Biomedical subjects

R Suenaga

Publications and source records attributed to R Suenaga.

At least 19 recordsLinked to original sources

PubMed2001

In vitro-activated human lupus T cells express normal estrogen receptor proteins which bind to the estrogen response element.

We have shown that estrogen receptor (ERalpha, ERbeta) transcripts are expressed in SLE and normal T cells. In this study, T cell nuclear extracts from female lupus patients and normal donors were tested for biologically active ER proteins capable of binding to the human estrogen response element (hERE) by electrophoretic mobility shift assays. When peripheral blood T cells were stimulated with 17beta-estradiol (E2), PMA and ionomycin, two major retarded bands in T cell nuclear extracts exhibited a migration pattern similar to slow migrating protein-ERE complexes in human breast cancer cell extracts. T cells cultured only with E2 did not have these complexes. The formation of the complexes was inhibited by competition with the hERE cold oligonucleotide and partially with anti-ERalpha antibodies. There was no notable difference in the migration pattern of ERE-binding proteins between the SLE and normal T cell extracts. Together, these results suggest that activated human T cells, whether lupus-derived or normal-derived, contain biologically active ERalpha proteins. Other factors may be responsible for differential sensitivity of lupus T cells to estrogen.

Adult↗

PubMed1998

V gene sequences of lupus-derived human IgM anti-ssDNA antibody: implication for the importance of the location of DNA-binding amino acids.

Binding and structural characteristics of human IgMk anti-ssDNA antibody 7B3 were determined. 7B3 was derived from Epstein-Barr virus-transformed peripheral blood B cells of a lupus nephritis patient. Purified 7B3 bound ssDNA from various species, but not dsDNA or structurally unrelated antigens. The relative avidity of 7B3 was high in comparison with IgM anti-DNA antibodies previously described by other investigators. Sequence analysis showed that 7B3 used VH26/D35/JH3 and Humkv328h5/JK1 germline genes, and had a few mutations in the complementarity determining regions (CDRs). No arginine was expressed in the heavy-chain CDR3. However, the putative DNA contact sites, based on the previous crystallographic and computer modeling studies, were occupied by mutated or germline-derived basic and polar amino acids. These results suggest that a minimally mutated IgM anti-ssDNA antibody with a paucity of arginines could display monospecificity and high avidity if DNA-binding amino acids are enriched at the critical DNA contact sites.

Amino Acid Sequence↗

PubMed1998

Gender differences in autoimmune diseases: estrogen increases calcineurin expression in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) predominantly affects women (9:1 compared to men) of childbearing age and often decreases its intensity in postmenopausal women, suggesting that sex hormones play a role in its pathogenesis. Comparison of steady-state levels of calcineurin mRNA using RNase protection assays revealed increased calcineurin expression in response to estradiol in cultured T cells from nine female lupus patients. Calcineurin mRNA levels did not increase significantly in T cells from eight age-matched normal control female volunteers. Estrogen-dependent calcineurin mRNA increased in a dose-dependent fashion, while progesterone and dexamethasone did not increase calcineurin mRNA in patient cells. Lupus T cell calcineurin mRNA increased in response to estradiol at 6 h but not at 3 h. Calcineurin phosphatase activity increased in lupus T cell extracts after incubation of cells with estradiol, while phosphatase activity in normal T cells was unaffected by estrogen. Calcineurin expression in T cells from patients with vasculitis and rheumatoid arthritis taking medications similar to those taken by the lupus patients was unaffected by estradiol. This study provides the first evidence for a molecular marker of estrogen action in lupus patients and suggests that estrogen-dependent changes in lupus T cell calcineurin could alter proinflammatory cytokine gene regulation and T-B cell interactions.

Adult↗

PubMed1998

Isolation of anti-nucleosome antibodies from the plasma of lupus nephritis patients.

