Idiotypic mimicry of a cell surface DNA receptor: evidence for anti-DNA antibodies being a subset of anti-anti-DNA receptor antibodies.

Anti-idiotypic anti-DNA antibodies (anti-anti-DNA) have previously been described in both patients with systemic lupus erythematosus and healthy individuals. Jerne's hypothesis predicts that such antibodies would bear a paratope reactive with non-sequence specific DNA binding proteins. Here we have explored the notion of a molecular mimicry between anti-anti-DNA antibodies and antibodies to a previously described 28-29 kD cell surface DNA binding molecule. It was shown that affinity purified anti-anti-DNA antibodies inhibit the binding of DNA to cells and that MoAb to the 28-29 kD receptor react with anti-DNA antibodies. These findings indicate that a subset of anti-anti-DNA antibodies are idiotypically related to antibodies reactive with a cell surface DNA binding molecule. It is hypothesized that anti-DNA antibodies may arise when a convergence of genetic and environmental influences favours an unrestrained anti-idiotypic response to cell surface DNA binding molecule(s).     

Non-DNA-Binding Antibodies in Patients with Lupus Nephritis Could Recognize Membrane Proteins of Glomerular Mesangial Cells

Lupus nephritis (LN) is a prototypic autoimmune disease, however, the precise immuno-pathogenesis of LN remains to be elucidated. In our previous studies, autoantibodies against mesangial cells had been identified in sera from patients with lupus nephritis and could bind the membrane proteins of human mesangial cells (HMC) directly through antigen-antibody interaction without DNA bridge. The current study is to investigate whether the autoantibodies were associated with anti-DNA antibodies and their target antigens distribution in different cell types. Sera from nine patients with renal biopsy proven lupus nephritis with positive anti-dsDNA antibodies and four healthy subjects were collected. IgG was isolated by Protein G affinity chromatography and then non-DNA-binding IgG fractions were obtained after deletion of anti-DNA antibodies using a DNA-cellulose affinity column. Membrane proteins, obtained from HMC, human umbilical vein endothelial cells (HUVEC), peripheral mononuclear cells by sonication and sequential centrifugation, were solubilized and applied in Western-blot analysis to characterize the target antigens. In results, the non-DNA-binding IgG fractions from sera of patients with lupus nephritis could blot the protein(s) of HMC membrane at 74, 63, and 42 kD. However, only a similar 74-kD protein could be blotted on membrane of HUVEC, and the target antigens on membranes of mononuclear cells were heterogeneous. In conclusion, our preliminary study had demonstrated that non-DNA binding autoantibodies against mesangial cells could be found in sera from patients with lupus nephritis. Although the target antigens might not be cell specific, the roles of these autoantibodies in the pathogenesis of lupus nephritis need further investigation.     

Anti-IL-1 alpha autoantibodies in patients with rheumatic diseases and in healthy subjects.

Antibody reactivity against the 'mitochondrial M2 antigen' was determined in sera from 10 patients with primary biliary cirrhosis (PBC), using Western blotting after SDS-PAGE separation of rat liver mitochondria (RLM) and plasma membrane proteins. The molecular weights of the major M2 antigens in rat liver mitochondria were 67 and 50 kD. Two of the 10 PBC patients did not react to any of these major antigens, eight reacted to the 67-kD and four of those also to the 50-kD antigen. The 67- and 50-kD antigens were present in both plasma membrane and RLM and had affinity to concanavalin A. Antibody reactivity against the 67-kD antigen could be detected in both IgG and IgA as well as in the IgM class. The reactive IgG subclasses to both types of antigen preparations were mainly of the G1 and G3 isotypes. This reactivity was always stronger with antigens from the plasma membrane preparations. Sera from two patients with high antibody titres against mitochondria also reacted with IgG2 against the 50-kD antigen from plasma membrane, but not to the corresponding antigen in mitochondria. Reactivity of antibodies in PBC sera to the periphery of viable hepatocytes and radioactive surface labelling of the 50-kD component are both consistent with a plasma membrane localization of M2. Serum from healthy controls and several patients with different diseases did not contain antibodies reactive against any of the antigens described. We suggest that antigens, partly identical to the mitochondrial M2, are located in the plasma membrane compartment. The PBC pathogenetical consequences of these findings are discussed.     

