PCR-RFLP genotyping of murine MHC haplotypes.
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Animals↗
Biomedical subjects
J Craft
Publications and source records attributed to J Craft.
MHC class I polymorphism in lupus-prone MRL/Mp mice.
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Amino Acid Sequence↗
Pattern of anti-small nuclear ribonucleoprotein antibodies in MRL/Mp-lpr/lpr mice suggests that the intact U1 snRNP particle is their autoimmunogenic target.
MRL/Mp-lpr/lpr (MRL/lpr) mice develop anti-Sm Abs that bind the B and D proteins of the U1 and other U small nuclear ribonucleoproteins (snRNPs). Humans with SLE also develop anti-Sm, but in contrast to MRL/lpr mice, anti-Sm Abs in humans are virtually always accompanied by anti-snRNP Abs that bind the A and 70 kDa proteins of the U1 snRNP. In this study, we identified anti-U1 snRNP Abs in 60% of MRL/lpr mice (40 of 66 animals), with the earliest and most frequent response directed against the A protein. This response was either accompanied or closely followed by Abs to other snRNP proteins, including the 70-kDa protein of the U1 particle and the B and D proteins that represent the anti-Sm response. Other U snRNPs were rarely bound by these sera, analogous to previous observations in human SLE. Using in vitro translated 5' and 3' deletion mutants, we found that these early Abs principally bound an epitope on the COOH-terminal of the murine A protein. As shown previously, human sera with anti-U1 snRNP reactivity bind a similar epitope. In summary, we have shown that anti-snRNP Abs in MRL/lpr mice are composed of a linked group of specificities against proteins of the U1 snRNP particle, similar to human SLE. These observations, in conjunction with our previous findings that intact snRNPs are necessary for diversification of Abs in normal mice immunized with snRNPs, strongly suggests that intact U1 particles may be targets of the immune response in this disease.
Animals↗
B cells process and present lupus autoantigens that initiate autoimmune T cell responses.
Antibodies against U small nuclear ribonucleoprotein (snRNP) particles are a common finding in the sera of humans with SLE and in certain strains of mice with murine lupus. It is likely that Th cells are important in amplifying this autoantibody response. The focus of this work was to investigate events that might initiate autoimmune B and T cell response in non-autoimmune mice to native snRNP particles. Mice that were immunized and boosted with native mouse snRNPs failed to produce any detectable specific anti-snRNP antibody or T cell responses, suggesting that these autoreactive cells were deleted from the repertoire or were anergic to stimulation with this self Ag. In contrast, immunization with native foreign (human) snRNPs elicited both T cells and cross-reactive anti-snRNP antibodies; the latter predominantly were directed toward the A protein of the U1 snRNP. When mice were immunized with human and mouse snRNPs together in adjuvant, T cells specific for mouse snRNPs could be elicited. The results of these experiments suggested that the mechanism of breaking T cell tolerance to self snRNPs was dependent on the ability of cross-reactive B cells to process and present these autoantigens. To address this hypothesis, B cells purified from mice immunized with recombinant human A protein were transferred into naive mice. Upon boosting with native mouse snRNPs, autoreactive CD4+ T cells specific for mouse Ags, and not cross-reactive with human snRNPs, were observed. These studies support a model of molecular mimicry whereby autoantigen-presenting B cells are generated by foreign cross-reactive determinants that can, in turn, elicit an autoimmune T cell response.
Animals↗
The expression of self antigenic determinants: implications for tolerance and autoimmunity.
T lymphocytes respond to small peptides in the context of major histocompatibility molecules and a host of other cell-surface proteins on antigen-presenting cells. By design, therefore, T-cell responses are dependent on the efficient and accurate processing of both foreign and self peptides by antigen-presenting cells. This review examines the functions of T cells that may be specific for self peptides processed and presented under less than ideal conditions or outside the normal pathways of antigen processing. Do these T cells survive selection events and remain in the repertoire of normal lymphocytes? Moreover, can these cells become activated and are they important in the pathogenesis of autoimmunity?
Animals↗
Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles.
