Protein heterogeneity in the human Ro/SSA ribonucleoproteins. The 52- and 60-kD Ro/SSA autoantigens are encoded by separate genes.

Two cDNA clones encoding the 52-kD form of a protein present in human Ro/SSA ribonucleoprotein complexes were cloned from a lambda gt11 human thymocyte cDNA library. These clones reacted with lupus patient sera which had anti-52-kD Ro/SSA antibodies, and with affinity-purified anti-52-kD Ro/SSA antibodies. Moreover, affinity-purified antibodies isolated from isopropyl-beta-D-thiogalactopyranoside-induced proteins of these clones reacted only with the 52-kD protein of lymphocytes in Western blots and precipitated Ro/SSA hY RNAs, confirming that the clones encode a 52-kD Ro/SSA antigen. The cDNA contains a single open reading frame of 1,425 nucleotides and encodes a predicted 475-amino acid polypeptide with a molecular mass of 54,108 D. This protein appears unique in that both a zinc finger and leucine zipper motif are present on this protein. Surprisingly, no homology was found between the 52-kD Ro/SSA gene or protein and three published 60-kD Ro/SSA sequences. However, significant similarity of the 52-kD Ro/SSA was detected with human rfp and mouse rpt-1. These three proteins each contain similar zinc finger motifs in approximately their first 145 amino acid residues. The cDNA and the protein expressed therefrom are useful in the analysis of the structural and functional properties of this autoantigen.

Anti-Ro(SSA) autoantibodies are associated with T cell receptor beta genes in systemic lupus erythematosus patients.

Several of the heterogeneous clinical manifestations of systemic lupus erythematosus have been associated with specific autoantibodies. Associations between HLA class II antigens and autoantibodies to the ribonucleoproteins Ro(SSA) and La(SSB) have been reported in these patients. Because HLA class II molecules present antigen to T cell receptors (TCRs), we have searched for a TCR gene associated with the production of anti-Ro(SSA) antibodies. A pair of restriction fragment length polymorphisms (RFLPs), one of which hybridizes to the TCR constant region C beta 1 and the other to the C beta 2 gene, has been identified, suggesting these may be genotypic markers for an extended region of the TCR beta locus. This RFLP pair occurs in 76% of patients with Ro(SSA) precipitins, 84% of anti-Ro(SSA)-positive patients lacking La(SSB) precipitins, but only 41% of the patients lacking both precipitins (P = 0.0004). This disproportionate occurrence in a subset of lupus patients indicates that these RFLPs are not disease susceptibility markers, but rather are important markers for TCR genes whose products are involved in the production of anti-Ro(SSA) antibodies. The majority of patients who have these RFLPs and HLA class II antigens previously associated with the anti-Ro(SSA) response make this antibody, suggesting that interactions between products of these loci occur in response to Ro(SSA).

HLA-DQ gene complementation and other histocompatibility relationships in man with the anti-Ro/SSA autoantibody response of systemic lupus erythematosus.

A strong gene interaction between HLA-DQ1 and DQ2 alleles has been associated with anti-Ro/SSA autoantibodies (Harley, J.B., M. Reichlin, F. C. Arnett, E. L. Alexander, W. B. Bias, and T. T. Provost. 1986. Science [Wash. DC]. 232:1145-1147; Harley, J. B., A. S. Sestak, L. G. Willis, S. M. Fu, J. A. Hansen, and M. Reichlin. 1989. Arthritis Rheum. 32:826-836; Hamilton, R. G., J. B. Harley, W. B. Bias, M. Roebber, M. Reichlin, M. C. Hochberg, and F. C. Arnett. 1988. Arthritis Rheum. 31:496-505). To test a gene complementation mechanism for these results, restriction fragment length polymorphisms (RFLP) of the DQ alpha and DQ beta genes have been related to Ro/SSA precipitins in patients with systemic lupus erythematosus. In this study Ro/SSA precipitins are related to the simultaneous presence of a particular pair of RFLPs. A DQ alpha RFLP associated with HLA-DQ1 and a DQ beta RFLP associated with HLA-DQ2 predict that the alpha beta heterodimer in HLA-DQ1/DQ2 heteroxygotes is most closely related to anti-Ro/SSA autoantibodies, thereby supporting a gene complementation mechanism. Beyond this effect, an RFLP associated with HLA-DQ2 and/or DR7 is also related to Ro/SSA precipitins. Multiple molecular histocompatibility mechanisms are implicated, therefore, in the production of anti-Ro/SSA autoantibodies in autoimmune disease. For anti-Ro/SSA autoantibodies in SLE, and perhaps more generally, these data show that the histocompatibility antigens are among the elements that confer autoimmune response specificity and restrict the production of particular autoantibodies among patients with systemic lupus erythematosus.

Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.

Anti-PM-Scl antibodies are associated with polymyositis-scleroderma overlap or either disease alone. Among sera from 39 patients with anti-PM-Scl, 23 recognized the 100-kD band in immunoblot against HeLa cell extract, 16 of which also stained the 70-kD band. A human thymocyte lambda gt11 cDNA expression library was screened with anti-PM-Scl serum, and two clones were identified whose products reacted with 33 and 37 of 39 anti-PM-Scl sera, respectively, but none of 26 negative control sera. Affinity-purified antibody reacting specifically with plaques of the clone stained the 100-kD band on immunoblot, reacted with nucleoli of HEp-2 cells, and immunoprecipitated the PM-Scl protein complex. Partial sequences of both inserts were identical. One insert was fully sequenced, and additional 5' and 3' sequence was obtained using a gene-specific primer to form a cDNA with HeLa cell RNA as template followed by PCR. The complete nucleotide sequence included 2,739-bp coding for a predicted full-length protein of 98,088 D. There was no homology with the PM-Scl 75-kD protein and no significant homology with other proteins. A mixed-charge cluster was identified, with 22 charged amino acids of 37. In conclusion, the full-length cDNA sequence was determined coding for the PM-Scl 100-kD protein, the most commonly antigenic protein of the PM-Scl complex.

Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen.

Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature.

Multiplex serum cytokine monitoring as a prognostic tool in rheumatoid arthritis.

OBJECTIVE: Early optimized therapy of rheumatoid arthritis (RA) results in improved outcomes. The initiation of optimized therapy is hindered by the difficulty of early diagnosis and the limitations of current disease activity and therapeutic response assessment tools. Identifying patients requiring early combination DMARD/biologic therapy is currently a significant clinical challenge given the lack of definitive prognostic criteria. Since cytokines are soluble intracellular signaling molecules that modulate disease pathology in RA, we tested the recent conjecture that en mass serum cyto-kine measurement and monitoring will provide a useful tool for effective therapeutic management in RA. METHODS: We assayed the levels of 16 serum cytokines in 18 RA patients treated prospectively with methotrexate and from 18 unaffected controls. Specific mechanistic aspects of inflammatory pathology in the periphery could be discerned on a patient-specific basis from patients' serum cytokine profiles, information that may aid in the design of anti-cytokine biologic therapy. A serum Cytokine Activity Index (CAI) was also created using multi-variant analysis methods. RESULTS: Distinct cytokines were significantly elevated in RA patients relative to controls, and three distinct clusters with correlations to disease activity were identified. The Cytokine Activity Index correlated well with the therapeutic res-ponse; responders and non-responders in this cohort were distinguishable as early as one month post initiation of methotrexate therapy, well before clinical assessments of response are commonly completed. CONCLUSION: Clinical assessment tools could be derived from this approach that may provide a means to continually track patients, allowing intervention strategies to be better evaluated on a patient-specific basis and to identify residual cytokine activity that could be used to guide combination therapy.

Two pseudogenes among three rat immunoglobulin lambda chain genes.

