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Biomedical subjects

J Rauch

Publications and source records attributed to J Rauch.

At least 55 records · Page 3Linked to original sources
PubMed1994

A human rheumatoid factor C304 shares VH and VL gene usage with antibodies specific for ubiquitous human viral pathogens.

Analysis of the variable region gene sequences of a human hybridoma rheumatoid factor (RF), derived from a patient with rheumatoid arthritis (RA), revealed the expression of genes from the V lambda I and VH3 families. Specifically, the C304 RF had rearranged the DPL8/Humlv1042 and VH26 germline VL and VH genes, respectively. This gene usage has been observed in the rearrangement of human anti-viral antibodies specific for the herpes group of viruses. This overlap between the autoimmune and anti-viral antibody gene repertoires suggests a possible structural relationship between the immune response directed against ubiquitous pathogens and the induction of RF production.

Amino Acid Sequence↗
PubMed1993

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis.

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Arthritis, Rheumatoid↗
PubMed1993

Molecular characterization of the GM 4672 human lymphoblastoid cell line and analysis of its use as a fusion partner in the generation of human-human hybridoma autoantibodies.

The GM 4672 lymphoblastoid cell line has been used in cell hybridization experiments with peripheral blood lymphocytes (PBLs) in order to generate human-human hybridomas that secrete immunoglobulins directed against a number of different autoantigens. The GM 4672 cells were fused with PBLs isolated from patients with rheumatoid arthritis or systemic lupus erythematosus, or from normal individuals, and the resulting hybridomas were screened for reactivity to platelets, erythrocytes, DNA, cardiolipin, human IgG-Fc, phosphatidylethanolamine, and for lupus anticoagulant activity. This report analyzes the results from 149 fusion experiments completed over a period of nine years. Fifty to sixty-six percent of the fusion experiments resulted in immunoglobulin-secreting clones, with an average of 15 clones/fusion. The hybridoma antibodies were predominantly of the IgM heavy chain isotype, and 67% expressed kappa light chains. Although most hybridoma antibodies (78%) recognized a single autoantigen, 22% recognized more than one autoantigen and were considered polyreactive. In addition, the light and heavy chain variable regions of the antibody secreted by the GM 4672 cell line were amplified by the polymerase chain reaction technique and sequenced. The GM 4672 light chain was encoded by a VkI gene and used a Jk4 minigene. The GM 4672 heavy chain was derived for the rearrangement of a gene from the VH4 subgroup and used a JH4 minigene. The 8 amino acid long diversity region was generated by the fusion of the DK1 and DLR2 genes. The hybridomas generated in fusion experiments, when examined, did not appear to secrete antibodies using the immunoglobulin variable regions derived from the GM 4672 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Binding of human monoclonal IgG rheumatoid factors to Fc is influenced by carbohydrate.

OBJECTIVE: The precise nature of the epitope on the Fc portion of the IgG molecule to which IgG rheumatoid factors (RF) bind has not been identified. As patients with rheumatoid arthritis (RA) have abnormal glycosylation of the Fc portion of IgG, we investigated the impact of the sugar present in the Fc on the binding of IgG RF. METHODS: Binding of monoclonal IgG RF to Fc fragments with varying oligosaccharide chains was detected using an immunoblot assay. RESULTS: We demonstrated that the binding of human hybridoma derived monoclonal IgG RF was strongly influenced by the presence of carbohydrate and was maximal when the carbohydrate molecule was intact. The RF did not bind directly to the carbohydrate moiety itself. CONCLUSION: This suggests that conformational changes in the polypeptide chain induced by the carbohydrate are responsible for the observed binding patterns.

Adult↗
PubMed1992

Suppression of renal disease and arthritis, and prolongation of survival in MRL-lpr mice treated with an extract of Tripterygium wilfordii Hook f.

OBJECTIVE: Treatment of MRL-lpr/lpr mice with Tripterygium wilfordii Hook f (TWHf) to evaluate its effects on mortality, renal disease, and arthritis. METHODS: Mice were fed water (group A, control), TWHf (group B), or first water and then TWHf (group C) from age 7 weeks until age 21 weeks. RESULTS: Arthritis and glomerulonephritis were decreased in groups B and C mice, and survival was increased in group B mice. CONCLUSION: TWHf decreases the mortality rate, and severity of glomerulonephritis and arthritis in MRL-lpr/lpr mice.

Animals↗
PubMed1992

The nature of antiphospholipid antibodies.