Autoantibodies to Vascular Heparan Sulfate Proteoglycan in Systemic Lupus Erythematosus React with Endothelial Cells and Inhibit the Formation of Thrombin-Antithrombin III Complexes

Vascular heparan sulfate proteoglycan (vHSPG) is an important functional component of the microvasculature. Previous studies have demonstrated autoimmunity to vHSPG in systemic lupus erythematosus (SLE). In the current studies, we further investigated the immunospecificity of anti-vHSPG antibodies in SLE sera by enzyme-linked immunoassay (ELISA). In direct binding assays, SLE sera contained IgG antibodies reactive with native vHSPG and with heparan sulfate (HS) glycosaminoglycan in significantly higher titers than controls. Employing purified SLE IgG in liquid-phase competitive immunoinhibition ELISAs, SLE IgG anti-HS antibodies cross-reacted with heparin and DNA, but not with other glycosaminoglycans or anionic phospholipid antigens. Immunochemical studies demonstrated that the immunodominant site on HS recognized by SLE IgG contained 2-O-sulfated uronic acid. Removal of N-sulfated and 6-O-sulfated residues primarily on N-acetylglucosamine had no effect on antigenicity, further demonstrating that nonspecific charge interactions which are the result of sulfation do not solely account for the antigenicity of HS. SLE IgG from patients with active SLE was further affinity purified on DNA-cellulose and HS-Sepharose columns for immunospecificity studies. After affinity purification of both anti-DNA and anti-HS antibodies, significant enhancement of direct binding reactivity with HS was noted. In addition, anti-DNA and anti-HS IgG antibody reacted with the cell surface of endothelial cells by a cellular ELISA (CELISA). Immunoinhibition studies of CELISA reactivity confirmed that affinity-purified SLE IgG anti-DNA anti-HS antibody were reactive with endothelial cell surface HS antigens. Furthermore, SLE IgG anti-DNA antibody reactivity with endothelial cells was not reduced by DNase treatment of the cells, but was significantly reduced by heparitinase digestion. Since HS plays an important role in the maintenance of normal anticoagulation on the endothelial cell surface by binding antithrombin III, we investigated the inhibition of heparin-accelerated thrombin-antithrombin III complex formation by SLE IgG. Purified IgG from patients with active SLE, but not from normal controls, inhibited heparin-accelerated formation of TAT complexes. These studies demonstrate the presence of IgG autoantibodies to HS in patients with SLE. Anti-HS antibodies recognize an antigenic site also present in heparin, but not other glycosaminoglycans, bind to the endothelial cell surface, and inhibit the formation of TAT complexes. SLE IgG anti-HS antibodies recognize a sulfated uronic acid epitope containing 2-O-sulfate which is important in certain functions of HS, including antithrombin III binding. Thus, anti-HS antibodies may promote a procoagulant state at the endothelial cell surface. Anti-DNA antibodies which cross-react with high avidity with anticoagulant sites on cell surface HS may represent a subpopulation of anti-DNA antibody with the capacity to cause thrombosis.     

Direct Binding of Monomeric Anti-DNA Antibodies to Raji Cells

The Raji-cell test is one of the most widely used methods for the detection and quantitation of immune complexes. Immune complexes and not 7 S IgG bind via C3 to complement receptors on the cell membrane of the Raji cell. During sucrose gradient fractionation of human and murine systemic lupus erythematosus sera, with a high Raji cell-binding activity, we could not demonstrate immune complexes in these sera. Subsequent analysis showed that the major part of the Raji cell binding was used by 7 S IgG with an anti-DNA specificity. Blocking experiments wilh complement-bearing aggregated IgG revealed that complement and Fc receptors were not involved in the binding of these anti-DNA antibodies to Raji cells. We conclude that the Raji cell test is not suitable for the detection and quantitation of immune complexes in sera containing anti-DNA antibodies.     