Anti-nucleosome antibodies, which recognise conformational epitopes consisting of histone and DNA in chromatin, have been described in autoimmune diseases. In this study, an attempt was made to isolate anti-nucleosome antibodies from the anti-DNA-depleted plasma IgG of two lupus patients either with or without nephritis by nucleohistone affinity chromatography. The purified nucleohistone-binding antibodies bound to nucleohistone in a specific manner and contained enriched anti-histone antibodies. However, adsorption of the purified antibodies with histone revealed that the nephritis patient-derived antibodies contained nucleohistone-specific antibodies. Although such purified antibodies may not recognise native structures of nucleosomes, this chromatography may provide a method to isolate and determine the fine specificity of anti-nucleosome antibodies in various autoimmune diseases.

Antibodies, Antinuclear↗

PubMed1998

Peripheral blood T cells and monocytes and B cell lines derived from patients with lupus express estrogen receptor transcripts similar to those of normal cells.

OBJECTIVE: To identify and characterize estrogen receptor (ER) transcripts expressed in immune cells of patients with systemic lupus erythematosus (SLE) and healthy donors. METHODS: Peripheral blood monocytes and T cells were prepared from patients with SLE (n = 6) and healthy donors (n = 8). T cells were separated into CD4 and CD8. Some monocytes and T cells were stimulated with estradiol, PMA, and ionomycin. Epstein-Barr virus-transformed B cell lines (n = 7) and B cell hybridomas (n = 2) established from patients with SLE and a healthy individual were used as a B cell source. These cells were examined for ER mRNA by reverse transcription nested polymerase chain reaction. Amplified cDNA were sequenced by standard methods. RESULTS: In all cells tested, ER mRNA was expressed without prior in vitro stimulation. Partial sequences from exons 1-8 were nearly identical to the published sequence of the human ER mRNA. There were no notable differences in the ER transcripts between patients and healthy controls. Variant receptor transcripts lacking exon 5 or exon 7, which encodes the hormone binding domain, were identified in the majority of the cells. Precise deletion of the exons suggests that they are alternatively spliced transcripts. Whether the detected transcripts are translated into functional receptor proteins remains to be determined. In vitro stimulation did not affect ER mRNA expression. The presence of variants did not correlate with disease activity or medication. CONCLUSION: Monocytes, T cells, and B cells in patients express transcripts of the normal wild type ER and the hormone binding domain variants in vivo.

Adult↗

PubMed1996

V gene sequences of human anti-ssDNA antibodies secreted by lupus-derived CD5-negative B cell hybridomas.

V gene sequences encoding two lupus-derived human monoclonal IgMk anti-ssDNA antibodies (2F7 and 1A6) and CD5 mRNA expression by the corresponding hybridomas were investigated. Both antibodies displayed V gene sequences nearly in germline configuration compared with their putative germline counterparts. It appeared that 2F7 used hv3019b9/HUD-3/JH6 and 12La/Jk2, while 1A6 utilized HHG19/D31-HUD-3/JH2 and Humkv328h5/Jk1. Assessment of R/S mutation ratios suggested that 2F7 and 1A6 have not undergone the antigen-driven somatic mutation. The HCDR3 featuring arginine appeared to be important in determining the anti-ssDNA specificity. CD5 mRNA was negative in both hybridomas. Since 2F7 was previously shown to be monospecific and of high affinity, these results provide the molecular basis of such unique immunochemical characteristics of the IgM anti-ssDNA antibody. Germline V genes and N sequences may be selected to confer such anti-ssDNA specificity during V gene rearrangement, which might involve CD5-negative B cells.

Amino Acid Sequence↗

PubMed1996

Binding affinity and quantity of estrogen receptor in peripheral blood monocytes of patients with systemic lupus erythematosus.

