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K B Elkon

Publications and source records attributed to K B Elkon.

At least 19 recordsLinked to original sources
PubMed1992

Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3.

Although proteinase 3 (PR3) has been identified as a major autoantigen in Wegener's granulomatosis, the precise antibody specificity(ies) and requirements for epitope recognition have not been characterized. We analyzed 11 sera containing antineutrophil cytoplasmic antibodies (cANCA) for binding to azurophilic granule proteins extracted from neutrophils under various conditions and for binding to native or rPR3. Ten of 11 (91%) of the cANCA sera bound to PR3 extracted by nonionic detergents when tested by immunoprecipitation or by IEF followed by capillary immunoblotting. Antibody binding to PR3 was retained when IEF was performed under dissociating conditions (8 M urea) indicating that PR3 is the major autoantigen in azurophilic granules and that association with other proteins is not required for antigenicity. In contrast, antigenicity was totally destroyed by exposure of PR3 to reducing agents or to low pH (less than 3.0) and was either lost or considerably diminished after boiling in SDS. cANCA sera also showed little or no binding to rPR3 expressed as a fusion protein in Escherichia coli or synthesized by wheat germ ribosomes in vitro. Inasmuch as PR3 enzymatic activity was partially retained after acid extraction, these findings indicate that cANCA bind to a limited number of conformational epitopes on PR3. In addition, IEF followed by capillary immunoblotting appears to be a sensitive and specific method to detect anti-PR3 antibodies in Wegener's granulomatosis.

Antibody Specificity↗
PubMed1992

Cloning and expression of Trypanosoma cruzi ribosomal protein P0 and epitope analysis of anti-P0 autoantibodies in Chagas' disease patients.

Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a major cause of heart failure in endemic areas. Antigenic mimicry by T. cruzi antigens sharing epitopes with host macromolecules has been implicated in the pathogenesis which is thought to have a significant autoimmune component. We report herein on the cloning and characterization of a full-length cDNA from a T. cruzi expression library encoding a protein, TcP0, that is homologous to the human 38-kD ribosomal phosphoprotein HuP0. The T. cruzi P0 protein shows a clustering of residues that are evolutionarily conserved in higher eukaryotes. This includes an alanine- and glycine-rich region adjacent to a highly charged COOH terminus. This "hallmark" domain is the basis of the crossreactivity of the highly immunogenic eukaryotic P protein family. We found that T. cruzi-infected individuals have antibodies reacting with host (self) P proteins, as well as with recombinant TcP0. Deletion of the six carboxy-terminal amino acids abolished the reactivity of the T. cruzi infection sera with TcP0. This is similar to the specificity of anti-P autoantibodies described for a subset of patients with systemic lupus erythematosus (SLE) (Elkon, K., E. Bonfa, R. Llovet, W. Danho, H. Weissbach, and N. Brot. 1988. Proc. Natl. Acad. Sci. USA. 85:5186). These results suggest that T. cruzi P proteins may contribute to the development of autoreactive antibodies in Chagas' disease, and that the underlying mechanisms of anti-P autoantibody may be similar in Chagas' and SLE patients. This study represents the first definitive report of the cloning of a full-length T. cruzi antigen that mimics a characterized host homologue in structure, function, and shared antigenicity.

Amino Acid Sequence↗
PubMed1992

Use of synthetic peptides for the detection and quantification of autoantibodies.

The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins). Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B' synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity. Synthetic peptide antigens have been used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantigens↗
PubMed1992

Analysis of autoantibody production in SCID-systemic lupus erythematosus (SLE) chimeras.

Mice with SCID disease have previously been successfully engrafted with human peripheral blood mononuclear cells (PBMC) obtained from normal individuals and from patients with various diseases. To determine whether SCID mice engrafted with SLE PBMC produced autoantibodies with specificities similar to those in the SLE donor, and to investigate which variables influence autoantibody production in the SCID recipients, we injected PBMC from 16 SLE patients into SCID mice and tested the recipients for autoantibodies to DNA and to five recombinant autoantigens. Ten out of 16 (68%) lupus and six out of nine (67%) normal grafts were successful as determined by the presence of human IgG greater than or equal to 5 micrograms/ml of SCID serum post-transfer. Autoantibodies to La/SSB, Ro/SSA, and RNP were detected in five out of 10 SCID-SLE recipients by ELISA and immunoblotting up to 22 weeks post-engraftment. The detection of autoantibodies in SCID-SLE mice was more closely related to autoantibody levels in donor sera than to total IgG concentrations in the SCID recipients. Autoantibody activity/mg IgG was similar in the donor and recipient sera. Histological evaluation of eight SCID-SLE mice killed 4-22 weeks post-transfer revealed population of the SCID thymus and spleen with mononuclear cells, but no evidence of lupus nephritis or dermatitis. These findings indicate that SCID mice can be engrafted with PBMC from patients with lupus and that specific autoantibodies are produced up to 5 months post-transfer. Failure to develop glomerulonephritis may be explained by low or absent anti-DNA antibodies or by changes in the cellular composition of the PBMC grafts.

