sepharose and Arthritis--Rheumatoid

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

Topics: Arthritis, Rheumatoid; Calorimetry, Differential Scanning; Circular Dichroism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin G; Inflammation; Light; Microscopy, Fluorescence; Oxygen; Peroxynitrous Acid; Protein Structure, Secondary; Scattering, Radiation; Sepharose; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Temperature; Tryptophan; Tyrosine

Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.

Topics: Arthritis, Rheumatoid; Autoantibodies; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Magnetics; Reproducibility of Results; Sepharose; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly destroys cartilages or bones at the joints. This inflammatory disorder is initiated by self-attack using own immune system, but the detail of pathological mechanism is unclear. Features of autoantigens leading to autoimmune disease are also under veil although several candidates including type II collagen have been suggested to play a role in pathogenesis. In this report, we tried to identify proteins responding to antibodies purified from RA patients and screen proteins up-regulated or down-regulated in RA using proteomic approach. Fibronectin, semaphorin 7A precursor, growth factor binding protein 7 (GRB7), and immunoglobulin mu chain were specifically associated with antibodies isolated from RA synovial fluids. In addition, some metabolic proteins such as adipocyte fatty acid binding protein, galectin-1 and apolipoprotein A1 precursor were overexpressed in RA synovium. Also, expression of peroxiredoxin 2 was up-regulated in RA. On the contrary, expression of vimentin was severely suppressed in RA synoviocytes. Such findings might give some insights into understanding of pathological mechanism in RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Autoantigens; Collagen Type II; Female; Gene Expression Regulation; Humans; Inflammation; Male; Middle Aged; Proteomics; Sepharose; Synovial Fluid

The role of anti-IgE autoantibodies in IgE-related allergic diseases has not been elucidated sufficiently. For example, anti-IgE antibodies have been reported to cause both proallergic and antiallergic blocking reactions. Contrary to other authors, some authors revealed a positive correlation between total IgE and the amount of IgE/IgG complexes detected. By comparing the IgE levels of allergic patients with those of control persons and rheumatoid arthritis patients the present study contributes to our understanding of the role of anti-IgE autoantibodies. The sera were tested by means of ELISA concerning their content of free and complexed IgG and IgM anti-IgE. In addition, the amounts of IgE/IgG and IgE/IgM complexes were determined in the sera of allergic and control persons after Superose 6 column separation. We found that the allergic patients revealed significantly higher values of free IgM anti-IgE than patients with rheumatoid arthritis and than control persons (mean 0.470, p = 0.0164 and p = 0.0061, respectively). The corresponding values for IgE/IgM complexes also tend to be higher (mean 0.431, p = 0.0784 and p = 0.0601, respectively). However, the corresponding contents of IgG anti-IgE autoantibodies and IgE/IgG complexes did not differ significantly. After Superose 6 column separation, we detected IgE/IgG in fractions corresponding to MW of 150 to 180 kDa for the sera of both allergic and control persons. In contrast, the IgE/IgM complexes were found in fractions corresponding to MW 330 kDa. We conclude that the increased IgM anti-IgE autoantibody titer in the sera of allergic patients is not correlated with the high total IgE level. Moreover, we suggest that the IgE/IgG complexes form de nova during ELISA. Unlike the IgE/IgG complexes, the IgE/IgM complexes are assumed to occur already in circulating blood.

Topics: Adult; Antibodies, Anti-Idiotypic; Antibody Specificity; Arthritis, Rheumatoid; Biomarkers; Chromatography, Gel; Enzyme-Linked Immunosorbent Assay; Female; Germany; Humans; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Male; Middle Aged; Sepharose; Time Factors

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.