Binding affinity and quantity of the estrogen receptor in monocytes of patients with systemic lupus erythematosus (SLE) were studied. The tritiated-estradiol binding assay was performed using peripheral blood adherent cells (> 95% monocytes) derived from six lupus patients (SLEDAI score: 2-30) and five age-comparable normal women during the mid-follicular phase of the menstrual cycle. Dissociation constant (Kd) and number of binding sites (Ro) were estimated by Scatchard analysis. The specificity and sensitivity of the assay were verified by using estrogen receptor-positive ZR75-1 human breast cancer cells. Kd and Ro of the type I receptor for the SLE patients were 12.2 +/- 6.5 (nM) and 69.0 +/- 42.4 (x 1000/cell), respectively, while those of the normals were 14.5 +/- 3.7 and 86.8 +/- 23.4, respectively. Three patients displayed relatively low Kd or Ro values. While those low values fell within the mean -3s.d. of the normal controls, precise statistical comparison was not possible. No clear correlation between the receptor parameters and the SLEDAI scores was noted. Although further studies of a larger number of samples are needed to conclude, these results suggest that peripheral blood monocytes of SLE patients express the estrogen receptor whose Kd and Ro are similar to those of normals.

Adult↗

PubMed1996

Anti-(DNA-histone) antibodies in active lupus nephritis.

OBJECTIVE: To detect and characterize anti-(DNA-histone) antibodies in patients with active lupus nephritis. METHODS: Calf thymus double stranded DNA was reassociated with histone in vitro. Polynucleosomes were prepared from chicken erythrocyte nuclei, calf thymus nucleohistone, and human peripheral blood mononuclear cells. Anti-DNA activity was depleted from purified IgG using DNA-cellulose. Binding of the adsorbed IgG to various (DNA-histone) related antigens was measured by ELISA. Antigen specificity was assessed by inhibition assays and adsorption studies using histone and nucleosome conjugated sepharose beads. RESULTS: Anti-(DNA-histone) antibodies were detected in 3 of 5 patients with active lupus nephritis. They specifically recognized determinants consisting of both DNA and histone, but not DNA or histone alone. Anti-(DNA-histone) antibodies largely overlapped with antinucleosome antibodies and appeared to react with nucleosomes released by apoptotic human mononuclear cells. CONCLUSION: Anti-(DNA-histone) antibodies are present in active lupus nephritis and largely represent antinucleosome antibodies. They may contribute to the pathogenesis of nephritis by forming immune complexes with apoptosis related nucleosomes.

Adsorption↗

PubMed1995

Heparan sulphate-ELISA gives false positive results for anti-DNA-DNA/histone immune complexes in sera of patients with SLE.

Heparan sulphate-reactive antibodies in lupus sera have been suggested to be anti-DNA-DNA/histone immune complexes and to be associated with lupus nephritis. In this study, 23 anti-DNA-positive lupus sera including 13 active nephritis sera were tested for the presence of circulating anti-DNA-DNA/histone immune complexes by solid phase heparan sulphate-ELISA. Because of high background binding to protamine chloride-linked heparan sulphate plates, poly-L-lysine (PLL) was used as a linker and the remaining active sites of PLL were blocked with poly-L-glutamic acid. The ELISA was capable of detecting small amounts of anti-DNA IgG-DNA/histone immune complexes formed in vitro. However, only three active nephritis sera of the 23 sera tested showed significant binding to heparan sulphate plates. This binding was found to be non-specific, the result of high background binding of IgG to PLL. Anti-heparan sulphate ELISA using positively charged linkers detects non-specific binding when lupus sera are tested. Specific assays need to be developed for DNA/histone-related immune complexes present in lupus sera.

Animals↗

PubMed1993

Cationic and high affinity serum IgG anti-dsDNA antibodies in active lupus nephritis.

To investigate differences between cationic anti-dsDNA antibodies during active and inactive nephritis, low- and high-affinity IgG anti-dsDNA antibodies were prepared from sera of a lupus patient and compared for their binding affinity, spectrotype, and idiotype expression. The ratio of high-affinity to low-affinity anti-DNA antibodies and the relative avidity of the high-affinity anti-DNA antibodies decreased when active nephritis became inactive. Isoelectric focusing showed that cationic anti-dsDNA populations were present predominantly in the high-affinity fraction during active nephritis and in the low-affinity fraction during inactive nephritis. Idiotypic analysis by ELISA and Western blotting showed that the high-affinity cationic anti-DNA antibodies during active nephritis were idiotypically different from their low-affinity counterparts during inactive nephritis. The differences in binding affinity and idiotypy of the cationic anti-dsDNA antibodies suggest that certain serum IgG anti-dsDNA antibodies with both cationic charge and high affinity may be associated with active nephritis.

