DNA-abzymes and their clinical significance in systemic lupus erythematosis]

AIM: To evaluate occurrence of DNA-abzymes with catalytic (DNA-hydrolysing) and cytotoxic properties in patients with systemic lupus erythematosus (SLE) for examination of clinical value of DNA-abzymes in diagnosis of autoimmune syndrome and apoptosis level in different variants of immunopathology. MATERIAL AND METHODS: The study group consisted of patients with verified SLE diagnosis (n = 120). They were compared to 72 patients with rheumatoid arthritis (RA), 82 patients with scleroderma systematica (SS), 60 patients with discoid lupus erythematosus (DLE), 88 patients with focal scleroderma (FS) and 198 autoimmune uveitis (AU) patients. 128 donors served control. Catalytic and cytotoxic activity of DNA-abzymes were determined by methods of molecular biology and enzymology. All the patients were examined for blood levels of IgG, IgM and IgA, anti-DNA, anti-Sm and other IgG-autoantibodies, CIC titers, phagocyting activity, content of main D-cell subpopulations. Key immunoregulatory indices were also estimated. RESULTS: DNA-abzymes were detected more often in SLE and RA patients. In SLE, catalytic and cytotoxic activities of DNA-abzymes reached their maximum. There was a correlation with leading clinicoimmunological signs of SLE. The disease was most severe with apparent immunopathology in patients with maximal catalytic and cytotoxic activity of DNA-abzymes. With lowering cytotoxic activity of DNA-abzymes more patients demonstrate low SLE activity without severe organic lesions and alleviated symptoms of immunopathology. CONCLUSION: An important role of DNA-abzymes in pathogenesis of SLE is shown. They are also valuable tools in diagnosis of various clinicoimmunological variants of the disease.     

DNA-abzymes in rheumatoid arthritis: pathogenetic and clinical significance

AIM: To study possible pathogenetic role and clinical significance of DNA-hydrolysing autoantibodies (autoAB) or DNA-abzymes in patients with rheumatoid arthritis. MATERIAL AND METHODS: Prevalence of DNA-abzymes and their catalytic activity were studied in 400 patients with rheumatoid arthritis (RA) and 88 healthy donors matched by age and gender. RESULTS: Associated with DNA-binding autoAB DNA-hydrolysing activity was detected in 41.5% cases of RA. DNA-abzymes were maximally active in men with rheumatoid factor (RF) and women without RF, while it was minimal in men without RF and women with RF. By catalytic activity there was no significant differences between patients with RF and without it. The highest catalytic activity of DNA abzymes was detected in patients with distinct extraarticular pathology. DNA-abzymes were also active in patients with x-ray stage III-IV of the disease in association with high prevalence of catalytic autoAB. DNA abzymes were also active in patients with RA activity stage II and III. CONCLUSION: It is possible to use DNA-abzymes in clinical practice for monitoring of the disease activity in RA.     

Catalytic autoantibodies in autoimmune myocarditis: clinical and pathogenetic implications

AIM: To evaluate pathogenetic and clinical significance of autoantibodies (AAB) with catalytic activity in the serum of patients with autoimmune myocarditis (AM). MATERIAL AND METHODS: The study was made on the sera from 99 patients with AM of different course: malignant, benign, myocardiosclerosis (MCS). In addition to standard immunological parameters, the study was made of serum levels of anticardiomyosine-antiCM (protabzymes) and anti-DNA (DNA-abzymes) of AAB. After obtaining anti-CM and anti-DNA IgG-AT, we determined non-specific and specific proteolytic activity of anti-CM. RESULTS: Maximal specific activity of protabzymes was seen in 73% patients with malignant AM, it correlated with blood levels of anti-CM AAB, DNA-abzymes activity was very high in 45% patients. In MCS proteolytic activity of autoAT was absent in 61% patients. In benign AM occurrence of protabzymes was confirmed in 35% cases. Elevated DNA-hydrolyzing activity of DNA-abzymes occurred in 13% cases. The activity had no significant correlation with serum titers of AB. In MCS proteolytic activity of AAB was absent in 61% cases, but high activity of anti-CM AAB was in 28%. The activity of DNA-abzymes in 44% ranged considerably which, in seropositive cases, detected significant correlation with serum titers of DNA-binding autoAT. CONCLUSION: Evaluation of catalytic activity of AAB may be considered as a criterial test assessing the stage, clinical variants and severity of AM. It also permits formulation of the disease prognosis and its possible outcomes.     