The U1 small nuclear ribonucleoprotein (sn-RNP) particle, which consists of the U1 small RNA and multiple polypeptides, is a central target of the autoimmune response in systemic lupus erythematosus. Autoantibodies to the individual proteins of the U1 snRNP typically co-occur in patients with systemic lupus erythematosus, an observation reconciled by postulating that the intact RNA-protein complex serves as the autoimmunogen and that snRNP-specific autoreactive T cells are necessary for autoantibody production. In this study, we demonstrated that normal mice did not develop antibody responses following immunization with purified self (murine) snRNPs. However, when such mice were coimmunized with self snRNPs in conjunction with the human (foreign) U1 snRNP A protein, they developed autoantibodies directed against individual proteins of the U1 snRNP, in addition to anti-A antibodies; we have previously shown that such mice develop snRNP-specific, autoreactive T cells. Intact snRNPs as a co-immunogen were a prerequisite for antibody expansion, since this response was abrogated by disruption of snRNP particles with pancreatic RNase prior to immunization. These findings indicate that autoreactive helper T cells can drive autoantibody production to the individual proteins of snRNP particles and that such autoantibody responses may require the presence of intact snRNP particles that possess intrastructural B-cell and helper-T-cell determinants. These results also suggest that induction of an immune response to one component of an autoantigenic snRNP complex, possibly through priming with molecular mimics, can induce the diversification of autoantibodies that is characteristic of that found in patients with systemic lupus erythematosus.
Animals↗
Nucleotide sequence analysis of the A protein of the U1 small nuclear ribonucleoprotein particle: the murine protein contains a 5' amino-terminal tag.
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Amino Acid Sequence↗
Antimicrobial prophylaxis after tick bites.
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Animals↗
Autoantigenic epitopes of the B and D polypeptides of the U1 snRNP. Analysis of domains recognized by the Y12 monoclonal anti-Sm antibody and by patient sera.
Anti-Sm antibodies, a specific marker for SLE, are directed against the B'/B and D polypeptides of Sm small nuclear ribonucleoproteins. The Y12 monoclonal anti-Sm antibody (Y12 mAb), as well as many anti-Sm patient sera, recognize cross-reactive epitopes on the B'/B and D polypeptides. This immunoreactive site is of special interest since polypeptides B and D share little amino acid sequence homology. In the present study, we have sought to establish the autoantigenic domain of polypeptides B and D that accounts for this epitope. We tested the ability of the Y12 mAb and anti-Sm sera to immunoprecipitate truncated forms of polypeptides B and D translated in vitro from mRNA bearing 5' and 3' end deletions. Most anti-Sm sera bound epitopes at the carboxyl-terminus of polypeptide B, however, autoantigenic epitopes were also found at the amino-terminus (amino acids 1 to 83 and 104 to 115). Surprisingly, the Y12 mAb recognized nonoverlapping amino-terminal and carboxyl-terminal halves of polypeptide B. One putative Y12 mAb binding site (amino acids 104 to 115) indicated by carboxyl-terminal deletion studies was confirmed through recognition of a corresponding synthetic peptide. Deletion studies with polypeptide D demonstrated a major autoantigenic domain on the carboxyl-terminus (amino acids 85 to 119) that was necessary for recognition by the Y12 mAb and by 7/14 patient sera. These results indicate that a cross-reactive epitope on B'/B and D, as defined by the Y12 mAb, resides on at least two different domains of polypeptide B and localizes to the carboxyl-terminus of polypeptide D. From the shared homology of truncated forms of B and D polypeptides recognizable with the Y12 mAb, we suspect that some form of GRG motif is involved in developing the Y12 mAb epitope that may involve other residues and be largely conformational in nature.
Amino Acid Sequence↗
Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.
Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.
Animals↗
Comparison of the Drosophila melanogaster, human and murine Sm B cDNAs: evolutionary conservation.