Specificity and immunochemical properties of antibodies to bacterial DNA in sera of normal human subjects and patients with systemic lupus erythematosus (SLE)

To elucidate the mechanisms of anti-DNA production, we assessed the binding of sera of normal human subjects (NHS) and patients with SLE to a panel of bacterial and mammalian DNA. Using single-stranded DNA as antigens in an ELISA, NHS showed significant binding to some but not all bacterial DNA, while lacking reactivity to calf thymus DNA. Among bacterial DNA, the highest levels of binding were observed with DNA from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, SLE sera showed high levels of binding to all DNA tested. To evaluate further immunochemical properties of the anti-DNA antibodies, the subclass distribution of these responses was evaluated by subclass-specific reagents. While NHS showed a predominance of IgG2 antibodies to bacterial DNA, SLE sera had a predominance of IgG1 antibodies to these antigens. Together, these results provide further evidence for the antigenicity of bacterial DNA and suggest that NHS and SLE anti-DNA differ in the patterns of epitope recognition as well as mechanisms of induction.     

Cross-reaction of anti-DNA autoantibodies with membrane proteins of human glomerular mesangial cells in sera from patients with lupus nephritis

Anti-DNA autoantibodies were thought to play a major role in the pathogenesis of lupus nephritis (LN). A recent study revealed that affinity-purified anti-DNA antibodies had a cross-reaction with human glomerular mesangial cells (HMC). However, whether the cross-reaction was antigen-antibody-mediated was unclear. The aim of the current study was to investigate the binding of anti-DNA antibodies to HMC membrane proteins and to characterize the target antigens. Affinity-purified IgG anti-DNA antibodies were purified by DNA-cellulose chromatography in sera from nine patients with biopsy-proven active lupus nephritis. In vitro cultured primary HMCs were disrupted by sonication and HMC membranes were obtained by differential centrifugation. The membranes of human umbilical vein endothelial cells (HUVEC), human proximal renal tubular epithelial cell line (HK2) and peripheral mononuclear cells (PMC) were obtained as controls. Binding of anti-DNA antibodies to the membrane proteins was investigated by Western blot analysis using soluble membrane proteins as antigens. Both HMC membrane and affinity-purified anti-DNA antibodies were treated with DNase I to exclude DNA bridging. All nine affinity-purified anti-DNA antibodies could blot the HMC membrane proteins, and there were at least three bands at 74 kDa, 63 kDa and 42 kDa that could be blotted. Among the nine IgG preparations, all nine (100%) could blot the 74 kDa band; eight (88.9%) could recognize 63 kDa and 42 kDa protein bands separately. After DNase treatment, the same bands could still be blotted by most affinity-purified anti-DNA antibodies. Affinity-purified anti-DNA antibodies could also blot similar bands on membrane proteins of other cells, but some bands were different. In conclusion, anti-DNA autoantibodies could cross-react directly with cell membrane proteins of human glomerular mesangial cells and might play an important role in the pathogenetic mechanism in lupus nephritis.     

Rabbits produce SLE-like anti-RNA polymerase I and anti-DNA autoantibodies in responses to immunization with either human or murine SLE anti-DNA antibodies.

