Autoantibody to hLSm4 and the heptameric LSm complex in anti-Sm sera.
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OBJECTIVE: To characterize the 15-kd human SmD-like autoantigen and its associated proteins previously shown to be recognized by IgM antibodies in patients with Epstein-Barr virus (EBV)-induced infectious mononucleosis. METHODS: The full-length complementary DNA for the 15-kd protein was expressed as recombinant protein and analyzed for reactivity using biochemical analysis and immunoprecipitation (IP). RESULTS: The 15-kd protein was determined to be the human like-Sm protein LSm4 (hLSm4). Rabbit antibody raised against the C-terminal polypeptide immunoprecipitated a 68-kd complex composed of LSm4 together with a group of smaller proteins ranging in size from 6.5 to 14 kd, consistent with the reported heptameric LSm complexes involved in U4/U6 duplex formation and messenger RNA (mRNA) decapping/degradation. About 80% of all anti-Sm sera from patients with systemic lupus erythematosus (SLE) recognized the hLSm4 in vitro translated product, while 6.7% (29 of 434) immunoprecipitated from cell extracts hLSm4 together with the other members of the hLSm complex. Four sera (0.92%) showed apparently exclusive reactivity to the hLSm complex in the absence of reactivity to Sm core proteins in the IP assay. CONCLUSION: These findings document that while IgM, but not IgG, autoantibodies to LSm4 were found in sera from patients with EBV infection, IgG autoantibodies to hLSm4 are detected in a large number of anti-Sm-positive sera from patients with SLE. Importantly, in a small number of anti-Sm sera the LSm complex can be recognized independently of the Sm core protein antigens. Our data introduce the concept that "Sm" autoantigens include Sm as well as LSm complexes involved in the maturation and degradation of mRNA. (+info)
Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins.
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Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus. (+info)
Allele-specific expression of imprinted genes in mouse migratory primordial germ cells.
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In somatic cells, imprinted genes are expressed monoallelically according to parent-of-origin. In contrast, in 11.5 days post-coitum primordial germ cells (PGCs), and later stage germ cells, these same genes are expressed biallelically, suggesting that imprints inherited from the gametes are largely erased by this stage. To determine when in germ cell development this biallelic expression phenomenon commences, we isolated migrating PGCs by flow cytometry and determined the allele-specific expression of four imprinted genes - Snrpn, Igf2, H19 and Igf2r. The first three genes were expressed monoallelically, while the latter gene was expressed biallelically. These results show that inherited imprints regulating monoallelic expression are largely intact in migrating PGCs. (+info)
Allele-specific expression analysis by RNA-FISH demonstrates preferential maternal expression of UBE3A and imprint maintenance within 15q11- q13 duplications.
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15q11- q13 contains many imprinted genes, and undergoes duplicon-mediated rearrangements, including deletions, duplications and triplications, and generation of marker chromosomes. Abnormal phenotypes, including language delays and autism spectrum disorders, are primarily observed with maternal 15q11- q13 duplication. To determine possible epigenetic effects on expression within duplicated 15q11- q13 regions, we utilized RNA-FISH to directly observe gene expression. RNA-FISH, unlike RT-PCR, is polymorphism-independent, and it also detects relative levels of expression at each allele. Unamplified, gene-specific RNA signals were detected using cDNA probes. Subsequent DNA-FISH confirmed RNA signals and assigned parental origin by colocalization of genomic probes. SNRPN and NDN expression was detected primarily from paternal alleles. Control Dystrobrevin transcripts were detected equally from both alleles; however, maternal-UBE3A signals were consistently larger than paternal signals in normal fibroblasts and in neural-precursor cells. Larger UBE3A signals were also observed on one or both maternal alleles in a cell line carrying a maternal interstitial duplication, on both alleles of a maternally derived marker(15) chromosome, and occasionally on a paternal allele in a cell line carrying a paternal interstitial duplication. Expression of NDNL2, just distal to the duplicated region, was not markedly altered but paralleled changes in UBE3A expression. Excess total maternal-UBE3A RNA was confirmed by Northern blot analysis of cell lines carrying 15q11- q13 duplications or triplications. These results demonstrate that: (1) UBE3A is imprinted in fibroblasts, lymphoblasts and neural-precursor cells; (2) allelic imprint status is maintained in the majority of cells upon duplication both in cis and in trans; and (3) alleles on specific types of duplications may exhibit an increase in expression levels/loss of expression constraints. (+info)
T cell reactivity against the SmD1(83-119) C terminal peptide in patients with systemic lupus erythematosus.
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BACKGROUND: The SmD1(83-119) peptide is a major target of the B cell response in patients with systemic lupus erythematosus (SLE). OBJECTIVE: To investigate the T cell response directed against this peptide, its disease specificity, and possible impact on SLE pathogenesis. METHODS: Peripheral blood mononuclear cells derived from 28 patients with SLE and 29 healthy and disease controls were stimulated by the SmD1(83-119) and the recombinant (r)SmD1 protein, and [3H]thymidine incorporation was measured. Patients with SLE were simultaneously tested for autoantibodies, disease activity, clinical symptoms, and medical treatments. RESULTS: T cell reactivity against the SmD1(83-119) peptide was detected in 11/28 (39%) patients with SLE and against the rSmD1 protein in 10/28 (36%) patients. In contrast, only 2/29 (7%) controls exhibited SmD1 reactivity. An analysis of proliferation kinetics showed that SmD1 reactive T cells are activated in vivo, as additionally confirmed by cytometric analysis. Addition of mammalian dsDNA to rSmD1 enhanced the rSmD1-specific T cell response. SmD1(83-119)-specific T cell reactivity was significantly more common in patients with cardiac and pulmonary symptoms. No correlation between T and B cell responses and disease activity was seen. CONCLUSION: SmD1(83-119) is a major T cell epitope of SmD1, commonly recognised by T cells from patients with SLE and much less commonly found by healthy or disease controls. This strong T cell reactivity as well as the high frequency and specificity of anti-SmD1(83-119) antibodies in SLE suggest a possible role in SLE pathogenesis, at least in a subset of patients. (+info)
Predicted structure and phyletic distribution of the RNA-binding protein Hfq.