Methyltransferase and phospholipase A2 activity in the cell membrane of neutrophils and lymphocytes from patients with Beh?et's disease, systemic lupus erythematosus, and rheumatoid arthritis

Phospholipid methylation and phospholipase A2 activation in the membrane of neutrophils and lymphocytes, which participate in the induction of cell activation, were assessed in patients with Beh?et's disease, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). [3H-methyl] incorporation and phospholipase A2 activity of neutrophils from active cases of Beh?et's disease and RA were significantly increased compared with normal controls. In lymphocytes from the patients with active Beh?et's disease and RA, a significant increase in methyltransferase activity and a marked enhancement of phospholipase activity were found. A modest increase in these two membrane phospholipid enzyme activities was observed in lymphocytes of patients with active SLE. In addition, these enzyme activities were significantly enhanced in normal leukocytes preincubated with serum from patients with active SLE and malignant RA. The potentiated functions of neutrophils and lymphocyte abnormalities in the patients tested thus seem to be at least partly due to an increase in these enzymatic activities in the cell membrane.     

Poly-adenosine diphosphate ribose autoantibody in systemic lupus erythematosus and other related autoimmune diseases

The binding activities of poly-adenosine diphosphate-ribose (ADPR) and ds-DNA were measured in the sera of patients with systemic lupus erythematosus (SLE) and other collagen diseases in comparison with normal subjects. High polyADPR binding activity was detected in the SLE sera. The polyADPR binding assay was as sensitive as the DNA binding assay for diagnosing SLE. In SLE sera, the increased polyADPR binding activity was correlated with that of ds-DNA and negatively with the complement (CH50) titer. With improvement of clinical symptoms of SLE, the binding activity of polyADPR decreased in parallel to the binding activity of ds-DNA and opposite to the CH50 titer. The polyADPR binding activity was occasionally high in other collagen diseases. Effects of steroid treatment of SLE on the binding activities of polyADPR and ds-DNA, and CH50 titer were examined over half a year, indicating that both binding activities are reliable parameters for judgment of the clinical course.     

Pro- and antioxidant blood system in patients with rheumatoid arthritis and systemic lupus erythematosus

AIM: To estimate the levels of prooxidants (malonic dialdehyde-MDA and nitric oxide-NO), Zn, activity of antioxidant enzymes (superoxide dismutase--SOD and glutathione peroxidase--GPO) in the blood of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). MATERIAL AND METHODS: Pro- and antioxidant system was assessed in 45 RA patients and 32 SLE patients by content of MDA estimated by reaction with thiobarbituric acid, NO (Boehringer Manheim kits, Germany), Zn (Unicam SP 190/191, Great Britain), activity of SOD and GPO (kits Ransod, Ransel; Randox, Great Britain). RA activity was evaluated by DAS index, SLE--by SLEDAI. RESULTS: MDA and NO concentrations were found elevated while SOD and GPO activity low in RA and SLE. The level of Zn was subnormal in RA. The activity of RA and SLE did not influence the above indices significantly. CONCLUSION: RA and SLE patients have high levels of prooxidants (MDA and NO) whereas their antioxidant enzymes (SOD and GPO) activity was low. This may promote oxidant stress.     

Catalytic autoantibodies--a new molecular instrument in cardiology and ophthalmology

AIM: To develop a conceptual model of using catalytic autoantibodies as diagnostic and monitoring tools in organ-specific autoimmune disorders. MATERIAL AND METHODS: A total of 99 patients (56 males and 43 females aged 21-52 years) with autoimmune myocarditis (AM) and 198 patients (77 males and 121 females aged 8-79 years) with autoimmune uveitis (A U) participated in the study. AM patients were examined for anticardiomyosin and anti-DNA autoantibodies (ACM, ADNAab), AU patients - for autoantibodies to S-antigen, IRBP, redopsin, phosphocine, autoDNA. RESULTS: AM patients had double level of DNA-binding autoantibodies. In 1/3 of them there was hydrolysing DNA and cytotoxic activity. In AU patients maximal titers were in Behcet's disease, sympathic ophthalmia, generalized uveitis and viral uveitis. CONCLUSION: Autoantibodies with different specificity and function including DNA-abzymes can be additional diagnostic and prognostic markers.     