Anti-DNA and anti-DNA polymerase I (RPI) autoantibody responses are symptoms of systemic lupus erythematosus (SLE). To investigate the relationship between these antibodies (Ab), rabbits were immunized with one of the following preparations: human SLE anti-DNA Ab; human SLE anti-DNA IgG; normal human anti-DNA Ab; human Grave's disease anti-DNA Ab; murine SLE anti-DNA Ab or anti-DNA IgG Fab; various normal human, murine, or rabbit IgG preparations; or complete Freund's adjuvant (CFA), alone. All of the animals immunized with anti-DNA Ab (n = 14) generated Ab reactive in radioimmunoassay with: ssDNA, dsDNA, RPI, the soluble fraction of rabbit liver crude nuclear extract, and the immunogen. Induced rabbit anti-DNA Ab in turn induced these responses in a different rabbit: a rabbit immunized with rabbit anti-DNA IgG Ab which had been previously induced by immunization with human anti-DNA Ab, produced Ab reactive with ssDNA, dsDNA, RPI, and the soluble fraction of rabbit liver nuclear extract. Although an individual animal's antisera reacted consistently over the course of immunization with the same individual RPI subunit(s), antisera from different animals reacted with different subunits of the 9-subunit RPI complex in Western blot analyses: 190 kD (n = 6); 120 kD (n = 1); 62 kD (n = 4); 45 kD (n = 2); and, no reactivity (n = 2). In contrast, animals immunized with normal IgG or CFA produced responses only against the immunogen. Together, these data suggest that anti-DNA and anti-RPI responses are connected through an autoimmune network in SLE.     

Autoantibodies to anti-DNA with anti-allotypic and anti-idiotypic specificities in (NZB X NZW)F1 mice

The monoclonal A52 (IgG2b, kappa) anti-DNA autoantibody represents a major cross-reactive idiotype in the murine and human autoimmune response to DNA. Examination of sera and purified IgG derived from (NZB X NZW)F1 mice showed that these mice develop an age-dependent binding reactivity with the pure anti-DNA IgG. Three monoclonal antibodies possessing this reactivity were prepared from unprimed female (NZB X NZW)F1 mice. One of these monoclonal antibodies appeared to be directed against allotypic determinants present in the NZB IgG2b; the other two antibodies exhibited a marked preference for idiotypic determinants of the A52 IgG. The IgG anti-allotype and anti-idiotype activities in (NZB X NZW)F1 mice may, therefore, represent the products of a deregulated immune system and/or constitute the normal elements of a functional immune regulation system.     

Localization, molecular weight and immunoglobulin subclass response to Aspergillus fumigatus allergens in acute bronchopulmonary aspergillosis

A specimen of Aspergillus fumigatus was isolated from a patient with acute bronchopulmonary aspergillosis (ABPA). Cultures were allowed to propagate and were separated into spores and mycelium to greater than 95% homogeneity. Extracts of both the spores and mycelium were prepared and used for study by both dot blot analysis and immunoblotting to study the major allergens. By dot blot analysis, 22 of 22 patients with ABPA and none of 10 healthy controls reacted with mycelium when probed for both IgG and IgE anti-Aspergillus reactivity. Similar results were obtained when these extracts were separated on polyacrylamide gel electrophoresis and then probed; this included both IgG and IgE reactivity. Further, the reactivity to preparations of whole extract was absorbed with mycelium but not spores. Several distinct protein bands were detected with IgG, ranging from 30 to 110 kilodaltons (kD). In contrast, the majority of patients with ABPA, when studied for IgE reactivity, reacted only with a 70-kD protein, although 1 out of 22 patients reacted with a 30-kD protein. Subclass analysis of IgG reactivity demonstrated that 90% of reactive sera contained IgG2 and 73% IgG4 reactivity. IgG1 and IgG3 reactivity were present but less frequently detected. The use of immunoblotting will enable the identification of relevant allergens. Further, the demonstration that the allergenicity is located primarily in mycelium will allow more definitive studies of allergen isolation for studies of reactivity and, ultimately, cloning of the 70-kD protein and, finally, epitope mapping.     

Selective recognition of DNA antigenic determinants by murine monoclonal anti-DNA antibodies.