To analyze the evolutionary stability of the Sm B polypeptides, the cDNA nucleotide (nt) sequence was derived for the Drosophila melanogaster Sm B polypeptide and compared to the cDNAs encoding human and murine Sm B. The three cDNAs were transcribed and translated in reticulocyte lysates followed by analysis of the synthesized proteins by SDS-PAGE. D. melanogaster B migrated at approximately 25 kDa, in comparison to 28 kDa for the murine and human B proteins, although all three proteins were immunoprecipitated by human anti-Sm autoantibodies and by the Y12 anti-Sm murine monoclonal antibody (Y12 mAb). Immunoblots and immunoprecipitations of [35S]methionine-labeled D. melanogaster S2/M3 cells confirmed the smaller size of the D. melanogaster protein, and revealed that B' was absent in this cell line, as in murine cells. In comparison to the 231 amino acids (aa) of human and murine B, the deduced sequence for the D. melanogaster clone was 199 aa (predicted M(r) of 24,598) with two 5-aa deletions and a 19-aa truncation at the 3' end, compared to the other two clones. D. melanogaster protein B shared 65% aa sequence identity with the human and mouse clones, and 80% similarity when conservative aa substitutions were noted. The C-terminal portion of the D. melanogaster protein was the most evolutionarily variable in comparison to the deduced aa sequences for the other two proteins; however, autoantigenic epitopes bound by human anti-Sm antibodies and the Y12 mAb in this region of the protein were conserved across species lines.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III.
In this study, autoantibodies to RNA polymerase II from sera of patients with systemic sclerosis have been identified and characterized. These antibodies immunoprecipitated polypeptides of 220 kD (IIA) and 145 kD (IIC), the two largest subunits of RNA polymerase II, and bound both subunits in immunoblots. These polypeptides were immunoprecipitated by the anti-RNA polymerase II monoclonal antibody 8WG16, which recognizes the carboxyl-terminal domain of the 220-kD subunit, and their identity to the proteins bound by human sera was confirmed in immunodepletion studies. Sera with anti-RNA polymerase II antibodies also immunoprecipitated proteins that were consistent with components of RNA polymerases I and III. In vitro transcription experiments showed that the human antibodies were an effective inhibitor of RNA polymerase II activity. In indirect immunofluorescence studies, anti-RNA polymerase II autoantibodies stained the nucleoplasm, as expected from the known location of RNA polymerase II, and colocalized with the anti-RNA polymerase II monoclonal antibody. The human sera also stained the nucleolus, the location of RNA polymerase I. From a clinical perspective, these antibodies were found in 13 of 278 patients with systemic sclerosis, including 10 with diffuse and three with limited cutaneous disease, but were not detected in sera from patients with other connective tissue diseases and from normal controls. We conclude that anti-RNA polymerase II antibodies are specific to patients with systemic sclerosis, and that they are apparently associated with antibodies to RNA polymerases I and III. These autoantibodies may be useful diagnostically and as a probe for further studies of the biological function of RNA polymerases.
Adenoviridae↗
Prevention of Lyme disease after tick bites. A cost-effectiveness analysis.
BACKGROUND: In areas of endemic disease, the probability of Lyme disease after a tick bite ranges from about 0.012 to 0.05. Early treatment with oral antibiotics prevents most complications of Lyme disease, but antibiotics are generally not prescribed until rash or other symptoms develop. METHODS: We used decision analysis to evaluate the outcomes, costs, and cost effectiveness of three alternative strategies to treat patients bitten by ixodes ticks in areas of endemic Lyme disease: empirically treat all patients with two weeks of doxycycline, treat only patients in whom erythema migrans develops, and treat only patients with erythema migrans or a positive serologic test for Lyme disease one month after exposure. RESULTS: Empirical treatment is the least expensive strategy and results in the fewest cases of Lyme disease and the fewest complications when the probability of Borrelia burgdorferi infection after a tick bite is 0.036 or higher. For probabilities of infection below 0.036, empirical therapy prevents most major complications, sequelae, and adverse events, but it incurs additional minor complications, especially as the probability of infection falls below 0.01. CONCLUSIONS: Empirical treatment of patients with tick bites is indicated when the probability of B. burgdorferi infection after a bite is 0.036 or higher, and this treatment may be preferred when the probability of infection ranges from 0.01 to 0.035. When the probability of infection after a tick bite is less than 0.01, empirical therapy is not warranted.
Animals↗
The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.
The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.
Animals↗
Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease.
