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Biomedical subjects

K B Elkon

Publications and source records attributed to K B Elkon.

At least 73 records · Page 4Linked to original sources
PubMed1994

Autoantigen-specific T cell proliferation induced by the ribosomal P2 protein in patients with systemic lupus erythematosus.

A role for helper T cells in the induction of pathogenic lupus autoantibodies is increasingly supported by data from studies of murine lupus and patients with systemic lupus erythematosus (SLE). However, the poor in vitro function of SLE T cells has hampered the identification and characterization of autoantigen-specific T cells. We used recombinant fusion proteins to study the T cell proliferative response of 31 lupus patients and 27 healthy subjects to a well-characterized SLE autoantigen, the ribosomal P2 protein. Although PBMC from SLE patients showed marked impairment in the proliferative response to the common recall antigen tetanus toxoid when compared with normal subjects, a significantly greater proportion of SLE patients (32%) than normal individuals (0%) showed a T cell response to a recombinant P2 fusion protein. When the SLE patients were subgrouped according to the presence of serum anti-P autoantibody, 7 of 10 anti-P antibody-positive patients, but 0 of 20 anti-P antibody-negative SLE patients, demonstrated > 2,000 cpm [3H]thymidine incorporation and a P2 stimulation index > 5. The specificity of the T cell proliferative response for the P2 protein was confirmed by studies using a second recombinant human P2 fusion protein and by the specific activation of P2-primed T cells by recombinant P2 in secondary cultures. Moreover, the T cell proliferative response to the P2 autoantigen was mediated by CD4-positive T cells and was inhibited by anti-MHC class II antibodies. These data demonstrate the presence of autoantigen-specific T helper cells in patients with SLE and suggest that these T cells drive the production of autoantibodies by B lymphocytes.

Autoantigens↗
PubMed1994

Autoimmunity versus allo- and xeno-reactivity in SCID mice.

The ability of SCID mice to accept xenografts has been exploited to study the survival, function and potential of peripheral blood mononuclear cells (PBMC) from patients with autoimmune disorders to produce tissue injury in the mouse. Studies performed with PBMC obtained from patients with organ specific and multisystem autoimmune diseases indicate that human PBMC survive in SCID mice for several months, produce IgG and autoantibodies with the same specificities as are found in the donor. Tissue injury is not generally observed in the SCID mouse recipient. SCID mice have also been partially reconstituted with bone marrow from BB (diabetic) and MRL (lupus) mice. SCID mice injected with both spleen cells from mice with collagen induced arthritis together with native bovine collagen developed more severe arthritis than the donors. SCID mice have therefore proven to be a useful resource to study autoimmunity. In both xeno- and allografts of mature lymphocytes, graft versus host reactions occur. Further studies will be necessary to improve donor cell survival without aggravating graft versus host disease.

Animals↗
PubMed1993

Occurrence of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis.

OBJECTIVE: To determine the prevalence and specificity of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis and assess whether increased levels of antibodies to B. burgdorferi were attributable to rheumatoid factor. DESIGN: Retrospective case-control study. SETTING: Urban referral center in an area devoid of infected ticks as a source of endocarditis sera. PATIENTS: Sera from 30 consecutive patients with culture-proven subacute endocarditis between 1979 and 1981 were compared with 30 control sera collected between 1989 and 1990. In addition, sera from 20 consecutive patients with rheumatoid arthritis who were positive for rheumatoid factor were collected between 1991 and 1992. Sera were compared with a convenience sample from 15 patients who met the criteria for Lyme disease. MEASUREMENTS: Antibodies to B. burgdorferi were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. IgM rheumatoid factor was quantified using solid-phase radioimmunoassay or latex agglutination techniques. RESULTS: Thirteen of 30 patients with endocarditis (43%) compared with 3 of 30 normal controls (10%) had increased levels of antibodies to B. burgdorferi (P < 0.01). Of these 13 patients, only 1 had an immunoblot consistent with previous infection. The others had nonspecific immunoblots: 5 showed isolated 60-kd reactivity; 1 patient had isolated 41-kd reactivity; and 6 had no bands of reactivity. Immunoblots of the 3 controls with increased antibodies showed only isolated 41-kd reactivity. Thus, the specificity of the B. burgdorferi antibody test in patients with endocarditis was only 60% (95% CI, 42% to 78%), compared with 90% (CI, 79% to 100%) in controls. No correlation was noted between IgM rheumatoid factor and antibodies to B. burgdorferi in patients with endocarditis (r = 0.2; P > 0.2). Only 1 of 20 patients with rheumatoid arthritis without known bacterial infections had antibodies to B. burgdorferi. CONCLUSIONS: Although a positive ELISA test for B. burgdorferi may be a "true positive," a positive serologic test alone does not ensure that the clinical problem is due to Lyme borreliosis. Cross-reactive antibodies to shared epitopes between B. burgdorferi and the endocarditis organism may account for the high false-positive results.