Animals↗
PubMed1992

MRL/lpr-->severe combined immunodeficiency mouse allografts produce autoantibodies, acute graft-versus-host disease or a wasting syndrome depending on the source of cells.

MRL/lpr (lpr) mice spontaneously develop a lupus-like illness as well as massive lymphadenopathy. Attempts to transfer autoimmunity by adoptive transfer or radiation bone marrow chimeras have been unsuccessful. Since severe combined immunodeficiency (SCID) mice have been engrafted with human and rat xenografts without apparent graft-versus-host disease (GVHD), we subjected SCID mice to low-dose irradiation and reconstituted the mice with spleen cells from young or old lpr mice or with lpr bone marrow. Fourteen out of twenty (70%) of SCID mice engrafted with spleen cells from old lpr mice produced autoantibodies (anti-DNA and anti-Sm) without evidence of the severe lymphoid atrophy previously described for lpr spleen-->+/+ chimeras. SCID mice engrafted with spleen cells from young lpr mice developed acute GVHD and 5/6 (83%) died within 4 weeks post-transfer. Although 8/11 (73%) of lpr-->SCID bone marrow allografts survived for at least 4 months, these mice developed a wasting disease characterized by lymphoid atrophy and fibrosis without the production of autoantibodies. None of the lpr-->SCID grafts resulted in the transfer of double negative T cells or the lymphoproliferative syndrome characteristic of MRL/lpr mice. These findings indicate that SCID mice can be engrafted with splenocytes from old MRL/lpr mice and that B cells continue to secrete autoantibodies for several months in the SCID recipients. This study also demonstrates that, unlike i.p. transplant of xenogeneic cells, acute GVHD is a consistent feature of i.p. transplants of normal allogeneic mononuclear cells into SCID mice.

Animals↗
PubMed1992

Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE.

Adult↗
PubMed1992

Induction of antiphospholipid autoantibodies by immunization with beta 2 glycoprotein I (apolipoprotein H).

A subset of patients with systemic lupus erythematosus has autoantibodies to acidic phospholipids. Since lipids are poor immunogens, the mechanism responsible for the induction of these antibodies is unclear. Immunization of a normal rabbit and normal mice with purified human beta 2-glycoprotein I (apolipoprotein H) resulted in the production of high levels of two non-cross-reactive antibody populations, anti-apolipoprotein H, and antiphospholipid. The antiphospholipid antibodies had binding specificities indistinguishable from autoantibodies obtained from human and murine lupus. These findings suggest a novel mechanism for the induction of antiphospholipid autoantibodies.

Animals↗
PubMed1992

Antiribosomal antibodies in systemic lupus erythematosus.

ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease. Anti-P antibodies appear to be a highly specific diagnostic marker for SLE because they are rarely detected in other multisystem autoimmune disorders. ARA are most frequently directed against the P proteins, and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested. Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances. This may be particularly true of patients with affective disorders. The clinical utility of serologic tests for anti-P in central nervous system lupus must await large, prospective studies. Other ARA antibodies have been detected in patients with SLE. These antibodies include anti-28S rRNA, anti-S10, and anti-L12. In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P. The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome. The L12 protein is the mammalian homologue of the E. coli and yeast proteins known to bind to the 28S rRNA epitope. These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome. This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production. Despite these findings, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful. It therefore seems likely that the ribosomal components must be altered to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production.

Animals↗
PubMed1991

Ribosomal proteins P0, P1, and P2 are phosphorylated by casein kinase II at their conserved carboxyl termini.

A potential casein kinase II (CK II) recognition site is located within the conserved carboxyl (COOH) terminus of the ribosomal P (phospho) proteins P0, P1, and P2. To determine whether the COOH termini of the P proteins are physiological substrates for CK II, we studied the phosphorylation of the P proteins in vitro and in intact cells. The results show that the addition of exogenous purified CK II and ATP to intact ribosomes in vitro resulted in the relatively selective phosphorylation of all three P proteins. A synthetic peptide corresponding to the COOH-terminal 22 amino acids of P2 (C-22) was also phosphorylated by CK II with a Km of 13.4 microM. An endogenous ribosome-associated, CK II-like enzyme also phosphorylated the P proteins relatively selectively in the presence of 10 mM Mg2+ and ATP. The endogenous kinase was inhibited by heparin, utilized either ATP or GTP as a phosphate donor, and phosphorylated casein. A CK II-specific peptide (Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu) and the C-22 peptide inhibited the phosphorylation of the P proteins by the endogenous kinase, providing further evidence for its CK II-like properties and for localization of the CK II phosphorylation site to the COOH termini of the P proteins. Tryptic phosphopeptide maps of P1 and P2 phosphorylated by exogenous CK II and the endogenous ribosome-bound kinase were virtually identical. These phosphopeptides comigrated with the tryptic digest of C-22 and with the tryptic phosphopeptides derived from P1 and P2 isolated from intact cells metabolically labeled with [32P]orthophosphate in vivo. These studies demonstrate that exogenous CK II and a ribosome-bound, CK II-like enzyme phosphorylate the ribosomal P proteins in vitro and localize the target site for phosphorylation to the COOH terminus. The incorporation of phosphate into the same target site in intact cells indicates that the P proteins are in vivo substrates of CK II.

