Biomedical subjects
Publications and source records attributed to K B Elkon.
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.
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.
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.
Anti-Sm and anti-ribosomal P protein antibodies show a high degree of specificity for the disease SLE. To determine whether a relationship between these two autoantibodies existed, the frequency of anti-P was determined in sera with and without anti-Sm activity. Of sera from lupus patients with anti-Sm 18/65 (28%), and 6/55 (11%) of sera without anti-Sm had anti-P as determined by an ELISA using a recombinant P2-beta-galactosidase fusion protein as Ag (p less than 0.05). The levels of anti-P were significantly higher in sera containing anti-Sm (0.37 +/- 0.45) than in sera without anti-Sm antibodies (0.18 +/- 0.20) (p less than 0.01). Similarly, a significantly higher proportion of anti-P positivity was found in autoimmune MRL/Mp-lpr/lpr mice positive for anti-Sm (11/53 = 21%) compared to age- and sex-matched mice without anti-Sm (3/53 = 6%) (p less than 0.05). The IgG subclass distributions for anti-Sm and anti-P antibodies were similar in the MRL mice (IgG2a greater than IgG2b greater than IgG3 greater than IgG1). The association did not reflect polyclonal B cell activation in a proportion of MRL mice because no significant differences were observed in anti-DNA, antichromatin or total serum IgG levels in mice with and without anti-Sm or, in mice positive for both anti-P and anti-Sm compared to mice positive for anti-Sm alone. Cross-inhibition experiments excluded the possibility that the Sm and P protein Ag shared a common epitope. Longitudinal measurement of anti-P and anti-Sm antibody levels by ELISA in three mice indicated that both antibodies first appeared at about 3 to 4 mo of age and fluctuated two- to threefold over 3 to 8 mo with independent peaks of activity. Recent observations regarding a relationship between anti-Sm and autoantibodies to other ribosomal proteins suggest that the association may be explained by an immune response to epitopes coassociated on the ribosome.
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.
The frequency and nature of IgG anti-cardiolipin and anti-ds-DNA antibodies among MRL/lpr, MRL/+ and NZB/W F1 mice (murine lupus strains) and non-autoimmune inbred strains of mice (NIH/Swiss and Balb/c) were analysed by ELISA. High titres of anti-ds-DNA were detected in autoimmune strains (MRL/lpr, 69%; MRL/+, 50%; and NZB/W, 80% positive), whereas anti-cardiolipin antibodies were detected only in MRL/lpr (69%) and MRL/+ (17%) mice. IgG subclass analysis of these antibodies in 20 MRL/lpr sera revealed that all four subclasses were represented. When tested for fine antigenic specificity, anti-cardiolipin antibodies in MRL/lpr and MRL/+ mice bound to acidic phospholipids rather than to neutral phospholipids and were not inhibited by DNA. In MRL/lpr mice, anti-cardiolipin antibodies were first detected at 2 months, peaked around 5 months and then declined preterminally. To determine whether complications associated with anti-cardiolipin antibodies were present in MRL/lpr mice, blood counts were performed and litter sizes were determined. Although no significant decreases in the red and white blood cell counts were observed in MRL/lpr mice, platelet counts were significantly lower compared with NIH/Swiss (P < 0.001) and Balb/c (P < 0.005) mice. MRL/lpr mice had significantly fewer pups per delivery compared with a normal strain (MRL/lpr, 5.3+2.6; NIH/Swiss, 72 +/- 2.1; P < 0002). These observations indicate that the serological characteristics of IgG anti-cardiolipin antibodies in MRL/ lpr mice are similar to those of anti-cardiolipin antibodies in humans with lupus. Whether these autoantibodies are pathogenetically related to thrombocytopenia and a small litter size in MRL/lpr mice remains to be determined.
The cDNA encoding the ribosomal protein P2 antigen was cloned from a human cDNA library constructed in the lambda gt11 expression vector. A beta-galactosidase-P2 fusion protein was purified to near homogeneity and used to develop an ELISA which was highly specific for anti-P antibodies produced in murine and human SLE. The median concentration of human IgG anti-P antibodies in serum was estimated to be 100 micrograms/ml (range 6-450 micrograms/ml). Pre-incubation of human anti-P sera with a synthetic peptide, corresponding to the C-terminal 22 amino acids of P2, completely inhibited reactivity with the fusion protein in the ELISA. These findings confirm that lupus anti-P sera show a striking restriction in epitope specificity and indicate that the P2 fusion protein is a useful alternative to the synthetic peptide antigen for detection and quantification of anti-P antibodies. To investigate the possibility that anti-P antibodies were induced by 'altered-self', cDNA encoding P2 were also cloned from lupus patients and control mononuclear cells. The predicted amino acid sequences of the patients' P2 were identical to that of the normal controls indicating that a primary structural abnormality of the P2 autoantigen was unlikely.