In order to examine the number and organization of the immunoglobulin lambda light chain genes of the rat, we have used mouse lambda chain cDNA probes to isolate hybridizing fragments from a partial EcoRI rat liver DNA library. We have determined the partial nucleotide sequence of two such clones. One clone, containing a 5.8 kb EcoRI insert which hybridizes to both mouse C lambda 1 and C lambda 2 probes, includes an apparently expressible C lambda 2-like gene as well as a C lambda 1-like pseudogene (psi C lambda 1.1), arranged similarly to the mouse C lambda gene complexes. Sequence analysis of a second cloned EcoRI fragment (1.15 kb in length) revealed part of a second C lambda 1-like pseudogene (psi C lambda 1.2), the coding regions of both pseudogenes being interrupted by multiple frame-shifting size differences. In the case of psi C lambda 1.2, the degree of sequence identity with mouse C lambda 1 drops abruptly immediately following the termination codon, suggesting that translocation events have played a role in its generation. These two rat pseudogenes, and the mouse C lambda 4 pseudogene, clearly have been rendered unexpressible by separate evolutionary events. Comparisons between C lambda coding and non-coding regions of rats and mice indicate that some of the unusual patterns of divergence we have observed in recently diverged Ck genes may exist in C lambda genes as well.

Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

The structure and evolution of immunoglobulin kappa chain constant region genes in the genus Rattus.

We have cloned and sequenced the C-kappa (Ck) genes from seven species and subspecies of rats which have diverged over the past few million years in Australia. Comparisons of these sequences with each other and the Ck genes of the laboratory rat, Rattus norvegicus, indicate noncoding regions have accumulated fewer mutations than adjacent coding sequences, and amino acid replacing nucleotide substitutions in the coding regions have accumulated at a rate at least as great as silent changes. Exactly the opposite of both of these findings is observed when comparisons are made between Ck or other genes from more distantly related species, indicating that these features may be characteristic of Ck short-term evolutionary gene divergence. Changes in the coding regions of these genes result in a non-random distribution of amino acid substitutions on the three-dimensional alpha-carbon backbone of the Ck domain in the most serologically distinct forms of Ck. While phylogenetic relationships inferred from the Ck nucleotide sequences are in general agreement with those derived from other data, considerable differences are seen in rates of accumulation of Ck gene nucleotide substitutions vs rates of accumulation of enzyme polymorphisms.

Autoantibodies of neonatal lupus erythematosus.

The most common manifestations of neonatal lupus erythematosus (NLE) are cutaneous lupus and congenital heart block. Autoantibodies to Ro/SSA occur in almost all cases of NLE. The autoantibody response to Ro/SSA is complex, and antibodies may be detected to 60-kD Ro/SSA, 52-kD Ro/SSA, La/SSB, and U1 ribonuclear protein in anti-Ro/SSA-positive sera. Which of these anti-Ro/SSA-related autoantibody specificities are important in the clinical expression of NLE is not conclusively established. We examined the autoantibody specificities in 20 maternal NLE sera to determine whether autoantibody specificities correlate with the clinical findings and to evaluate the relative importance of autoantibodies to the different Ro/SSA-associated proteins. Autoantibodies were examined using immunodiffusion, immunoblotting, and enzyme-linked immunosorbent assay. Eleven babies had NLE skin disease, 11 had heart block, and two had both skin disease and heart block. All 20 maternal sera had antibodies to 60-kD Ro/SSA. Eighteen of the 20 had antibodies to 52-kD Ro/SSA, nine had antibodies to La/SSB, and one had antibodies to U1 ribonuclear protein. The prevalence of anti-La/SSB was the same in the skin-disease and heart-block subsets of NLE. Titers of anti-60-kD Ro/SSA were significantly (p < 0.02) lower in NLE skin disease maternal sera than in the NLE heart-block maternal sera. These results point out the importance of 60-kD Ro/SSA as a potential target in NLE. We speculate that the lower titers of anti-60-kD Ro/SSA in the sera from mothers of babies with skin disease may be due to substantial deposition of antibodies in the mothers' and babies' skin, leading to lower circulating titers, or may reflect a lower threshold for development of skin disease than for heart block.

Polymorphisms of coagulation factor XIII subunit A and risk of nonfatal hemorrhagic stroke in young white women.