Antibodies, Antinuclear↗

PubMed1992

Shift of private and not of cross-reactive anti-DNA idiotypes in systemic lupus erythematosus.

The shift of private idiotype (Id) and cross-reactive Id (CRI) on anti-DNA antibodies in a lupus patient KE was investigated during a 7-year period. Anti-private Id and anti-CRI activities were separated by affinity chromatography from rabbit (R)-anti-Ids raised against KE anti-DNA antibodies during active (1/84) and inactive (4/90) stages of the disease. Anti-CRI isolated from the 84 R-anti-Id appeared to recognize binding site-related Ids that are shared with KE non-anti-DNA antibodies, unrelated lupus patients' sera, and certain normal sera. Id expression on serial serum samples of KE using these fractionated R-anti-Ids as probes showed that the 1/84 private Id expression declined while the 4/90 private Id expression gradually increased. Expression of the CRI showed a relatively stable pattern. These results suggest that anti-DNA populations detected by anti-private Id can shift, while populations expressing CRI may stay stable.

Antibodies, Antinuclear↗

PubMed1992

Expression of inactive stage anti-dsDNA idiotypes on anti-ssDNA antibodies in a lupus patient during active stage of lupus cerebritis.

The possibility that idiotypes (Ids) defined on anti-double stranded DNA (dsDNA) antibodies during active and inactive stages of lupus (1/84 Id and 4/90 Id, respectively) were expressed on anti-DNA antibodies during a subsequent active period (9/90) of the disease was investigated in a lupus patient with lupus cerebritis. Using rabbit (R)-anti-Ids specific to 1/84 Id and 4/90 Id in inhibition assays, the 4/90 Id was shown to be expressed on the framework regions of anti-single stranded DNA (ssDNA) but poorly on co-existing anti-dsDNA antibodies of active (9/90) stage. The 1/84 Id was poorly expressed on both types of 9/90 anti-DNA antibodies. While the 9/90 anti-ssDNA significantly bound to immobilized ssDNA and several single-stranded polynucleotides, only ssDNA inhibited the binding of the anti-ssDNA to ssDNA, suggesting its monospecificity toward ssDNA. Western blot analysis following isoelectric focusing showed that a spectrotype pattern of 4/90 Id-positive 9/90 anti-ssDNA IgG was similar to that of the 4/90 anti-dsDNA, suggesting that they are of related clonal origin. The present study suggests the idiotypic heterogeneity of anti-DNA antibodies and the shift of antigen specificity within an idiotypically related anti-DNA population during exacerbation of the disease.

Animals↗

PubMed1992

Lupus-derived human monoclonal IgM anti-DNA antibody displays monospecificity, high affinity and private idiotype specificity.

A human monoclonal IgM k anti-DNA antibody, designated 2F7, was prepared by somatic hybridization of peripheral blood lymphocytes from a lupus patient with a human-mouse heterohybridoma cell line, K6H6/B5. 2F7 was tested for its antigen binding and idiotypic specificity by direct binding and inhibition enzyme-linked immunosorbent assays. 2F7 had a high binding activity to single-stranded DNA (ssDNA) but not to double-stranded DNA. It cross-reacted with single-stranded homopolymers with pyrimidine bases and double-stranded polynucleotides containing those homopolymers, suggesting that 2F7 recognizes a conformational determinant made up of both deoxyribose-phosphate backbone and specific nucleotide base. 2F7 did not cross-react with eight structurally unrelated self-antigens. Dissociation constant (Kd) of 2F7 for sonicated ssDNA was approximately 4.5 x 10(-9) M, indicating its relatively high affinity. Idiotypic characterization with rabbit anti-idiotype raised against 2F7 suggested that 2F7 expressed an idiotype at or near its antigen-binding sites that was not detected in sera from 20 unrelated lupus patients, 10 lupus family members and 10 normal individuals. These results suggest that certain IgM class anti-DNA antibodies in human systemic lupus erythematosus may arise by antigen stimulation and not simply by polyclonal B-cell activation.

