Biomedical subjects

R A Roubey

Publications and source records attributed to R A Roubey.

27 records · Page 2Linked to original sources

PubMed1992

Autoantibodies to nucleolin cross-react with histone H1 in systemic lupus erythematosus.

IgM autoantibodies to nucleolin and histone H1 are strongly associated in the serum of patients with systemic lupus erythematosus. IgM eluted from immobilized nucleolin specifically stained histone H1 blotted to nitrocellulose; conversely, IgM eluates prepared from immobilized histone H1 stained nucleolin blots. We conclude that the linkage of anti-nucleolin and anti-histone H1 autoantibodies in SLE is due, at least in part, to immunologic cross-reactivity between these two autoantigens, which share certain similar structural features.

Adult↗

PubMed1992

Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I.

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.

Animals↗

PubMed1991

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.

Alkaloids↗

PubMed1991

Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases.

The 110-kDa intracellular phosphoprotein (110K) described previously by this laboratory as a common IgM autoantigen in SLE and certain other systemic autoimmune disorders and viral infections is identified as nucleolin in the present investigation. Using rabbit antiserum to rat nucleolin as a probe, IgM autoantibody-reactive 110K co-migrated with human lymphocyte nucleolin in one- and two-dimensional immunoblots. Rabbit anti-nucleolin also specifically depleted autoreactive 110K from detergent lysates of human cells. Because nucleolin shares amino acid sequence similarity and/or forms dynamic particles with other prominent autoantigens, the present observation raises the possibility that the nucleolin/anti-nucleolin system may be of special significance for the development of humoral autoreactivity to nuclear Ag.

Antibody Specificity↗

PubMed1990

Reactivity of autoantibodies and DNA/anti-DNA complexes with a novel 110-kilodalton phosphoprotein in systemic lupus erythematosus and other diseases.

Utilizing nonionic detergent lysates of human lymphoid and non-lymphoid cells as substrate, IgM and/or IgG antibodies to a 110-kDa/isoelectric point 5.4 phosphoprotein (110K) was demonstrated in serum from patients with SLE or certain other systemic autoimmune disorders by immunoblotting and immunoprecipitation. Ig of this specificity was not demonstrable in serum from normal individuals, but, in a limited survey, was detected in serum from patients with acute hepatitis A or infectious mononucleosis. 110K shares a number of properties with nucleolin, i.e., identical Mr and isoelectric point, localization in both the nucleus and the cytosol, increased expression in rapidly dividing cells, and shown to be distinct from already defined autoantigens of similar size, i.e., topoisomerase I, PM-Scl, and RNA polymerase I. Because 110K could bind denatured DNA, as demonstrated by its specific absorption by DNA-cellulose and by its reactivity with monoclonal anti-ssDNA antibody in the presence of denatured DNA, special efforts were made to distinguish reactivity of pre-formed DNA/anti-DNA complexes in SLE serum from that due to specific anti-110K autoantibodies. Although binding to 110K could be mediated by DNA and anti-DNA in some SLE sera, the accumulated evidence supports the existence of a major new autoantibody system in SLE, other autoimmune diseases, and certain virus infections.

Antibodies, Antinuclear↗

PubMed1990

Synthesis of complement component C5 by human B and T lymphoblastoid cell lines.

Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lymphoblastoid cells with both distributions of C5 were shown to synthesize a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A)+ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by immunoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but had an alpha subunit of slightly smaller relative mass.

B-Lymphocytes↗

PubMed1989

Acquired congenital heart block. Pattern of maternal antibody response to biochemically defined antigens of the SSA/Ro-SSB/La system in neonatal lupus.

The molecular basis of autoantibody reactivity with components of the SSA/Ro-SSB/La particle exhibited by sera of mothers of infants with severe and permanent manifestations of neonatal lupus (NLE) was investigated using immunoblotting and immunoprecipitation. The characteristics of NLE that were studied included congenital complete heart block (CCHB), second degree heart block, and hepatic fibrosis. Antibodies specific for one or more components of the SSA/Ro-SSB/La particle were found in sera from all 20 mothers of permanently affected infants. However, no antibody specific for a single peptide of this particle was common to all sera. Using tissue extracts from a human cell substrate, 80% of these sera had antibodies to one or more components of the SSA/Ro particle demonstrable by immunoblotting. The predominant antibody response in the NLE group was to the newly recognized 52-kD SSA/Ro peptide component. In contrast, antibodies to the 60-kD SSA/Ro component although present, were the least represented and not significantly increased in frequency among mothers of these infants, compared with a group of 31 mothers with autoimmune diseases such as systemic lupus erythromatosus (SLE) but who had healthy offspring. Antibodies directed to the 48-kD SSB/La antigen were demonstrated in 90% of the NLE mothers often accompanying antibodies against the 52-kD SSA/Ro component. The combination of antibodies to 48- and 52-kD structures was significantly increased in the NLE group, with an odds ratio of 35. The type of cell or tissue substrate was shown to influence detectability of antibodies. The 52-kD SSA/Ro peptide and the 48-kD SSB/La peptide were abundant in cardiac tissues from fetuses aged 18-24 wk, further supporting the possible relevance of these peptides to heart block.

Autoantibodies↗

PubMed1985

Immunoassay for IgG rheumatoid factor with a murine monoclonal anti-Fd antibody.

Conventional radioimmunoassays for IgG rheumatoid factors (RF) detect the binding of IgG RF (or their F(ab')2 fragments) to solid-phase human IgG Fc fragments or rabbit IgG. Binding is detected with a radiolabeled antibody that is IgG-specific but is nonreactive with human Fc (i.e., an alpha-Fd reagent) or with rabbit IgG. Anti-Fd reagents are quite laborious to produce. We developed a murine monoclonal alpha-Fd antibody, confirmed its specificity, and compared it with conventional rabbit alpha-Fd in the IgG RF radioimmunoassay. We also adapted the assay to an enzyme-linked assay with both human Fc and rabbit IgG as antigen. We compared the four assay methods with each other and examined their relationship to certain clinical and laboratory features of rheumatoid arthritis.

Animals↗

PubMedDate not supplied

Anticardiolipin and anti-beta 2glycoprotein I immunoassays in the diagnosis of antiphospholipid syndrome.

Autoantibodies directed to phospholipids or to phospholipid binding proteins are now studied using a growing number of laboratory tests. However, the history of the interest in this area goes back to the identification of the so-called false positive reactions in the non-treponemal serological test for syphilis (STS) and to the subsequent description of an in vitro coagulation defect called lupus anticoagulant (LAC). In the 1980s the introduction of the anticardiolipin antibody (aCL) immunoassay was a determining factor in the definition of the antiphospholipid syndrome (APS). In addition, lupus prone mice spontaneously producing aCL antibodies and normal mice passively infused with these antibodies provided useful models for the study of the pathogenic role of aPL. When in 1990 a phospholipid binding protein (beta 2glycoprotein I, beta 2GPI) was identified as the cofactor required for aCL antibody binding, the true antigenic target of the antibodies was first discussed. Soon afterwards an anti-beta 2GPI ELISA was developed that has proved to be of great clinical significance. We will discuss here the similarities and differences between these various assays (LAC, aCL, and anti-beta 2GPI), focusing on their specificity, sensitivity and practical applications.

Animals↗