Despite the striking clinical manifestations associated with antiphospholipid antibodies (aPL) the role of these autoantibodies in disease and the nature of their true "inducing" and "target" antigens remain elusive. To address these issues, we investigated the immunogenic potential of phospholipid structures. To date, phospholipid immunogens have included hexagonal (II) forms of phosphatidylethanolamine and mixtures of apolipoprotein H (beta 2-glycoprotein I) with cardiolipin. Both hexagonal (II) phosphatidylethanolamine and the cardiolipin/apolipoprotein H mixture were capable of inducing aPL with lupus anticoagulant activity. Bilayer phosphatidylethanolamine and cardiolipin in the absence of apolipoprotein H were nonimmunogenic. Our data support our views that specific phospholipid structures are recognized by the immune system and that such structures serve as inducing and/or target antigens in the pathogenesis of aPL in vivo.

Animals↗
PubMed1992

Binding characteristics of human hybridoma IgG and IgM rheumatoid factors: influence of IgG isotype and carbohydrate content.

The interaction of human monoclonal rheumatoid factors (RF) with the Fc portion of IgG is complex. We have investigated the influence of the nature of the antigen (Fc) on the binding of hybridoma-generated RF derived from patients with rheumatoid arthritis or systemic lupus erythematosus. For IgM RF, the interaction is strongly influenced by the primary structure of the Fc with little or no effect of the carbohydrate, which is positioned at amino acid 297 in the gamma-2 domain. In contrast, our preliminary data suggest that IgG RF binding is affected both by the primary structure and the nature of the carbohydrate of the Fc. These results suggest that the antigen selection events which lead to the induction and production of IgG RF are likely to be different from those that induce IgM RF production.

Carbohydrate Metabolism↗
PubMed1991

Molecular cloning of gp 80, a glycoprotein complex secreted by kidney cells in vitro and in vivo. A link to the reproductive system and to the complement cascade.

cDNA clones coding for the gp 80 heterodimeric glycoprotein complex secreted constitutively at the apical surface of Madin-Darby canine kidney (MDCK) cells have been isolated from MDCK cDNA libraries in lambda gt11 and lambda gt10. The cloned sequences encode a polypeptide chain of 445 amino acids. The deduced amino acid sequence of the gp 80 protein reveals 80% homology to rat SGP-2, a major secretory protein of the testes epithelium and 83% homology to SP-40,40, a human complement-associated protein. SGP-2 and SP-40,40 have been proposed to be serum and seminal forms of the same protein. The sequence homology as well as the results of Southern and Northern blot analyses and immunological studies suggest that gp 80 is the canine homolog of the rat SGP-2 and the human SP-40,40. The protein is expressed in the embryonic kidney already early during organogenesis. In the adult kidney the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells.

Amino Acid Sequence↗
PubMed1991

A systemic lupus erythematosus-derived human hybridoma autoantibody reactive with antigens expressed on ADP-activated platelets.

Hematological complications seen in systemic lupus erythematosus (SLE) patients may be caused by the binding of specific autoantibodies to platelets, but the epitopes on platelets responsible for antibody binding and the mechanisms by which autoantibodies induce hemostatic abnormalities in SLE patients remain unknown. We have previously demonstrated that polyspecific platelet-binding antibodies can be derived from SLE patients. In the present study, we have characterized an SLE-derived polyspecific hybridoma antibody, 9604, which was previously shown to be strongly cytotoxic to platelets in vitro and to have weak lupus anticoagulant activity. We demonstrated that this antibody does not bind to fixed intact platelets in an enzyme-linked immunoassay (ELISA), but does react with lysed, washed or ADP-activated platelets. By Western blotting analysis, antibody 9604 was unique among other platelet-binding autoantibodies in that it reacted mainly with polypeptides of approximately 200,000 and 32,000 molecular weight (MW) in platelets. In blots of endothelial cell proteins, 9604 reacted with a band of approximately 200,000 MW, but no 32,000 MW reactive band was observed. Based on these findings, we postulate that antibody 9604 may bind to a protein or proteins of 32,000 MW exposed on the platelet surface during activation. To our knowledge, this is the first demonstration of a human hybridoma monoclonal antibody derived from an SLE patient which distinguishes between activated and resting platelets. Further characterization of the proteins recognized by this autoantibody may provide insight into the mechanisms responsible for the production and pathogenesis of anti-platelet autoantibodies in patients with SLE.

Adenosine Diphosphate↗
PubMed1991

Detection of endothelial cell-reactive immunoglobulin in patients with anti-phospholipid antibodies.