Antibodies, Bacterial↗
PubMed1993

The Fas protein is expressed at high levels on CD4+CD8+ thymocytes and activated mature lymphocytes in normal mice but not in the lupus-prone strain, MRL lpr/lpr.

The lymphoproliferation (lpr) mutation in the MRL strain of mice is caused by the insertion of the early transposable element ETn in the Fas gene. The insertion causes a striking decrease in Fas mRNA expression and is associated clinically with marked acceleration of the lupus-like disease. To further explore the role of the Fas protein in T-cell selection in the thymus and tolerance in the peripheral immune system, we produced a monospecific polyclonal anti-murine Fas antibody that binds to a polymorphic region of the protein. Fas protein expression was detected on approximately 90% of BALB/c and MRL +/+ thymocytes, and the expression was highest on CD4+CD8+ thymocytes, the stage at which most thymocytes die by apoptosis. In contrast to the high level of expression of Fas on thymocytes, Fas was detected on < 10% of normal splenic T cells. After activation of splenic T cells with Con A or anti-CD3 and interleukin 2, Fas expression increased approximately 10-fold. Fas expression on splenic B cells was also markedly up-regulated after activation with lipopolysaccharide or anti-mu antibodies. The Fas protein was not detected on resting or activated lymphocytes obtained from MRL lpr/lpr mice. Together, these findings suggest that Fas plays a role in both thymic selection and T-cell survival in the periphery and that the accelerated autoimmunity in MRL lpr/lpr mice results from a defect in both of these pathways.

Amino Acid Sequence↗
PubMed1993

The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn.

Fas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/lpr mice results in massive lymphoproliferation (lpr) and accelerated autoimmunity. To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wild-type Fas message was produced at approximately 10-fold lower levels in the lpr compared with the ++ substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts containing an insertion of the early retrotransposon (ETn). Molecular cloning and nucleotide sequencing of a Fas-ETn chimeric cDNA suggested that the striking reduction in wild-type Fas mRNA levels and the presence of aberrant transcripts in MRL/lpr mice are most likely explained by the insertion of the ETn retrotransposon into an intron of the Fas gene and induction of alternative splicing involving the 5' ETn long terminal repeat.

Animals↗
PubMed1993

The SCID mouse as a vehicle to study autoimmunity.

Peripheral blood mononuclear cells (PBMC) injected into severe combined immune deficiency (SCID) mice continue to secrete human immunoglobulin and respond to immunization with recall antigens. PBMC for patients with autoimmune diseases produce autoantibodies of the same specificities but at lower levels compared to the donor. SCID recipients of patients' PBMC fail to develop clinical disease although some histological lesions suggestive of autoimmunity have been reported. Transfer of autoimmunity from rodents to SCID mice has been successful in some instances. Despite obstacles related to limited survival and varying degrees of graft vs host disease (GVHD), SCID mice should prove to be a useful vehicle to explore autoantibody regulation.

Animals↗
PubMed1993

Penetration of autoantibodies into living epithelial cells.

The ability of autoantibodies to penetrate living cells is controversial. We have identified immunoglobulin G (IgG) antibodies capable of penetrating an epithelial cell line, COLO-16, in five of 36 (14%) antinuclear antibody positive sera from patients with SLE. Thirty minutes following incubation of cells with dilutions of either whole sera, globulin fractions, or F(ab')2 fragments of IgG, approximately 80-90% of cells demonstrated intranuclear IgG by indirect immunofluorescence. Viability of cells prior to assay was > 98% as determined by trypan blue staining and penetration of IgG into the nuclei did not affect viability or DNA synthesis of the cells in short-term culture. Intracellular IgG could not be detected following exposure of the cells to high-titer reference autoantibodies of known specificities (against DNA, Ro, La, Sm, RNP, or ribosomes). Furthermore, absorption of the sera with either DNA or chromatin failed to abolish intranuclear penetration, indicating that the autoantibodies were not directed against DNA receptors or nucleosomes on the cell surface. Antibody uptake was relatively selective for epithelial cell lines, because intranuclear IgG was not detected in cell lines of lymphoid origin exposed to the sera. Two of the five sera immunoprecipitated proteins of molecular weight 88 kD with or without a 68-kD protein from COLO-16 cells labeled with 125I at the cell surface. These findings indicate that a subset of SLE patients have IgG capable of penetrating a cell line of epithelial origin. These antibodies, most likely, bind to cell surface proteins and are translocated into the cell nucleus. Although direct immunofluorescence of a skin biopsy obtained from one of the five patients with "penetrating IgG" also showed intranuclear staining for IgG, the biologic relevance of these findings remains to be determined.

Antibodies, Antinuclear↗
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↗