BACKGROUND AND PURPOSE: Although family studies have suggested a genetic influence on hemorrhagic stroke, the underlying genetic risk factors remain poorly defined. Coagulation factor XIII, which is involved in hemostasis, fibrinolysis, vascular remodeling, and tissue repair, represents a candidate gene for hemorrhagic stroke. We assessed the potential role of 3 factor XIII subunit A coding-sequence polymorphisms, along with a promoter polymorphism of plasminogen activator inhibitor-1 (PAI-1, which is also involved in fibrin stabilization and vascular remodeling), in young white women with hemorrhagic stroke. METHODS: Genotype analysis for factor XIII subunit A Val34Leu, Tyr204Phe, and Pro564Leu and for PAI-1 -675 4G/5G was performed in a population-based case-control study of 42 white women aged <45 years with nonfatal hemorrhagic stroke and 345 demographically similar control subjects. RESULTS: Compared with the respective homozygous wild-type genotypes, the Tyr204/Phe204 genotypes (age-adjusted odds ratio [OR] 2.9, 95% 95% CI 1.1 to 7.5) and the Leu564/Leu564 genotype (OR 4.3, 95% CI 1.4 to 13.7) were each associated with an increased risk of nonfatal hemorrhagic stroke. The risk estimate associated with the Phe204 variant was highest in women with subarachnoid hemorrhage and in nonsmokers, whereas the risk estimate of the Leu564/Leu564 genotype was highest in women with intracerebral hemorrhage and in smokers. Women who carried either the Phe204 allele or the Leu564/Leu564 genotype in combination with the PAI-1 5G/5G genotype had a nearly 20-fold increased risk of hemorrhagic stroke (OR 18.9, 95% CI 3.8 to 95.1). CONCLUSIONS: Our findings suggest that the Phe204 and Leu564 variants of coagulation factor XIII may be markers for genetic susceptibility to hemorrhagic stroke in women aged <45 years.

Autoantibodies to the Ro/SSA autoantigen are conformation dependent. II: Antibodies to the denatured form of 52 kD Ro/SSA are a cross reacting subset of antibodies to the native 60 kD Ro/SSA molecule.

The immune response to the Ro/SSA particles is conformation dependent. In sera with only anti-Ro/SSA precipitins, the autoantibodies to the 60 kD Ro/SSA are largely to the native 60 kD Ro/SSA while autoantibodies to the 52 kD Ro/SSA particle when present are exclusively to the denatured 52 kD Ro/SSA particle. Antibodies eluted from a recombinant 52 kD Ro/SSA fusion protein reacted in a sandwich ELISA which only measures antibody to native 60 kD Ro/SSA antigens and this reaction is largely inhibited by native homogeneous 60 kD Ro/SSA. In addition, antibody binding to the 52 kD Ro/SSA antigen in Western blot is also strongly inhibited by native 60 kD Ro/SSA. These experiment strongly suggest that reactivity of denatured 52 kD Ro/SSA antigen represents a cross reaction with autoantibodies directed to the native 60 kD Ro/SSA antigen. As a corollary of these experiments, data are presented that suggest the hY-RNAs are not associated with the 52 kD Ro/SSA protein but only with the 60 kD Ro/SSA protein. These data are consistent with the hypothesis that the autoanti-Ro/SSA response is driven by native 60 kD Ro/SSA and the immune response to denatured 52 kD Ro/SSA is largely a cross-reactive subset of the immune response to native 60 kD Ro/SSA.

The autoantibody response to Ro/SSA in cutaneous lupus erythematosus.