Belimumab,an anti-BLyS human monoclonal antibody for potential treatment of inflammatory autoimmune diseases

Background: B lymphocyte stimulator (BLyS) is a factor determining the survival of B cells, and elevated levels in serum or locally have been observed in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients. Belimumab (LymphoStat-B), a human monoclonal antibody that inhibits BlyS, was developed for the treatment of these diseases. Objective: To summarize preclinical development, efficacy and safety of belimumab in treatment of RA and SLE. Methods: Articles found in a PubMed search and data presented in abstract form at international conferences up to August 2008 are described. Results/conclusions: Belimumab was well tolerated in treatment of RA over 24 weeks and SLE over 3 years. It significantly decreased rheumatoid factor (RF) levels, and modestly reduced symptoms of RA, especially in some subgroups such as patients with high disease activity, positive RF and no anti-TNF treatment experience. It also significantly reduced symptoms of SLE, and decreased anti-double-stranded DNA autoantibodies among patients with positive baseline anti-double-stranded DNA or antinuclear antibodies during a long-period treatment. These results suggest that careful patient selection is necessary to achieve optimal outcomes.     

Serial estimation of anti-RNP antibody titers in systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis

Anti-nuclear ribonucleoprotein (RNP) antibody titers were estimated serially in 19 anti-RNP antibody-positive patients, which included 10 patients with systemic lupus erythematosus (SLE), 6 patients with mixed connective tissue disease (MCTD) and 3 patients with rheumatoid arthritis (RA). Counterimmunoelectrophoresis was used for antibody detection and a calf thymus extract was utilized as an antigen. Anti-RNP antibody titers in most patients with MCTD and RA did not fluctuate significantly, whereas those in patients with SLE fluctuated by more than a four-fold serum dilution. The anti-RNP antibody titers in SLE seemed to correlate with disease activity. Previous reports have stated that anti-RNP antibody titers do not fluctuate easily throughout the disease course. The present report shows that they do fluctuate in patients with SLE. The use of high dose of steroids in SLE might be responsible for the fall in antibody titer.     

Antineutrophil cytoplasmic antibodies in patients with diffuse diseases of connective tissue

AIM: To investigate the significance of antibodies against neutrophil cytoplasmic antigens (ANCA) in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SS), their relations with the syndromes and laboratory indices. MATERIAL AND METHODS: The sera from 277 patients suffering from connective tissue disease and 31 healthy persons were examined. Of patients, 103, 126 and 48 had RA, SLE and SS, respectively. The patients in each group were subdivided into ANCA positive (ANCA+) and ANCA negative (ANCA-). The patients were matched within the groups by age, sex and disease duration. There were 41 such pairs in RA group, 23 in SLE and 13 in SS group. Questionnaires and laboratory tests (ANA, RF, a-DNA, a-MPO, a-Scl-70, a-PR3, a-CL) were used in the examination. RESULTS: The sensitivity of ANCA in SLE patients group was as high as 58.7%, specificity--93.5%. In other groups ANCA were less frequent. ANCA were significantly associated with skin vasculitis and ANA prevalence but the disease activity in SLE was not related to this feature. Anemia and antibodies against cardiolipin were found significantly more frequently in ANCA positive RA group. In SS group the inverted clinical association with kidney damage was seen but a-DNA were more prominent in ANCA+ group. Subspecificity for a-MPO and a-PR3 and lactoferin by ELISA were revealed less often than a-ANCA by immunofluorescence. Only two SLE patients with a-lactoferin antibodies were evidently different in prognosis while the other ones did not differ in the disease course within their group. CONCLUSION: The ANCA pattern in connective tissue diseases is found rather often but only few clinical and laboratory associations could be established such as skin vasculitis and ANA domination in SLE group, anemia and a-CL in RA group and a-DNA in SS ANCA+ groups. The validity of ANCA test is not so significant as it is in vasculitis patients.     

Human-human hybridoma autoantibodies with both anti-DNA and rheumatoid factor activities.

Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.     