Individuals with anti-phospholipid antibodies are at increased risk for the development of thrombosis and fetal loss. The pathogenesis of these syndromes is unknown, but may involve antibody-mediated alterations in endothelial cell coagulant activity. To address this possibility, we determined the incidence of endothelial cell-reactive antibodies in 76 patients whose plasma contained anti-phospholipid antibodies, but who had no clinically-evident immune disorder. Plasma from 47 patients deposited significantly more immunoglobulin on cultured endothelial cells than control plasma. Positive tests were more frequent in patients with a history of thrombosis than in those without (17/19 v 23/48; P = 0.004). However, we observed no correlation between immunoglobulin deposition on cardiolipin and endothelial cells by individual plasmas. Furthermore, endothelial cell reactivity was not diminished by adsorption of anti-cardiolipin antibodies from patient sera using liposomes. Immunoglobulin fractions prepared from 5/6 patient sera immunoprecipitated a approximately 70 kDa endothelial cell surface protein; 4/5 of these fractions also induced the release of von Willebrand factor from endothelial cells. These results demonstrate that plasma from many patients with anti-phospholipid antibodies, but no clinically-evident autoimmune disease, also contains endothelial cell-reactive antibodies. Detection of such antibodies might help identify individuals in this patient population at greatest risk for thrombosis.

Antibodies↗
PubMed1990

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells.

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.

Amino Acid Sequence↗
PubMed1990

Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding.

We investigated the influence of carbohydrate on the binding of human rheumatoid factors (RF) to the Fc fragment of IgG. The monoclonal RF studied were derived from the serum of patients with mixed cryoglobulinemia or from hybridomas generated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. Polyclonal RF were derived from patients with RA. The carbohydrate located on the Fc fragment, regardless of whether it contained different amounts of mannose or reduced amounts of galactose, or was removed, did not affect the binding of the RF. In contrast, the isotype of the Fc was found to be critical. Two groups of hybridoma RF could be delineated. One group bound preferentially to IgG1 and/or IgG2, and a second group (primarily from patients with RA) bound preferentially to IgG3 and/or IgG4. Our results indicate that the isotype of the Fc fragment, and not the extent of galactosylation, influences the binding of the RF.

Arthritis, Rheumatoid↗
PubMed1990

Phospholipid in the hexagonal II phase is immunogenic: evidence for immunorecognition of nonbilayer lipid phases in vivo.

Immunization of mice with phosphatidylethanolamine in the hexagonal II phase but not the bilayer phase resulted in the induction of anti-phospholipid antibodies. These antibodies, which were strongly reactive with phosphatidylethanolamine and crossreactive with cardiolipin, had functional lupus anticoagulant activity and were characteristic of autoantibodies common in patients with autoimmune disease. Recognition of the hexagonal II phase by the afferent limb of the immune system suggests that nonbilayer phospholipids can arise in the course of membrane remodeling and induce the autoantibodies of disease.

Animals↗
PubMed1990

Differences between human hybridoma platelet-binding antibodies derived from systemic lupus erythematosus patients and normal individuals.

The binding and functional activities of platelet-binding hybridoma autoantibodies from SLE patients were compared with those derived from normal individuals. Twenty-nine SLE-derived hybridoma antibodies and 20 normal-derived hybridoma antibodies were analyzed for binding to glutaraldehyde fixed platelets, dDNA and phospholipids, and for lupus anticoagulant activity. Twenty-four of the 29 SLE-derived antibodies and 9 of the 20 normal-derived antibodies showed one or more activities in these assays. Of the 24 SLE-derived antibodies, 8 (33.3%) were reactive in only one assay (monospecific), while the other 16 (66.7%) had more than one of these activities (polyspecific). In contrast, none (0%) of the 9 normal-derived antibodies with known activities were monospecific, while all 9 (100%) showed polyspecificity. Statistical analyses demonstrated that there was no correlation of anti-DNA activity with anti-platelet and most anti-phospholipid activities for the SLE-derived antibodies, and strong positive correlations between these reactivities for the normal-derived antibodies. Similarly, differences were observed in Western blotting analyses; SLE-derived antibodies bound more specifically to individual platelet proteins than normal-derived antibodies. Moreover, in chromium-51 release assays, all of the SLE-derived platelet-binding antibodies were cytotoxic to platelets, while none of the normal-derived platelet-binding antibodies showed significant cytotoxicity. Our results suggest that hybridoma platelet-binding autoantibodies derived from SLE patients exhibit greater antigen specificity and functional activity than similar antibodies derived from normal individuals.

Antibodies, Antinuclear↗