Ralph Heimer

TGF-β modulates the synthesis of proteoglycans by myocardial fibroblasts in culture

In this study we examined the production of proteoglycans by fibroblasts cultured from the left ventricular myocardium of normal adult rats. Various molecular species of proteoglycan were detected, either by labeling glycosaminoglycan chains with 35SO4 or by labeling the proteoglycan core protein with [35S]methionine. The medium of the cell cultures, which contained quantitatively most of the proteoglycans, appeared to consist mainly of biglycan, lesser amounts of decorin and proteoglycans of higher molecular weight. Biglycan and decorin were identified not only by the characteristic mobility of the intact protein and the core protein but also by immunolocation on Western blots. TGF-beta upregulated the synthesis of all these proteoglycans, coincident with elongation of glycosaminoglycan side chains observed for biglycan and decorin. The apparent molecular weight of the core protein of the two proteoglycans remained unaffected by TGF-beta. The results of these experiments suggest that with regard to proteoglycan synthesis and its regulation by TGF-beta, cultured fibroblasts originating from the myocardium share to a large extent the properties of cultured fibroblasts of other organs.

On the distribution of rheumatoid factors among the immunoglobulins.

Abstract Six rheumatoid sera were selected for studying the possible presence of rheumatoid factors belongings to all three major immunoglobulin classes. Indirect evidence by immunoelectrophoresis for IgA rheumatoid factors is presented for all six sera. A more detailed examination established the presence of IgA rheimatoid factors in some preparations. Indirect evidence for the existence of IgA rheumatoid factor in saliva is also presented. The IgA rheumatoid factors appear to be reactive in the latex fixation test as well as in tests utilizing rabbit and human incomplete Rh antibody. These rheumatoid factors appear to resemble other IgA antibodies in their molecular weight and sensitivity characteristics. IgG rheumatoid factors were demonstrated by isolation of the IgG fraction in two sera. The IgG rheumatoid factors were demonstrable by latex fixation test but not the other two tests used in this study.

Igm and igg anti‐f(ab )2 antibodies in rheumatoid arthritis and systemic lupus erythematosus

Radioimmunoassays for anti-F(ab )2 antibodies, which feature the use of goat anti-human Fc antibody for correcting potentiation of IgM anti-F(ab )2 antibody titers by endogenous IgM anti-Fc antibodies (rheumatoid factors), are described. Individuals with classic rheumatoid arthritis had significantly more IgM anti-F(ab )2 antibody (P < 0.001) and IgG anti-F(ab )2 antibody (P = 0.05) than did individuals with systemic lupus erythematosus or normal volunteers. There is some similarity in patterns of isotype distribution of anti-F(ab )2 antibodies and rheumatoid factors.

The hyperviscosity syndrome, polysynovitis, polymyositis and an unusual 13S serum IgG component

A patient is described in whom chronic polysynovitis, polymyositis and the hyperviscosity syndrome developed, and whose serum contained an unusual IgG that circulated mainly as a polyclonal 13S component, with rheumatoid factor-like activity. The 13S serum component, which at times represented more than 40 per cent of the serum total protein, was the major factor responsible for serum hyperviscosity. A striking correlation was observed among the following factors: intensity of the synovitis, intensity of the myositis, the concentration of serum IgG and 13S serum component concentration. Each seemed to be decreased by the administration of prednisone. The possible significance of these various interrelationships is discussed.

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.

Detection of glycosaminoglycans at the one-nanogram level by 125I-cytochrome c.

The basic protein cytochrome c forms stable ionic complexes with all known glycosaminoglycans. When labeled with 125I, cytochrome c is capable of detecting exceptionally small quantities of glycosaminoglycans. Subsequent to electrophoresis on cellulose acetate strips using pyridine formate buffer at pH 3, followed by ethanol fixation, and treatment with 125I-cytochrome c, all the known glycosaminoglycans are detected at minimum levels of 1 ng/0.25-microliter application. The method can be used for quantification of glycosaminoglycans in other electrophoretic buffer systems also.