BACKGROUND AND DESIGN: Seventeen patients with subacute cutaneous lupus erythematosus (SCLE) were compared with 15 patients with discoid lupus erythematosus (DLE) to evaluate the relationship of 60- and 52-kd Ro/SSA autoantibodies to the clinical diagnosis and to evaluate assays for anti-Ro/SSA. RESULTS: All serum samples from patients with SCLE had precipitating anti-Ro/SSA antibodies in immunodiffusion, and all had high titer anti-60-kd Ro/SSA in enzyme-linked immunosorbent assay. Immunoblotting was inadequately sensitive for detecting anti-60-kd Ro/SSA. Fifteen patients with SCLE had anti-52-kd Ro/SSA (11 high titer, four low titer). Only one of the 15 patients with DLE had precipitating, high-titer anti-Ro/SSA. Nine other patients with DLE had low-titer anti-60-kd Ro/SSA, and four had low-titer anti-52-kd Ro-SSA. Low-titer anti-Ro/SSA did not confer an increased risk for photosensitivity in the DLE group. CONCLUSIONS: High-titer, precipitating antibodies to Ro/SSA are typical of SCLE and unusual in DLE. Low-titer, nonprecipitating antibodies to Ro/SSA are common in DLE and could be an indication of pathogenic factors shared with SCLE. However, low titers of anti-Ro/SSA do not confer a significant risk for SCLE skin lesions. For the purpose of clinical evaluation of skin disease, immunodiffusion assays for anti-Ro/SSA are cost-effective and informative.

5alpha-Androstane-3alpha,17beta-diol activates pathway that resembles the epidermal growth factor responsive pathways in stimulating human prostate cancer LNCaP cell proliferation.

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is considered to have no androgenic effects in androgen target organs unless it is oxidized to 5alpha-dihydrotestosterone (5alpha-DHT). We used microarray and bioinformatics to identify and compare 3alpha-diol and 5alpha-DHT responsive gene in human prostate LNCaP cells. Through a procedure called 'hypervariable determination', a similar set of 30 responsive genes involving signal transduction, transcription regulation, and cell proliferation were selected in 5alpha-DHT-, 3alpha-diol-, and epidermal growth factor (EGF)-treated samples. F-means cluster and networking procedures showed that the responsive pattern of these genes was more closely related between 3alpha-diol and EGF than between 5alpha-DHT and 3alpha-diol treatments. We conclude that 3alpha-diol is capable of stimulating prostate cell proliferation by eliciting EGF-like pathway in conjunction with androgen receptor pathway.

Ablation of Stat3 by siRNA alters gene expression profiles in JEG-3 cells: a systems biology approach.

Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.

Genome-scale assessment of molecular pathology in systemic autoimmune diseases using microarray technology: a potential breakthrough diagnostic and individualized therapy-design tool.

Systemic autoimmune rheumatic diseases are of complex aetiology, characterized by an intricate interplay of various factors. A myriad of genes lies behind the heterogeneous manifestations of these diseases, and the overexpression and repression of particular genes form a specific gene-expression profile (genetic fingerprints) that is characteristic to the given disease phenotype. Besides the description of various cell types by using gene-expression profiling, the data should be directly applicable to the design of individual therapeutic protocols for patients suffering from various autoimmune diseases. In this review, we summarize the gene-expression profile, various genetic signatures of different autoimmune diseases and give an overview on the possible interpretations of the data. The application of recent breakthroughs in high-throughput molecular profiling technologies, such as microarray technology has been the basis for a revolution in biomedical research, as well as diagnostics and pharmaceutical development. It is easy to envision a day when personalized medicine, which is the diagnosis and treatment of a given patient with agents and procedures tailored to that patient's genetics, physiology and pathology, will become the standard of care.

A genome-scale assessment of peripheral blood B-cell molecular homeostasis in patients with rheumatoid arthritis.

OBJECTIVE: While rheumatoid arthritis (RA) is considered a prototypical autoimmune disease, the specific roles of B-cells in RA pathogenesis is not fully delineated. METHODS: We performed microarray expression profiling of peripheral blood B-cells from RA patients and controls. Data were analysed using differential gene expression analysis and 'gene networking' analysis (characterizing clusters of functionally inter-relelated genes) to identify both regulatory genes and the pathways in which they participate. Results were confirmed by quantitative real-time polymerase chain reaction and by measuring the levels of 10 serum cytokines involved in the pathways identified. RESULTS: Genes regulating and effecting the cell-cycle, proliferation, apoptosis, autoimmunity, cytokine networks, angiogenesis and neuro-immune regulation were differentially expressed in RA B-cells. Moreover, the serum levels of several soluble factors that modulate these pathways, including IL-1beta, IL-5, IL-6, IL-10, IL-12p40, IL-17 and VEGF were significantly increased in this cohort of RA patients. CONCLUSIONS: These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

Transcriptional modulation of TCR, Notch and Wnt signaling pathways in SEB-anergized CD4+ T cells.