Topics: Adsorption; Arthritis, Rheumatoid; Cellulose; DNA; Humans; Immunosorbents; Lupus Erythematosus, Systemic; Particle Size; Sepharose

Topics: Adult; Aged; Arthritis, Rheumatoid; Carcinoma, Hepatocellular; Cell Line; Chromatography, Affinity; Concanavalin A; Female; Humans; Liver Neoplasms; Male; Orosomucoid; Protein Binding; Sepharose; Tumor Cells, Cultured

Rheumatoid arthritis patients were found to have CD4+ T cells that proliferate in response to autologous synovial fluid and plasma. T cell clones and polyclonal T cell lines were found to respond to antigen(s) eluted from protein A Sepharose and anti-human immunoglobulin (Ig) antibody Sepharose. The antigen(s) was further resolved to fractions that contained intact Ig or Ig heavy chain since the T cells responded to > 100 kDa and 40-60 kDa polypeptides derived from purified Ig under nonreducing and reducing conditions, respectively. These results indicated that the antigen(s) is either Ig heavy chain or Ig-binding proteins that copurify with Ig and Ig subunits. Pepsin and papain digestion of the antigenic fractions eluted from protein A destroyed the T cell reactivity. Since most Fab regions are resistant to these enzymes, further analyses are required to localize the antigenic epitope(s). The presence of Ig- or Ig-antigen complex-reactive T cells in arthritic joints implies that B cells expressing anti-Ig antibody (i.e. rheumatoid factor) may play an important role in antigen presentation to autoreactive T cells.

Topics: Antibodies, Anti-Idiotypic; Antigen Presentation; Arthritis, Rheumatoid; Autoantibodies; Carrier Proteins; HLA-DR Antigens; Humans; Immunoglobulin Heavy Chains; Knee Joint; Lymphocyte Activation; Receptors, Antigen, T-Cell, alpha-beta; Sepharose; Synovial Fluid; T-Lymphocytes

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.

Topics: Adolescent; Adult; Animals; Arthritis, Juvenile; Arthritis, Rheumatoid; Blood Physiological Phenomena; Cartilage, Articular; Cattle; Cell Division; Cells, Cultured; Culture Media; Extracellular Matrix; Humans; Reference Values; Sepharose; Sulfates; Synovial Fluid

Agarose-poly-L-lysine (Ag-(lys)n-DNA) has been used to bind DNA for assay of anti-DNA antibodies (ab). In this work, an algorithmic approach has been used to classify antinuclear ab (ANA) as being directed against native DNA (dsDNA), denatured DNA (ssDNA), DNA-protein complexes (deoxyribonucleoprotein; DNP), and against antigens which are independent of DNA (iDNA). These ab were subjected to Ag-(lys)n-DNA, and the selectivity of this adsorbent for the various specificities of ab was determined. The DNA on the columns was left untreated or treated with S1 nuclease, this being effected either by treating the DNA prior to introducing it onto the columns or by adding S1 nuclease to the columns after the DNA was bound. Ag-(lys)n-DNA adsorbs ab directed against ssDNA and DNP as well as ab to dsDNA; iDNA ab are not adsorbed. S1 nuclease treatment does not effectively remove ssDNA regions from the Ag-(lys)n-DNA, but it does result in the abolition of the adsorption of a population of ab which are in the anti-DNP sera and contribute to the total ANA load. While anti-iDNA ab are not adsorbed onto the columns, they do contribute to the ANA titer, unlike anti-ssDNA ab which are adsorbed onto the Ag-(lys)n-DNA but do not contribute to the ANA titer. We conclude that Ag-(lys)n-DNA bears antigenic sites for dsDNA, ssDNA, and DNP ab and suggest that our understanding of the characteristic ab-binding profile of this versatile immunoadsorbent may have applications in the study of autoimmune diseases.