To assess the immune recognition of DNA in systemic lupus erythematosus, the antigenic specificity of monoclonal anti-DNA antibodies from autoimmune MRL-lpr/lpr mice was investigated Determinant specificity was assessed by ELISA in terms of binding to a panel of ssDNA antigens including calf thymus, human placenta, Escherichia coli, Clostridium perfringens, Micrococcus lysodeikticus, salmon testes, chicken blood and murine DNA. Among the monoclonal antibodies, a variety of binding patterns was observed, although for all antibodies tested murine DNA was among the most reactive antigens. Binding to other DNAs varied markedly, with some antibodies showing only low reactivity to certain antigens in the test panel. Similar results were obtained with sera of individual MRL-lpr/lpr mice. These results suggest that anti-DNA antibodies bind specific antigenic determinants variably expressed by DNAs of various species. Furthermore, the preferential binding to mouse DNA by some MRL-lpr/lpr antibodies may suggest a role of self-DNA in the in vivo selection of anti-DNA antibodies for expression.     

Lupus anti-DNA antibodies bearing the 8.12 idiotype appear to be somatically mutated

Anti-DNA antibodies in systemic lupus erythematosus (SLE) sera were analyzed using an antiidiotype designated 8.12 which recognizes a determinant on lambda light chains highly expressed in SLE sera. Eight of ten normal individuals had peripheral blood lymphocytes which produced high-titered 8.12-positive antibodies, following transformation with Epstein Barr virus, implying that the 8.12-reactive sequence originates in the germline gene (GLG). Of 58 SLE sera, 32 contained elevated titers of 8.12-reactive antibodies. Twenty-three of these sera had 8.12-reactive anti-DNA antibodies, suggesting a strong correlation between 8.12 idiotype and DNA binding. Moreover, 20 of 26 8.12-reactive IgG antibodies and only 4 of 10 8.12-reactive IgM antibodies bound DNA (P<0.05). These observations strengthen our previous findings in myeloma sera that DNA binding is associated with IgG isotype in the 8.12 idiotype system and suggest that the acquisition of anti-DNA reactivity in antibodies bearing the GLG idiotype 8.12 is achieved by somatic mutation, a feature of an antigen-driven response.     

Crossreactivity of Human Anti-dsDNA Antibodies to Phosphorylcholine: Clues to Their Origin

The presence of anti-double stranded DNA (dsDNA) antibodies is a serological diagnostic feature of systemic lupus erythematosus (SLE), an autoimmune rheumatic disorder. Studies by several investigators have suggested that a response to a microbial antigen can lead to the induction of SLE-like autoimmunity, in both humans and mice, since anti-dsDNA antibodies have been shown to crossreact with foreign antigens. In particular, anti-DNA antibodies have been shown to crossreact with phosphorylcholine (PC), a dominant epitope on pneumococcal cell wall polysaccharide. We have investigated the binding characteristics of human polyclonal anti-DNA antibodies from the sera of SLE patients. In this study we show that the DNA binding of polyclonal serum derived antibodies can be partially inhibited by phosphorylcholine (PC). The binding of affinity-purified anti-DNA antibodies from the sera of patients with SLE was also found to be inhibited by PC. We further demonstrated that the serum IgG1 (T dependent) anti-DNA response was more likely to crossreact with PC than the IgG2 (T independent) response to DNA. The studies suggest there may be a T dependent and T independent response to DNA with the T dependent response displaying more crossreactivity with microbial antigen.     

Comparison of conidial and mycelial allergens of Alternaria alternata

Alternaria allergens, separated by SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, were identified using sera from Alternaria-allergic patients. 30 Alternaria components of spore and mycelium, ranging in molecular weight from more than 150 to 12 kD, bound IgE antibodies. 15 were detected by more than 25% of the sera and only 4 (85, 56, 42, 31 kD) by more than 50% of the sera. Among these four major allergens, the 56-kD component was present mostly in the spore extract, the 85- and 42-kD fractions in the mycelium extract and the 31-kD in both extracts. The 31-kD component showed the highest IgE binding and the highest frequency of binding (95% with mycelium extract and 79% with spore extract). It was composed of two subunits.     