Amino Acid Sequence↗
PubMed1991

The small nuclear ribonucleoproteins, SmB and B', are products of a single gene.

Nucleotide sequencing of HeLa genomic DNA amplified by the polymerase chain reaction revealed 696-bp and 841-bp introns within the gene encoding the proteins SmB and SmB', respectively. Since splice consensus sequences were observed flanking the 145-bp insertion detected in the cDNA encoding SmB, we conclude that SmB and SmB' are derived from alternative splicing of a single gene transcript.

Autoantigens↗
PubMed1991

Detection and quantification of human anti-Sm antibodies using synthetic peptide and recombinant SmB antigens.

Anti-Sm-positive sera from patients with systemic lupus erythematosus (SLE) recognize a major epitope located within the carboxyl-terminal 27 amino acids of a recombinant SmB fusion protein. To determine whether a synthetic peptide corresponding to this region could be used as an antigen to detect anti-Sm antibodies, sera were typed as anti-Sm positive or anti-Sm negative by counterimmunoelectrophoresis (CIE). Twenty-three SLE sera that were anti-Sm positive by CIE, 22 that were anti-Sm negative by CIE, and 42 sera from patients with other autoimmune diseases were tested for anti-Sm antibodies by enzyme-linked immunosorbent assay (ELISA), using either the synthetic peptide (C27) or a recombinant SmB (rSmB) fusion protein as the antigen. More than 90% of the sera that were anti-Sm positive by CIE were also positive by both the C27 and rSmB ELISAs, and an additional 2 SLE sera originally typed as anti-Sm negative were found to be positive (1 by the C27 ELISA, 1 by the rSmB ELISA), due to the greater sensitivity of the ELISAs. In the rSmB ELISA, anti-Sm antibodies were not detected in any of the sera from patients with other autoimmune diseases, whereas 3 patients with anti-U1 RNP antibodies (1 each with polymyositis, scleroderma, and mixed connective tissue disease) had a positive result in the C27 ELISA. These results indicate that both the C27 synthetic peptide and rSmB are excellent antigens for use in ELISAs to quantify anti-Sm antibodies.

Antibodies↗
PubMed1990

Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the polymerase chain reaction.

SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.

Amino Acid Sequence↗
PubMed1989

Antiribosomal S10 antibodies in humans and MRL/lpr mice with systemic lupus erythematosus.

Autoantibodies directed against a ribosomal small subunit protein of 20,000 molecular weight were found in sera from 5 of 44 patients with systemic lupus erythematosus (11%) and 5 of 48 MRL/lpr mice (10%). This ribosomal protein was identified as S10 on the basis of two-dimensional gel electrophoresis and immunoblotting, as well as immunoblots of the purified S10 protein. The S10 protein antigen was readily extracted from ribosomes at low salt (300 mM KCl) and low magnesium (0.5 mM) concentrations, consistent with the highly exposed location proposed for this protein on the 40S subunit. Anti-S10 antibodies were observed significantly more frequently in lupus sera containing both anti-Sm and antiribosomal P protein antibodies and in MRL/lpr sera with anti-Sm activity, suggesting a linked pattern of autoantibody response. Together with anti-Sm and antiribosomal P protein antibodies, anti-S10 represents a third autoantibody highly specific for lupus in humans and MLR/lpr mice.

Animals↗
PubMed1986

Sensory neuronopathy and small cell lung cancer. Antineuronal antibody that also reacts with the tumor.

An autoantibody highly restricted to the nuclei of neurons was found in four patients with subacute sensory neuronopathy and small cell carcinoma of the lung. The antibody was not found in normal subjects or patients with or without cancer who did not have subacute sensory neuronopathy. One- and two-dimensional immunoblotting analysis of nuclear extracts of rat and human brain identified an antibody reactive with a 35-38 kilodalton (kd) basic nucleoprotein. An identical protein lacking the 38 kd peptide was identified in tumor extract of one of the patients. All four patients' serum reacted with the same antigen in the brain and the tumor. In the postmortem study of one of the patients, IgG was found within the neurons of the dorsal root ganglia by direct immunofluorescence. These findings support the hypothesis that the subacute sensory neuronopathy is caused by an antibody that cross-reacts with a tumor cell antigen and a brain nucleoprotein.

Aged↗
PubMed1985

Lupus autoantibodies target ribosomal P proteins.

All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.

Antibody Specificity↗
PubMed1979

Hairy-cell leukaemia with polyarteritis nodosa.

In four patients a systemic vasculitis similar to polyarteritis nodosa developed within 2 years of the onset of hairy-cell leukaemia. Arteriographic studies in two patients revealed microaneurysms, and biopsy specimens in three patients revealed a vasculitis affecting medium-sized vessels. Blood neutrophilia and neutrophilic vascular infiltrate were absent. One patient had circulating immune complexes. Two patients responded to corticosteroids alone, one required cyclophosphamide as well as steroids, and one improved without chemotherapy. The association of vasculitis with hairy-cell leukaemia may provide insight into the pathogenesis of arteritis.

Adult↗