Gene expression changes in CD4 + Vbeta8+ T cells energized by in vivo exposure to staphylococcal enterotoxin B (SEB) bacterial superantigen compared to CD4 + Vbeta8+ non-energic T cells were assessed using DNA microarrays containing 5184 murine complementary DNAs. Anergy in splenic T cells of SEB-immunized BALB/c mice was verified by dramatically reduced proliferative capacity and an 8 x overexpression of GRAIL mRNA in CD4 + Vbeta8+ T cells taken from mice 7 days after injection. At an Associative t-test threshold of P<0.0005, 96 genes were overexpressed or detected only in anergic T cells, while 256 genes were suppressed or not detected in anergic T cells. Six of eight differential expressions tested using real-time quantitative PCR were validated. Message for B-Raf was detected only in non-anergic cells, while expression of the TCR signaling modulator Slap (Src-like adapter protein) and the TCR zeta-chain specific phosphatase Ptpn3 was enhanced. Modulation of multiple genes suggests downregulation of Wnt/beta-catenin signaling and enhanced Notch signaling in the anergic cells. Consistent with previous reports in a non-superantigen in vivo anergy model, mRNA for CD18 and the transcription factor Satb1 (special AT-rich-binding protein 1) was increased in SEB-energized T cells. This is the first report of global transcriptional changes in CD4+ T cells made anergic by superantigen exposure.

Characterization of DNA binding properties and sequence specificity of the human 52 kDa Ro/SS-A (Ro52) zinc finger protein.

The Ro52 protein is an autoantigen in Sjögren's Syndrome and systemic lupus erythematosus. Although its function is not yet known, sequence similarities to other proteins suggest that it binds to DNA. In this study, the hypothesis that Ro52 recognizes particular nucleic acid sequences was tested. Ro52 bound to double stranded but not single stranded DNA. 1,10-Phenanthroline, a chelater of zinc, was found to inhibit this interaction, suggesting that the zinc fingers of Ro52 are functional. DNA sequences were selected from an oligonucleotide library by binding to Ro52 followed by amplification by Taq DNA polymerase in order to characterize the DNA sequence-binding motif for this protein. These studies support the hypothesis that Ro52 is functionally a member of a family of zinc finger proteins, many of which are known to bind to DNA or regulate gene expression. We speculate that Ro52 functions as a transcription factor, and that its disregulation may have important consequences in the expression or susceptibility of certain autoimmune diseases.

Biochemical and biologic characterization of lymphocyte regulatory molecules. VIII. Purification of interleukin 2 from a human T cell leukemia.

The ability of interleukin 2 (IL 2) to stimulate and maintain growth in IL 2-dependent cell lines distinguishes this substance from interleukin 1 and other T cell lymphokines. Jurkat-FHCRC, a human T cell leukemia cell line, has been shown to produce relatively high quantities of IL 2 after stimulation with the T cell mitogens phytohemagglutinin or concanavalin A. In this paper we describe techniques used to isolate human IL 2 from Jurkat supernatants and the biochemical properties of the resulting material. IL 2 produced by this cell line is eluted from a DEAE-Sephacel ion-exchange column with 0.05 M NaCl and has a m.w. determined by AcA54 gel chromatography in the range of 9000 to 18,000. Isoelectric focusing yields active material with a pI value of 7.75. Elution of IL 2 from a SDS-polyacrylamide gel shows it to have a m.w. of approximately 13,500. This material is capable of stimulating mouse as well as human IL 2-dependent cell lines.