Characterization of a 58- and a 78-kD Monocytic Membrane Protein with Affinity to the Acetylcholine Receptor in Myasthenia Gravis Patients

The autoimmune disease myasthenia gravis (MG), caused by the effect of specific antibodies, directed towards the nicotinic acetylcholine receptor, is triggered by autoantigen-specific T cells. In order to investigate cellular parts of the immune response in MG, the authors investigated the binding of the nicotinic acetylcholine receptor (AChR) to peripheral blood mononuclear cells (PBMC) from MG patients. AChR binding cells were identified by resetting experiments using AChR-coated fluoresceine beads. Applying this technique, a significant percentage of PBMC (21.2 × 7.65%) from MG patients formed rosettes with AChR-coated beads.
Membrane preparations of nycodenz- or percoll-separated monocytes from MG patients or T-cell depleted monocytic subpopulations were applied to SDS-PAGE under reducing conditions. Ligand-blotting studies with biotinylated AChRs revealed two cell-membrane proteins with molecular weights of 58- and 78-kD. In parallel the same results were obtained by affinity chromatography of monocytic membrane proteins using AChR-sepharose. A possible interference of anti-AChR IgG was excluded. The 58- and the 78-kD proteins are detectable under reducing conditions by ligand blotting with AChR-biotin, while under non-reducing conditions only the 58-kD protein can be detected. Furthermore, in experiments using Endoglycosidase-H, the 58-kD protein appears to be non-glycosylated, while the 78-kD protein bears carbohydrates.
These findings suggest that monocytes which bind the AChR via specific membrane proteins on their surface might act as antigen-presenting cells and may lead to an induction of the T-cell response, in the early phase of the disease.     

Murine and human B cell epitope mapping of the Mycobacterium tuberculosis 10-kD heat shock protein using overlapping peptides.

The human immune response to the 10-kD M. tuberculosis protein was studied by a competition ELISA using monoclonal antibody (MoAb) SA-12. Twenty-five per cent of the sera from 20 patients with tuberculosis and none from 21 control subjects inhibited binding of SA-12 to the 10-kD antigen. To characterize the antigenic parts of the 10-kD antigen, overlapping decapeptides according to the amino acid sequence of the 10-kD protein were synthesized. In total, 91 sequential decapeptides, with an overlap of nine amino acids, were tested in ELISA with MoAb SA-12, human and murine sera (PEP scan). SA-12 recognized the amino acid sequence WDEDGEK (amino acid 50-56). All human sera, from patients with tuberculosis as well as from control subjects, gave almost identical undulating patterns of reactivity with the decapeptides. No relationship was found between the ability of the patients' sera to inhibit binding of MoAb SA-12 and the binding of these sera to the decapeptides comprising the epitope recognized by SA-12 in the PEP scan. Apparently, antibodies in patients' sera against the 10-kD protein are predominantly directed against discontinuous epitopes and, consequently, the continuous epitopes as presented in the PEP scan are not suitable to discriminate between patients with tuberculosis and control subjects. In the PEP scan, sera from BALB/c mice, both non-immunized and immunized with either live M. tuberculosis or the 10-kD protein gave similar patterns of reactivity, albeit different from the patterns obtained with the human sera. However, after immunization of the mice, clearly increased levels of antibodies to primary structures of the 10-kD protein were observed.     

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins and by immunoblotting of electrophoretically separated cell, granule or nuclear extracts. Forty-two sera (68%) reacted with ethanol-fixed neutrophils. In the ELISAs 32% of the 28 medium to strongly IIF-positive sera were negative, while 43% were weakly positive for more than one antigen. Immunoblots of whole neutrophils showed IgG reactions at 25–35 kD, in the 55-kD region, at 80 kD, and at 110kD. Most sera reacted with more than one band. Except for the 55-kD antigen, none of the antigens appeared in lymphocytes. The most notable reactivity in subcellular fractions was with lactoferrin and with bands of 25–35 kD from nuclei. In conclusion, anti-neutrophil autoantibodies from RA patients recognize different antigens in the cytoplasm and in the nucleus. Lactoferrin is one of the common antigens recognized, but also unknown nuclear antigens of 25–35 kD mol. wt are involved.     

Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals.

The cross-reaction between anti-DNA and anti-cardiolipin IgG antibodies and its relation to the standard test for syphilis was studied with sera and monoclonal antibodies derived from human patients and mice with systemic lupus erythematosus (SLE). Syphilitic sera of humans and rabbits infected with the spirochete Treponema pallidum were also tested in this study. In addition, rabbits were immunized with ssDNA and cardiolipin and the cross-reactions of the induced antibodies were studied in two different assay systems. The results of these experiments suggest: that the anti-DNA and anti-cardiolipin IgG autoantibodies in SLE sera constitute separate antibody populations and, therefore, cardiolipin cannot play a role in the induction of immune response to DNA in SLE; that in immunized experimental animals there is a significant level of cross-reaction between anti-DNA and anti-cardiolipin-the detection of this cross-reaction depends on highly amplified solid phase assay systems which measure low affinity antibodies and that there is no correlation between the activity of syphilitic sera in the serologic test for syphilis and their binding to pure cardiolipin-this implies that cardiolipin may not be the dominant ingredient in this test as previously proposed.     

Interpretation of the cross-reactivity of anti-DNA antibodies with cell surface proteins: the role of cell surface histones

The putative cross-reaction of anti-DNA antibodies with "lupus-associated membrane proteins (LAMP)" on the surface of intact Raji cells was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses. Cell surface proteins of 14, 17, 18, 33 and 34 kDa were detected by monoclonal anti-double-stranded (ds) DNA antibodies and the sera of patients with systemic lupus erythematosus (SLE) in active states, but were not detected by the sera of SLE patients in inactive states, nor in healthy controls. However, pre-treatment of these anti-DNA antibodies with DNase I markedly reduced the reactivity to the cell surface proteins. Judging from the electrophoretic mobility, these proteins were identical with histones, and purified histones inhibited the reaction of anti-DNA antibodies with the cell surface proteins. Moreover, affinity-purified antihistone antibodies could demonstrate histones in the Raji cell surface proteins. Thus, we conclude that "cross-reaction" of anti-DNA antibodies with LAMP is due to DNA-anti-DNA immune complexes which could react with cell surface histones.     

Human antibody response to a group B serotype 2a meningococcal vaccine determined by immunoblotting

The antibody response of 30 volunteers vaccinated with a complex of group B polysaccharide and outer membrane vesicles (OMV) from serotype 2a Neisseria meningitidis and of 3 individuals who received a placebo vaccine was determined by immunoblotting. OMV were separated by sodium dodecyl sulfate-gel electrophoresis and electrotransferred to nitrocellulose filters. Binding of immunoglobulin G (IgG), IgA, and IgM antibodies in the human sera to OMV components was detected with class-specific peroxidase-conjugated antibodies. The immunoblotting results were also related to the bactericidal activity of the sera and the meningococcal carrier status of the volunteers. Before vaccination weakly reactive bands in the molecular weight range of 140,000 to 10,000 were observed on the blots. Sera from carriers showed more marked bands. Individual patterns of increased reactivity were seen 6 weeks after vaccination. The main immunoreactive components of OMV corresponded to a molecular weight of 43,000 (class 1 protein), 30,000 (class 5 proteins), and 22,000. IgG antibodies in postvaccination sera of high bactericidal titers showed distinct binding to the 43,000-molecular-weight antigen. Meningococcal carriers had antibodies against an antigen of 22,000 molecular weight; in polyacrylamide gels this component did not stain with Coomassie brilliant blue or silver. The marked binding of IgG antibodies to the class 5 proteins decreased considerably between weeks 6 and 25 after vaccination. Periodate oxidation of OMV abolished the binding of IgG antibodies to the class 5 proteins, whereas the antigenicity of the 43,000-molecular-weight (class 1 protein) and 22,000-molecular-weight antigens was unaffected.