A latex test for the detection of human IgG aggregates and IgG anti-IgG antibody

Abstract Latex particles (0·2 μ Lytron, Monsanto) were used for the measurement of certain types of human IgG. At pH 8·4 agglutination of latex particles occurred in the presence of purified 7 S IgG anti-IgG antibody and also of a 13 s IgG aggregate isolated from the serum of a patient with polymyositis, polyarthritis and hyperviscosity syndrome. A mixture of 10 S and 7 S pooled human IgG prepared by DEAE cellulose chromatography and Sephadex G-200 gel filtration was also reactive at certain restricted concentrations with latex particles. Agglutination of latex particles by 7 S IgG anti-IgG antibody and by the 13 S IgG aggregate was strongly inhibited when pooled IgG was present in a concentration of 20 μg/ml. Agglutination of latex particles was used for determining IgG aggregates and/or 7 S IgG anti-IgG antibody in human serum. Sera were treated with mercaptoethanol and diluted 30 fold. The solutions were exposed to an immunoadsorbent prepared from heated human IgG. The immunoadsorbent was eluted at pH 4·5, and the eluates were exposed to a suspension of latex particles not previously coated with human IgG. Preliminary screening tests show that serum IgG aggregates and 7 S IgG anti-IgG antibody and more prevalent in individuals with rheumatoid arthritis than in normal healthy adults.

Proteoglycan profiles obtained by electrophoresis and triple immunoblotting

Using synovial fluid of individuals with osteoarthritis as a prototypic biologic fluid containing one part proteoglycan per 100 to 1000 parts of other protein, profiles of proteoglycan were produced without preliminary purification through agarose-acrylamide gel electrophoresis, transfer to nitrocellulose, and triple immunoblotting. Chondroitin sulfate, keratan sulfate, and a hyaluronic acid binding region appear to be present on individual synovial fluid proteoglycans in variable amounts, and consequently a triple immunoblot using monoclonal antibodies to these three epitopes has the potential for developing a proteoglycan profile. The profile is assembled by means of densitometric scans of autoradiograms obtained after use of 125I-labeled anti-mouse immunoglobulin. By contrast to the profile of a relatively homogeneous proteoglycan purified from articular cartilage extracts, the proteoglycans of synovial fluid appeared to be quite heterogeneous with the bulk of keratan sulfate epitopes migrating ahead of the bulk of the chondroitin sulfate epitopes. Most of the proteoglycans appeared to possess a hyaluronate binding region.

The measurement of IgG anti-IgG antibody in rheumatoid arthritis☆

Abstract Serum IgG anti-IgG antibody was measured in patients with rheumatoid arthritis and osteo-arthritis and in healthy young adults. The antibody was isolated by immunoadsorption and evaluated by titration with latex particles. After establishing the methods specificity for IgG anti-IgG, its reproducibility and a means for converting titer to IgG concentration, IgG anti-IgG was found in greatest amounts in rheumatoid arthritis when compared with two other groups. The concentration of IgG anti-IgG, however, was consistently lower in our study than in studies by other workers. This discrepancy appears to be related to our procedure, namely dilution of serum prior to exposure to immunoadsorption, the latter tending to dissociate immune complexes (removal of IgG antigen from IgG anti-IgG antibody). The eluate obtained in this manner contains antibody IgG freed of IgG antigen, as manifested by mainly 7 S IgG, rather than 7 S IgG admixed with complexes of higher sedimentation coefficients. The method reported in this article, therefore, appears to measure IgG anti-IgG antibody with a greater degree of accuracy than previously reported techniques.

Glycosaminoglycans and Chondroitin/Dermatan Sulfate Proteoglycans in the Myocardium of a Non-Human Primate

Lyophilized mid-wall left ventricular myocardial tissues of the long-tailed non-human primate, Macaca fascicularis, were examined for the presence of glycosaminoglycans and chondroitin/dermatan sulfate proteoglycans. Mean uronic acid concentration in all samples was 0.97 +/- 0.27 micrograms per mg dry weight myocardium. The distribution of the glycosaminoglycans in the myocardium, determined by cellulose acetate strip electrophoresis was 62 +/- 4% heparan sulfate, 20 +/- 6% hyaluronan, and 16 +/- 5% chondroitin/dermatan sulfate. The analysis of chondroitin/dermatan sulfate proteoglycans, done directly on the extracts of lyophilized myocardium using agarose-acrylamide gel electrophoresis and Western blotting with monoclonal antibodies to various carbohydrate epitopes and with polyclonal antibodies to the protein core, showed the presence of biglycan and decorin. That these two and no other chondroitin/dermatan sulfates were present was established by core protein analysis using SDS PAGE and Western blotting. Quantification of chondroitin/dermatan sulfate proteoglycans uncovered high individual specific variability of the chondroitin/dermatan sulfate epitopes, but only moderate variability of biglycan and decorin core proteins. The variability of the chondroitin/dermatan sulfate epitopes is most likely related to individual specific differences in chain number, iduronate content and sulfation patterns of biglycan and decorin.