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Hfq, a bacterial RNA-binding protein, was recently shown to contain the Sm1 motif, a characteristic of Sm and LSm proteins that function in RNA processing events in archaea and eukaryotes. In this report, comparative structural modeling was used to predict a three-dimensional structure of the Hfq core sequence. The predicted structure aligns with most major features of the Methanobacterium thermoautotrophicum LSm protein structure. Conserved residues in Hfq are positioned at the same structural locations responsible for subunit assembly and RNA interaction in Sm proteins. A highly conserved portion of Hfq assumes a structural fold similar to the Sm2 motif of Sm proteins. The evolution of the Hfq protein was explored by conducting a BLAST search of microbial genomes followed by phylogenetic analysis. Approximately half of the 140 complete or nearly complete genomes examined contain at least one gene coding for Hfq. The presence or absence of Hfq closely followed major bacterial clades. It is absent from high-level clades and present in the ancient Thermotogales-Aquificales clade and all proteobacteria except for those that have undergone major reduction in genome size. Residues at three positions in Hfq form signatures for the beta/gamma proteobacteria, alpha proteobacteria and low GC Gram-positive bacteria groups. (+info)
Lack of autoantibody production associated with cytomegalovirus infection.
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To confirm an association between cytomegalovirus (CMV) infection and the presence of antibodies to Smith (Sm), to ribonucleoprotein (RNP), and to a component of the U1 ribonucleoproteins (U1-70 kD), we measured antibodies to these protein antigens using an enzyme immunoassay and an immunoblot. The antibodies were measured in the sera of 80 healthy subjects, one-half of whom were naturally CMV seropositive and one-half were CMV seronegative, and in eight subjects immunized with a live attenuated strain of CMV. None of the vaccinees developed antibodies to Sm, to RNP, or to U1-70 kD at either 4 or 12 months after immunization. Additionally, there was no statistically significant association between levels of antibodies to Sm or to RNP and between sera obtained from vaccinees, natural CMV seropositive individuals, and CMV seronegative individuals. One CMV seropositive serum and one CMV seronegative serum tested positive for antibodies to U1-70 kD. These data indicate that neither wild-type infection nor the live-attenuated Towne vaccine frequently induce autoantibody production. (+info)
Familiality and co-occurrence of clinical features of systemic lupus erythematosus.
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OBJECTIVE: To evaluate familiality of 15 clinical and laboratory features in systemic lupus erythematosus (SLE)-affected sibpairs, and to estimate correlations with the age at SLE diagnosis in affected sibpairs and parent-offspring pairs. METHODS: Concordance rates and sibling risk ratios were used as indicators of familiality for 15 manifestations of SLE. Pearson's correlations and paired t-tests were used to compare the age at SLE diagnosis in affected sibpairs and in parent-offspring pairs. RESULTS: Increased sibling risk ratios (1.9-3.9) for thrombocytopenia, discoid rash, neurologic disorder (defined as seizure or psychosis), and hemolytic anemia were observed in 159 SLE-affected sibpairs. Among these clinical features, paired expression of hemolytic anemia plus thrombocytopenia and hemolytic anemia plus neurologic disorder appeared to be more frequent in 709 SLE patients than would be expected by chance (P < 0.00001 and P < 0.007, respectively). The ratio of the presence of both hemolytic anemia and neurologic disorder was approximately 13 times higher in the younger affected sib than in the older affected sib (P < 0.02). Familiality of patient age at SLE diagnosis, as observed by relative correlations, was greater in 125 affected sibpairs (r = 0.67, P < 0.0001) than in 37 affected parent-offspring pairs (r = 0.47, P = 0.003). The median +/- SD age at SLE diagnosis was significantly lower in offspring (21.5 +/- 10.1 years) than in their parents (41.6 +/- 15.8 years) (P < 0.0001) but was not different in sibpairs. The combined non-Caucasian sibpairs had a younger mean age at SLE diagnosis compared with Caucasian sibpairs (P = 0.014). CONCLUSION: Evidence for familiality of thrombocytopenia, discoid rash, neurologic disorder, hemolytic anemia, and co-occurring neurologic disorder plus hemolytic anemia in SLE was observed in 159 affected sibpairs. Familiality of the age at SLE diagnosis in relative pairs suggests that shared genes and/or shared environmental exposures impact disease susceptibility. Shared immediate environmental triggers appear less compelling, because the average time between dates of diagnosis was 11 years in parent-offspring pairs and 7.5 years in affected sibpairs. The significantly earlier age at disease diagnosis in offspring compared with their parents suggests that some forms of anticipation might play a role in susceptibility to SLE. Stratifying families by subphenotypes that are familial may reduce heterogeneity and facilitate identification of genetic risk factors for SLE. (+info)