Antibody binding to total protein extracted from a mammalian source (HeLa cells) and from a prokaryotic source (Escherichia coli) was compared in sera from patients with systemic lupus erythematosus (SLE) and sera from normal subjects. When the average numbers of peptides or proteins recognized by IgG antibodies were compared on immunoblots, SLE sera bound to a significantly greater number of proteins from the HeLa cell extract than did sera from normal individuals (P less than 0.001). In contrast, SLE sera actually bound to fewer E coli proteins than did the sera obtained from normal controls, although the difference was not statistically significant. There was no correlation between the number of E coli proteins and HeLa proteins recognized by individual SLE sera, and there was no trend toward reactivity with a larger number of antigens in sera containing higher levels of IgG. IgG from SLE sera did not bind to 6 purified eukaryotic protein standards (selected solely on the basis of differences in size and charge) either in their denatured state or in their native state. These findings indicate that the high levels of IgG antibodies against selected eukaryotic intracellular proteins in patients with SLE cannot be explained by a random polyclonal B cell activation.
Improved methods for high resolution composite gel electrophoresis under dissociating conditions and electrophoretic transfer of immunoglobulins (Mr 150,000-1,800,000) to nitrocellulose have been developed. Using these techniques and highly specific antisera to detect either light or heavy chains on nitrocellulose transfers, the immunoglobulin and clonal composition of washed cryoglobulins (3 micrograms each) could be determined within two days. The results were confirmed by isoelectric focusing and immunoblotting of selected samples. In addition, the method was used for detection of an idiotypic determinant and rheumatoid factor activity within components of the cryoglobulins.
Immunoglobulin G (IgG) antibodies reactive with intracellular components of transformed cells were detected in 26/35 sera from patients with melanoma using immunofluorescence and/or Western blotting. By extracting cellular proteins with either sodium dodecyl sulphate or moderate concentrations of salt (400 mM NaCl), the protein antigens were partially characterized by immunoblotting procedures. Although considerable heterogeneity in the molecular weights of the protein antigens was observed, two common groups were delineated. The anti-Pol antibodies reacted with 30 kd cytoplasmic protein and the anti-Ca antibodies recognized acidic high molecular weight (75-95 kd) proteins. These antigens were detected in all transformed cell lines tested, but were not restricted to them. Anti-Ca and anti-Pol antibodies were not found in sera from patients with other solid tumors or in systemic lupus erythematosus.
The IgG subclass and light chain distribution of anticardiolipin and anti-DNA antibodies were determined in serum samples from patients with systemic lupus erythematosus. With an enzyme linked immunosorbent assay (ELISA) and mouse monoclonal antibodies to individual subclasses, significant differences in the distributions of IgG2, IgG3, and IgG4 subclasses were observed between anticardiolipin and anti-DNA antibodies. Whereas anti-DNA antibodies were predominantly IgG1 and IgG3, all subclasses of anticardiolipin were detected with a prevalence ranging from 34% (IgG3) to 57% (IgG1). Clinical complications were found slightly more frequently (83%) in patients with sera containing the non or weak complement fixing subclasses (IgG2 and IgG4) than in patients with sera containing complement fixing (IgG1 and IgG3) subclasses (62%). Light chain analysis by ELISA showed a trend towards use of kappa chains for anti-DNA and lambda chains for anticardiolipin antibodies. These findings further emphasise the differences between anti-DNA and anticardiolipin antibodies in terms of their origins and potential mechanisms for producing tissue injury.
In 18 of 20 patients with psychosis secondary to systemic lupus erythematosus (SLE), autoantibodies to ribosomal P proteins were detected by immunoblotting and measured with a new radioimmunoassay using a synthetic peptide as antigen. The frequency of anti-P was not increased in patients with other central nervous system manifestations of SLE (3 of 20, by radioimmunoassay), in patients with transient behavioral abnormalities due to SLE (none of 8), in patients with psychosis who did not have SLE (none of 13), or in normal controls (none of 20). In four of five paired serum samples, anti-P-peptide antibody levels increased 5-fold to 30-fold during the active phase of lupus psychosis. Longitudinal studies of anti-P activity in two patients with psychosis revealed that anti-P levels increased before and during the active phases of psychosis but not during sepsis or other exacerbations of SLE, and that the elevations were selective for anti-P antibodies, as opposed to anti-DNA antibodies. Longitudinal studies of anti-P activity in two patients with anti-P but without psychosis showed less than threefold changes in anti-P levels despite exacerbations of disease. We conclude that anti-P is associated with lupus psychosis and that synthetic peptide antigens may be useful for the detection and measurement of autoantibodies to intracellular proteins.