Animals↗

PubMed1991

Detection and purification of antiidiotypic antibody against anti-DNA in intravenous immune globulin.

Pooled normal human IgG for therapeutic use, following depletion of anti-DNA, anti-Fc, and anti-F(ab')2 of normal IgG, expressed antiidiotypic activity against anti-DNA derived from lupus sera. The antiidiotype enriched by elution from anti-DNA affinity columns bound directly to anti-DNA IgG and inhibited the binding of lupus sera to DNA but did not bind to normal IgG or inhibit the binding of anti-tetanus toxoid to tetanus toxoid. Antiidiotypes in pooled normal sera may have a role in the clinical improvement seen in patients with autoimmune diseases receiving intravenous immune globulin.

Antibodies, Anti-Idiotypic↗

PubMed1991

Idiotypic and immunochemical differences of anti-DNA antibodies of a lupus patient during active and inactive disease.

IgG anti-DNA antibodies of a lupus patient during active and inactive stages of her disease were studied. There were no significant differences in the amounts, in double-stranded DNA-binding activity, or in complement-fixing ability between purified IgG anti-DNA antibodies of both stages. However, their idiotype (Id) expressions were different as revealed by binding to rabbit anti-Ids raised against each of the anti-DNA antibodies. Moreover, the active stage anti-DNA antibodies had higher activity and less cross-reactivity with polynucleotides. Dominant anti-DNA populations during the active and inactive stages of systemic lupus erythematosus are clonally and qualitatively different.

Antibodies, Antinuclear↗

PubMed1989

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients.

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab')2 fragments. Family members' sera had higher binding to anti-DNA F(ab')2 and to normal IgG F(ab')2 fragments (P less than 0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab')2 than to normal IgG F(ab')2 fragments (P less than 0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The antiidiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.

Adult↗

PubMed1989

Lack of correlation between HLA types and anti-idiotypic production in family members of a lupus patient.

Correlations of anti-single-stranded (ss) DNA, anti-F(ab')2, and anti-idiotypes to HLA types of 16 healthy family members of a lupus patient were studied. High levels of anti-ss DNA (63%) and anti-F(ab')2 (69%) were detected. Of the 12 family members who expressed HLA-DR2 antigen, 8 had anti-ss DNA and anti-F(ab')2 antibodies. One out of 3 family members who shared the same HLA phenotypes, A1B8DR2, of the proband had high levels of anti-idiotype directed against the proband's F(ab')2 anti-DNA. Though a high prevalence of A1B8DR2, of anti-ss DNA, and of anti-F(ab')2 in healthy family members of a lupus patient was found, anti-idiotypes against anti-DNA were not dependent on HLA-A, B, Dr.

Antibodies, Anti-Idiotypic↗

PubMed1989

Characterization of two anti-DNA antibodies bearing distinct idiotypes. Correlation with clinical manifestations.

Spontaneous idiotype shift of anti-DNA antibodies in systemic lupus erythematosus was shown to be associated with changes in the clinical manifestations of the disease. Characterization of two anti-DNA antibody preparations from the same lupus patient showed that lupus nephritis was associated with antibodies showing higher specific antigen-binding activity and avidity to DNA and presenting more cationic quality upon isoelectric focusing and Western blot analysis than the ones found in patients without renal involvement. Lupus cerebritis was associated with anti-DNA antibodies with different idiotypic characteristics not shared by anti-DNA antibodies present in lupus nephritis. Lupus cerebritis anti-DNA antibodies had less specific antigen-binding activity and avidity to DNA and did not show cationic quality. Idiotypic and immunochemical changes of anti-DNA antibodies in lupus may be associated with various clinical manifestations.

Antibody Affinity↗