Topics: Antibodies, Antinuclear; Arthritis, Rheumatoid; DNA, Single-Stranded; Epitopes; Humans; Immunosorbent Techniques; Lupus Erythematosus, Systemic; Mixed Connective Tissue Disease; Polylysine; Sepharose

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Chemical Precipitation; Chromatography, Affinity; Complement Activating Enzymes; Complement C1q; Complement C3; Complement C3c; Complement System Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Immunoelectrophoresis; Immunoglobulins; Polyethylene Glycols; Sepharose

The solid-phase C1q binding assay for circulating immune complexes has been evaluated. The assay provides a rapid, sensitive (detecting as little as 1 microgram of aggregated IgG) and reproducible procedure for the detection of immune complexes in biological fluids. Using artificially prepared immune complexes, the assay detects complexes at four-times antigen-excess. Gel filtration over Sepharose 6B showed that these complexes were distributed over a range of molecular weights from greater than 4 x 10(6) to 300,000 daltons. Using radiolabelled anti-BSA, antigen (BSA) could be detected in these complexes. Screening of gel-filtered SLE showed that the assay detects complexes of both high and low molecular weight, but does not detect all complexes in the SLE sera. Clinical studies showed that immune complexes are frequently found in the sera of patients with SLE and measurement of the concentrations of complexes provides a more sensitive index of disease activity than either serum C3 or C4 concentrations or DNA binding capacity. In patients with RA concentrations of immune complexes were generally higher in synovial fluid than serum, although a patient with systemic rheumatoid disease with hypocomplementaemia had an extremely high level of circulating immune complexes. The assay only infrequently detects circulating immune complexes in glomerulonephritis and in renal transplant recipients. It is concluded that the assay provides a useful clinical tool, but detects only a limited species of immune complexes. It can be used in the detection of antigens in complexes.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Chromatography, Gel; Complement C1q; Complement System Proteins; DNA; Glomerulonephritis; Humans; Immunoassay; Kidney Transplantation; Lupus Erythematosus, Systemic; Molecular Weight; Reproducibility of Results; Sensitivity and Specificity; Sepharose; Serum Albumin, Bovine

Cytotoxic antibodies to B lymphocytes have been shown to be IgM. They are not absorbed by red blood cells and therefore not absorbed by red blood cells and therefore not directed against the I antigen of red cells. They are also not inhibited by mannose, as are certain natural cytotoxins against lymphocytes. Methods for producing purified eluates of IgM anti-IgM antibodies are given. These antibodies are postulated to be immunoregulative by acting on B lymphocytes.

Topics: Absorption; Antilymphocyte Serum; Arthritis, Rheumatoid; B-Lymphocytes; Binding Sites, Antibody; Cold Temperature; Humans; I Blood-Group System; Immunoglobulin G; Immunoglobulin M; Rheumatoid Factor; Sepharose; Serum Albumin

The applicability of affinity chromatography to the isolation of C1q-binding immune complexes (IC) in sera was explored. Purified human C1q was covalently coupled to agarose or adsorbed to IgG-agarose resins. Sera containing preformed virus-antibody complexes or rheumatoid arthritis (RA) sera were passed through the columns and C1q-bound IC, eluted off with 1,4-diaminobutan at mild basic conditions, were analysed by immunodiffusion, crossed immunoelectrophoresis, gel filtration and electron microscopy. Under conditions of antibody treatment which caused almost 100% inhibition of virus plaque formation, about 30% of formed 14C-labelled equine arteritis virus-antibody complexes was bound specifically to and desorbed from C1q-IgG agarose columns. Studies with RA-sera indicated the presence of both IgM-IgI and intermediate size IgG, C1q-binding, complexes in 3 out of 5 tested seropositive sera. In two sera only intermediate size IC were demonstrable. The results obtained in these two IC model systems suggested that the described methods could be useful for isolation of C1q-binding IC in general.

Topics: Antibodies, Viral; Antigen-Antibody Complex; Arthritis, Rheumatoid; Binding Sites, Antibody; Chromatography, Affinity; Complement C1; Complement System Proteins; Diamines; Humans; Immunoglobulin G; Immunoglobulin M; Immunologic Techniques; Protein Binding; Resins, Plant; Rheumatoid Factor; Sepharose