A 22-amino acid synthetic peptide (P peptide) containing the conserved, shared autoantigenic determinants of the ribosomal P proteins was conjugated to rabbit serum albumin and used to analyze anti-P heterogeneity in 17 lupus sera. Anti-P peptide antibodies demonstrated moderate restriction in isotype (IgM and IgG, but not IgA), subclass (predominantly IgG1 and IgG2), light chain type (predominantly kappa) and spectrotype. In one serum, almost exclusive use of IgG2 and the kappa light chain was observed. These findings indicate that there is a nonrandom selection of heavy and light chain constant region genes as well as limited variable region diversity in the anti-P peptide response.
A patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer had a clinical presentation of acute polyneuritis. The patient had an antibody (anti-Hu) restricted to nuclei of neurons identical to that reported in patients with subacute sensory neuronopathy and lung cancer. This finding further supports the hypothesis that PEM and subacute sensory neuronopathy are closely related disorders of autoimmune origin. PEM should be considered in patients with small cell lung cancer and clinical features limited to the peripheral nervous system.
Sera from 16 patients with falciparum malaria, 16 patients with vivax malaria and 31 patients with leprosy were tested for autoantibodies to intracellular proteins and nucleic acids. Precipitating antibodies to soluble protein extracts were not detected in any serum. Sera from malaria patients showed prominent immunofluorescence staining of the HEP2 nuclear membrane as well as frequent 75% (24/32) and intense Western blot reactivity. In contrast, only 20% and 36% of patients with leprosy had positive immunofluorescence or positive immunoblots respectively, and reactivity was weak in most cases. Neither the malaria nor leprosy sera contained autoantibodies with specificities similar to the characteristic lupus autoantibodies such as double stranded DNA (dsDNA), Ro/SSA, La/SSB, Sm, RNP and P proteins. Low levels of antibodies to single stranded (ssDNA) were however found in 11 (34%) malaria sera and in seven (23%) leprosy sera. Thirteen percent of patients with leprosy had anti-histone antibodies. These findings demonstrate considerable differences in the capacity of infectious agents to induce autoantibodies and also the infrequency with which autoantibodies characteristic of idiopathic systemic lupus erythematosus are induced.
Antibodies to the ribosomal P proteins (anti-P) were detected, by Western blot analysis, in the sera of 20 of 114 patients with various autoimmune disorders. Eighty-five percent of the patients with anti-P had systemic lupus erythematosus (SLE). Of 93 randomly selected patients, the frequency of anti-P was 7 of 59 SLE patients (12%) and 0 of 34 non-SLE patients. Approximately one-third of the patients with anti-P antibodies were male; approximately half were black. In contrast to the findings of some previous studies which used isolated ribosomes as antigen, an increased frequency of renal disease was not observed. Although the overall frequency of central nervous system lupus was similar in SLE patients with and those without anti-P, 6 of 6 patients with psychosis had anti-P antibodies. Western blotting was the most sensitive and specific method for the detection of anti-P antibodies; counter-immunoelectrophoresis and cytoplasmic indirect immunofluorescence were positive in only 47% and 65% of the anti-P-positive patients, respectively. Although 53% of the SLE patients with anti-P had concomitant anti-Ro antibodies, none had anti-La (as detected by counterimmunoelectrophoresis). Anti-P antibodies, therefore, appear to be relatively specific serologic markers for SLE and may be detected in the serum even when antibodies to double-stranded DNA are not found.
Autoantibodies may play an important role in the pathogenesis of central nervous system (CNS) disease in systemic lupus erythematosus (SLE). We obtained cerebrospinal fluid (CSF) and, in some cases, sera from 19 SLE patients with CNS lupus and from 12 SLE patients without CNS lupus. Autoantibodies to saline soluble cellular antigens were detected in the CSF of lupus patients and reflected those present in the serum. These antibodies were distinct from the previously described antineuronal antibodies. Analysis of the fine specificities of the anti-saline soluble cellular antigen antibodies revealed that the antiribosomal P protein antibody was present in 4 of 4 patients with lupus psychosis and was enriched in the CSF of 1 patient. Sera containing antiribosomal P protein showed prominent cytoplasmic staining of human cortical neurons, as well as an epithelial cell substrate. These observations, together with the increase in intrathecal IgG synthesis detected in 71% of patients tested, suggest that several populations of antibodies may contribute to the enhanced immunologic activity in the CSF of CNS lupus patients.
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.