OBJECTIVE: To elucidate the profile and clinical significance of autoantibodies to small nuclear and small cytoplasmic ribonucleoproteins in Japanese patients with polymyositis and dermatomyositis (PM/DM). METHODS: Radioimmunoprecipitation assays were performed with sera from 91 patients with various inflammatory muscle diseases and from 254 control patients with other rheumatic diseases. Patients with PM/DM were categorized according to autoantibody specificities, and clinical comparisons were made. RESULTS: Antibodies to aminoacyl transfer RNA (tRNA) synthetases and the signal recognition particle (SRP) were found to be specific for PM/DM: PM/DM patients with antibodies to 3 aminoacyl-tRNA synthetases (histidyl, threonyl, and glycyl) appeared to form a distinct clinical subset, associated with chronic interstitial lung disease, polyarthritis, and myositis, while 3 patients with antibodies that bound the SRP had severe myositis that was resistant to corticosteroid therapy. In general, the frequency and clinical correlations of anti-aminoacyl-tRNA synthetase antibodies in Japanese patients were similar to those in North American populations. In contrast, among this Japanese cohort, the occurrence of anti-PM/Scl appeared to be reduced in frequency, whereas anti-Ku and anti-U2 RNP antibodies were accentuated in patients with overlap syndromes. CONCLUSION: We conclude that racial groups differ somewhat in their characteristic autoimmune responses to individual autoantigens. These differences could reflect variation in immunogenetic background or different etiologic mechanisms which might occur at different geographic locations.
Asian People↗
Autoantigenic epitopes of the B polypeptide of Sm small nuclear RNP particles. Identification of regions accessible only within the U1 small nuclear RNP.
OBJECTIVE: To analyze the autoantigenic epitopes of the Sm B polypeptide of the U1 small nuclear RNP (snRNP) using complementary DNA (cDNA) clones. METHODS: Expression of Sm B fusion proteins in lambda phage vectors, immunoblots, immunoprecipitations, and affinity purification of antibodies. RESULTS: Immunoblots using antibodies affinity-purified from B fusion proteins demonstrated that there were cross-reactive epitopes between the B'/B and A polypeptides of the U1 snRNP. Immunoprecipitation assays suggested that there were at least 3 different autoantigenic epitopes on the B polypeptide that could be classified into 2 general groups based upon autoantibody reactivity. The first group of autoantibodies, which bound 2 separate autoantigenic epitopes (BU1-1, BU1-2), participated in immunoprecipitation of the U1 snRNP alone. The second group, which bound the third type of autoantigenic epitope (BSm-1), immunoprecipitated all the abundant Sm snRNPs. CONCLUSION: There is at least 1 region on the B proteins that is accessible to antibodies only within the structure of the U1 snRNP, as well as a region that is accessible on all Sm snRNPs. These data support the notion that the native U1 RNP, perhaps containing B proteins in a different conformation than those found on other Sm snRNPs, may drive the humoral immune response in systemic lupus erythematosus.
Amino Acid Sequence↗
Nucleotide sequence and genomic structure analyses of the p70 subunit of the human Ku autoantigen: evidence for a family of genes encoding Ku (p70)-related polypeptides.
cDNA encoding the p70 polypeptide subunit of the human Ku autoantigen was isolated. In vitro expression analysis of the cDNA demonstrates that it encodes the entire open reading frame. Nucleotide sequence analysis and comparison to other previously described sequences indicate the existence of several single-nucleotide and amino acid polymorphisms. Southern blot analyses demonstrate the presence of multiple copies of homologous DNA sequences in the human genome. These data support the hypothesis that multiple genes encode a family of Ku(p70)-related polypeptides.
Amino Acid Sequence↗
Autoantibodies against cyclophilin in systemic lupus erythematosus and Lyme disease.
Autoantibodies against cyclophilin, a cyclosporin A binding protein, were detected in sera of 29 of 46 (63%) patients with systemic lupus erythematosus and 14 of 40 (35%) Lyme disease patients. The antibodies are directed against the denatured form of both the major and minor isoform of cyclophilin and can be demonstrated in Western blots. Some first-degree relatives of lupus patients also express these antibodies. They are specific for cyclophilin and are not the consequence of hypergammaglobulinaemia. Four monoclonal IgM antibodies from a patient with lepromatous leprosy also bound to cyclophilin. The generation of these antibodies may be of special interest because they are against a protein involved in the control of the immune system not known to be directly associated with DNA or RNA.
Adolescent↗