类风湿关节炎和系统性红斑狼疮患者血清基质金属蛋白酶2、9检测的意义

目的探讨基质金属蛋白酶（MMPs）在类风湿关节炎（RA）和系统性红斑狼疮（SLE）中的重要作用。方法采用酶联免疫吸附试验和酶谱分析了48例RA和27例SLE患者及186例健康对照者血清中MMP-2和MMP-9的浓度和活性。结果酶谱分析结果显示RA和SLE患者血清MMP-2和MMP-9的活性显著高于对照组（P〈0.01）。ELISA检测结果也表明RA、SLE组的MMP-2和MMP-9浓度明显高于对照组（P〈0.05）。结论MMP-2和MMP-9在RA和SLE患者血清中的水平及活性明显升高,表明MMPs在这些自身免疫疾病起一定的作用,可作为临床辅助诊断指标。     

绝经前女性风湿病患者骨密度变化影响因素分析

目的:对绝经前女性系统性红斑狼疮(SLE)与类风湿关节炎(RA)患者进行骨密度(BMD)的监测,评估发生骨量丢失的原因。方法:采用超声骨密度仪测量89例绝经前女性患者(SLE49例,RA40例)及68例健康志愿者(对照组)右跟骨部位的BMD,同时对患者诸多影响BMD的因素(年龄、病程、疾病活动度、激素使用时间、激素累积剂量及激素日剂量)与BMD进行相关分析及多元回归分析。结果:患者右跟骨部位的BMD(SLE为-1.42±0.56,RA为-1.36±0.63)明显低于对照组(-0.58±0.52,P<0.01),骨质疏松的发生率(SLE为17.1%,RA为13.3%)明显高于对照组(P<0.01)。线性相关分析结果显示SLE患者的BMD与C3呈正相关(r=0.521,P<0.01),与激素累积剂量呈负相关(r=-0.398,P<0.05);BMD与RA患者的CRP呈负相关(r=-0.431,P<0.05)。多元回归分析提示C3下降与激素累积剂量增高为SLE患者BMD下降的独立危险因素,CRP升高为RA患者BMD下降的独立危险因素。结论:绝经前女性SLE与RA患者骨质疏松的发生率较正常人群明显增高。SLE与RA患...     

Correlations between IL-2 enhancing activity and clinical parameters in patients with rheumatoid arthritis and systemic lupus erythematosus.

In a previous paper (Tomura, K. et al. Tohoku J. Exp. Med., 1989, 159, 171-183), we discovered IL-2 enhancing factor(s) designated B cell derived-growth enhancing factor-2 (BGEF-2), which enhanced IL-2 dependent cell proliferation, and reported that BGEF-2 was produced by B cells of the patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) only when they were in the active stage of the disease. In this paper, we studied relationship between each IL-2 enhancing activity from B cell supernatant of the patients with these diseases and clinical parameters. IL-2 enhancing activities did not correlate with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), but correlated with plasma concentrations of gamma-globulin from the patients with RA and SLE in the active stages. IL-2 enhancing activities correlated with hypocomplementemia and leukocytopenia in the patients with SLE, and also correlated with RAHA titer in the patients with RA. Moreover, on several patients with RA or SLE in the active stages, diminution of IL-2 enhancing activity was found when they were in the remission stage after treatments. These findings suggested that IL-2 enhancing activity (i.e., BGEF-2 activity) correlated with activity of these diseases and supported the hypothesis that BGEF-2 played an important role in the polyclonal B cell activation and autoantibody production in patients with these diseases.     

Potential role of HBV DNA-induced CD8high T cell apoptosis in patients with systemic lupus erythematosus and rheumatoid arthritis

ObjectiveTo investigate the potential role of hepatitis B virus (HBV) DNA-induced CD8^high T cell apoptosis in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).MethodsThe activity and HBV seropositivity rates of patients with SLE and RA were determined. The proportions of T cell subgroups were detected by fluorescence-activated cell sorting. The apoptosis of T cell subgroups was detected after peripheral blood mononuclear cells were stimulated with HBV DNA.ResultsThe HBV infection rate was higher in patients with RA than in patients with SLE. Current or previous HBV infection was more common among patients with inactive SLE than among those with active SLE. Conversely, previous or current HBV infection was more common among patients with active RA than among those with inactive RA. CD4⁻CD8^high T cell counts were higher among patients with active SLE than in those with inactive SLE. However, CD4⁻CD8^high T cell counts were lower in patients with active RA patients than in those with inactive RA. HBV DNA increased the apoptosis of CD4⁻CD8^high T cells.ConclusionHBV DNA-induced CD8^high T cell apoptosis appears to play different roles in SLE and RA.     

