a rise of intracellular cathepsin B in macrophages.

The amyloid related protein SAA was isolated from the serum lipoproteins of three patients with connective tissue disease, one of them having amyloidosis, and from a pool of normal control sera. The bulk of SAA (apoSAA) was complexed to high density lipoprotein (HDL), but significant amounts of apoSAA were also detected in the other lipoprotein fractions. Electrofocusing revealed five-six subspecies of SAA which were distributed in similar proportions in HDL and LDL. Three of these SAA subspecies made up almost all of the apoSAA present in HDL and LDL in the sera from the patients as well as in the control. A small portion of SAA not complexed to lipoproteins was isolated corresponding to a molecular weight higher than 200,000. No particular 'amyloid prone' SAA was found in serum from the patient with amyloidosis.

Analysis of purified amyloid fibrils by gel filtration, polyacrylamide gel electrophoresis in SDS and 8 M urea, and immunodiffusion and immunoelectrophoresis showed that, in addition to the specific amyloid proteins AA and AL, the amyloid preparations all contain a high molecular weight complex. The latter protein complex contains fibronectin, a component which reacts with a non-AA specificity of an antiserum to degraded AA amyloid fibrils (termed the 'B' specificity), and a high molecular weight component excluded by a Sepharose 2BCL column. Similar components were found in aqueous extracts of normal tissues prepared by an identical procedure, and these form aggregates of different size in non-dissociating conditions. It is suggested that amyloid fibrils are complexes of a variety of macromolecules in addition to the specific proteins AA and AL.

In a prospective study of pregnant patients with rheumatic disease and healthy pregnant women, the effect of pregnancy serum on human blood monocytes was investigated. Intracellular activity of the collagen degrading enzyme cathepsin B was found to be significantly depressed in monocyte cultures exposed to pregnancy and cord sera, independent of the presence or absence of an inflammatory state in the pregnant woman. Enzyme inhibition developed in a dose-dependent fashion over 3 days in culture. In contrast to cord and pregnancy sera, non-pregnant serum and serum from oral contraceptive users displayed enzyme stimulating activity. The response of monocytes to the stimulating agent carrageenan was unaffected by inhibitory sera. The nature of possible inhibitory factors in pregnancy and cord serum is discussed.

Mouse peritoneal macrophages were stimulated by sera from patients with active rheumatoid arthritis (RA) to increased intracellular cathepsin B activity. By gel filtration of three RA sera, the stimulatory activity was found in the IgG and to a lesser extent in the IgM containing fraction. The DEAE-cellulose purified IgG preparations of five additional RA patients stimulated intracellular cathepsin B activity significantly above IgG from healthy controls. IgG and IgM antibodies to macrophages were detected in sera from RA patients but not from controls by indirect immunofluorescence (IIF) technique. Pepsin F (ab')2 fragments of IgG from the RA patients also gave clearcut membrane fluorescent staining of the macrophages which demonstrated the antibody nature of the binding. A good correlation between the cathepsin B assay and the IIF was found when serial dilutions of serum were compared.

In this study of protein composition of amyloid fibrils isolated from eight patients representative of the spectrum of amyloidosis found in Papua New Guinea has been investigated. All fibril preparations, including three from patients with amyloidosis secondary to lepromatous leprosy and one from an unusual juvenile case of primary amyloidosis, contained the non-immunogobulin amyloid protein, protein AA. However, only 44% of thirty-six amyloid patients had detectable levels of the protein AA-related serum component, protein SAA. Alkali-degraded material from each of the fibril preparations failed to react in double immunodiffusion test with antiserum to the amyloid-related light chain VgammaV, but evidence was found for this immunoglobulin light chain-specificity in the serum of one patient.

Human amyloid-related protein SAA has been prepared and purified by gel filtration, ion-exchange and affinity chromatography techniques. It was shown that SAA, even after extensive purification, is an electrophoretically heterogeneous protein. In addition, prealbumin and fragments of albumin were detected in the SAA preparation. Most of the SAA molecules and the fragments of albumin were present in a free form, but some SAA was also found to be complexed with albumin fragments.

Frozen sections of kidney tissue from 12 patients with systemic lupus erythematosus were examined for the expression of oncogene proteins. Polyclonal rabbit antibody to c-myc peptide or to whole 65,000 D c-myc protein, was used to identify c-myc protein within tissue sections. Patterns of nuclear staining in the Hep-2 cell line were similar to those seen with monospecific human SLE sera showing anti-Sm reactivity. In addition c-myc staining was not abolished by prior incubation of tissue sections with human serum containing anti-Sm antibodies. Five of 12 kidney biopsy tissues from patients with systemic lupus erythematosus (SLE) showed positive speckled c-myc protein staining within nuclei of monocyte/macrophage cells of glomerular tufts. Positive staining was in all instances completely abolished by prior absorption of anti-c-myc antibody with c-myc protein. No c-myc protein was identified within SLE immune complex deposits.

Amyloid obtained from the myocardium of a patient (Han) with familial amyloid cardiomyopathy of Danish origin was studied. Gel filtration and electrophoresis of purified and denatured amyloid fibrils Han revealed various fractions ranging in mol. wt from 40,000 to 8,000 daltons. Amyloid Han and fractions reacted with an antiserum against amyloid Han showing a reaction of identity with each other; partial identity between Han and human pre-albumin was observed, while no reaction was seen with AA or AL proteins. Cardiac tissue sections from Han showed reactivity with antisera to amyloid Han, pre-albumin and protein AP, but not with anti-AA or anti-AL in indirect immunofluorescence. Amino acid composition and sequence studies of a protein fraction of amyloid Han with mol. wt 15,000 daltons confirmed the structural relationship with pre-albumin.

Problems concerning isolation and characterization of the amyloid related serum protein SAA in a pure form prompted us to make monoclonal antibodies to the protein. Protein SAA isolated by gel filtration under dissociating conditions was used for immunization of BALB/c mice, and spleen cells from a mouse producing high titred antiserum to SAA were fused with cells from the mouse plasmacytoma line P3U1. Antibody specificity to various preparations of protein SAA was tested using an indirect enzyme-linked immunosorbent assay. Monoclonal antibodies with specificity for SAA were obtained in addition to antibodies which reacted with both SAA and the related amyloid protein AA. Antibodies specific for one of the apoC proteins of the lipoprotein fraction were also produced showing that the SAA preparation used for immunization was contaminated with apoC proteins.