Relationship between complement-fixing (hemolytic) antibodies to single-stranded and double-stranded DNA and the prognosis in systemic lupus erythematosus

Hemolytic (complement-fixing) antibodies to single-stranded (ss) or double-stranded (ds) DNA, measured by recently developed PHL assay, occurred closely correlated with renal activity in patients with systemic lupus (SLE). Approximately one third of the patients with renal disease had hemolytic antibodies to ss-DNA but never to ds-DNA. Hemolytic antibodies were scarcely detectable in patients with mild course. Serial studies also revealed that the estimation of the hemolytic antibodies to ds- and/or ss-DNA was particularly valuable in predicting the future course of the SLE. The emergence of hemolytic antibodies to DNA may be an ominous sign suggestive of grave prognosis in SLE.     

CTLA-4基因启动子区-318位点基因多态性与SLE的相关性研究

目的：探讨细胞毒性T细胞相关分子—4(cytotoxic T lymphocyte antigen4，CTLA—4)基因启动子区—318位点基因多态现象在中国南方人群中的分布及与SLE易感性的关系。方法：提取外周静脉血有核细胞基因组DNA，以聚合酶饺反应—限制性片段长度多态性(polymerase chain reaction—restriction fragment length polymorphism,PCR-RFLP)方法检测180例SLE患者和140名正常对照组CTLA—4基因启动子区—318位点基因型。结果：SLE患者CTLA—4基因启动子区—318位点基因型频率分别为TT型1.1％、TC型41.1％和CC型58．8％，等位基因频率为T等位基因21.7％、C等位基因78.3％。SLE患者各种基因型频率及等位基因频率与对照组比较均无统计学意义。CTLA—4基因启动子区—318位点各基因型与SLE患者临床表现及实验室检查结果无相关关系。结论：SLE患者CTLA—4基因启动子区—318位点基因多态现象与SLE发病无关。     

Circulating immune complex levels measured by new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies correlate with clinical activities of SLE but not with those of RA

The authors studied sera from both patients with SLE and from those with RA to evaluate clinical usefulness and significance of circulating immune complexes (CIC) detected with new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies. CIC values of patients with SLE significantly correlated to severity of disease activities evaluated by clinical symptoms and laboratory tests, especially for serum complement levels. Since substances detected with the ELISA kits were closely related to serum complement components, it was determined that a direct relationship exists between clinical activities and CIC values appearing in SLE patients with hypocomplementemia. In RA patients, CIC values did not correlate to clinical activities evaluated by Lansbury's index, anatomical bone damage with X-ray or functional assessment of activities of daily living, but did significantly correlate to levels of IgM-RF, serum IgG concentrations and some markers of systemic inflammation. Detection of IC after fractionation of RA sera revealed a broad range of molecular sizes detectable with the ELISA kits, which indicated that CIC in vivo were heterogenic and complicated in formation, degradation and interaction with serum complements.     

Frequency of lymphadenopathy in rheumatoid arthritis and systemic lupus erythematosus

This study aimed to assess the frequency of all palpable lymph nodes during active disease and remission in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Hospital records of 100 SLE patients, 100 RA patients, 100 spondyloarthropathy patients, and 150 osteoarthritis patients, treated in our rheumatology department, were evaluated retrospectively. Overall frequencies of enlarged lymph nodes in patients with active RA and SLE were 82% and 69%, respectively. Enlarged lymph nodes associated with RA were mostly located in the axillary region, and in SLE the nodes were smaller and lymphadenopathy was more generalized compared with RA. Palpable lymph nodes disappeared in the majority of patients during remission. Lymphadenopathy was significantly less frequent in patients treated with steroids before admission. Lymph node enlargement is an important physical finding associated with RA and SLE disease activity. Atypical locations and unusually large lymph nodes should raise clinical suspicion of another underlying disease.