Antibodies to centromere antigens measured by an automated enzyme immunoassay

BACKGROUND: Anticentromere antibodies (ACA) are frequently observed in patients with Raynaud's phenomenon and in the CREST syndrome, a subclass of systemic sclerosis. Likewise, ACA are also found in other autoimmune and non-autoimmune diseases. The objective of the present study was to evaluate the clinical utility of the measurement of antibodies to the best characterized centromere antigen (CENP-B) protein by an enzyme-linked immunosorbent assay (ELISA) that uses human recombinant CENP-B antigen and compare it with indirect immunofluorescence assay (IFA) on HEp-2 cells. METHODS: We have analyzed 128 sera samples from patients with the following diseases: systemic lupus erythematosus (SLE, n = 53), mixed connective tissue disease (n = 1), primary Sj?gren syndrome (n = 10), primary Raynaud's phenomenon (n = 10), primary systemic sclerosis (n = 7), polymyositis/dermatomyositis (n = 3), rheumatoid arthritis (n = 9), cutaneous lupus (n = 5), primary biliary cirrhosis (n = 9), chronic autoimmune hepatitis (n = 5) and ANA-positive non-autoimmune diseases (n = 16). RESULTS: The ELISA evaluated shows a good concordance with IFA, with the advantage of being an automatable quantitative technique. CONCLUSIONS: Measurement of anticentromere antibodies by this ELISA using human recombinant antigen is a useful alternative for the autoimmune laboratory checking for diseases associated with anticentromere antibodies.     

Prevalence and factors associated with headache in patients with systemic lupus erythematosus

Headache is common in systemic lupus erythematosus with reported prevalence as high as 70%. The aims of this study were: to estimate the prevalence and types of headache in a sample of patients with systemic lupus erythematosus comparing it with rheumatoid arthritis, to determine clinical and serological associations. Eighty-one systemic lupus erythematosus and 29 rheumatoid arthritis consecutive patients seen in our outpatient clinic were interviewed. Headache was evaluated using the diagnostic criteria proposed by the International Headache Society. Additional evaluations were carried out in the 81 systemic lupus erythematosus patients including depression, disease activity, lupus damage, function disability, quality of life, and severity degree using a validated scales. We analysed the following autoantibodies: anti-double stranded DNA, anti-nucleosomes, anti-histones, anti-ribosomal P, anti-cardiolipin antibodies, anti-beta2-glycoprotein-I (GPI), and antinuclear antibodies. Forty-one per cent of systemic lupus erythematosus and 17% of rheumatoid arthritis patients suffered from headache (P = 0.02). No significant difference for any primary headache type between the two groups was found. Frequency of headache types in systemic lupus erythematosus patients was: migraine 24%, tensional-type headache 11%, and mixed headache 5%. In systemic lupus erythematosus patients the risk factors associated with headaches were Raynaud's phenomenon (OR 3.6; 95% CI 1.3-9.5; P = 0.009) and beta2GPI antibody positivity (OR 4.5; 95% CI 1.2-16.2; p = 0.016). We conclude that headache is more common in systemic lupus erythematosus than in rheumatoid arthritis patients and was independently associated with Raynaud's phenomenon and beta2GP-I antibodies.     

Comparison of different methods for the detection of autoantibodies in autoimmune diseases

Summary Different immunological techniques were compared for their sensitivity in detecting some important autoantibodies in the sera of patients with rheumatic diseases. Sera of patients with systemic lupus erythematosus were screened for anti-dsDNA, Sm, and RNP autoantibodies byCrithidia luciliae assay, Farr assay, enzyme immunoassay, and immunoblotting. Sera of patients with Sj?gren's syndrome were screened for anti-SSA and anti-SSB antibodies by enzyme immunoassay, counter immunoelectrophoresis, and immunoblotting and sera of patients with scleroderma for SCL-70 autoantibodies by enzyme immunoassay counter immunoelectrophoresis, and immunofluorescence on Hep-2 cells. Enzyme immunoassay and counter immunoelectrophoresis gave the most positive results and the best agreement compared with the other techniques. Immunofluorescence gave few positive results for each antibody evaluated. Immunoblotting gave intermediate results for all autoantibodies except anti-SSA, where the prevalence was low. There was no relationship between levels of dsDNA, Sm, and RNP antibodies and disease activity score in systemic lupus erythematosus patients.     

抗核小体抗体对系统性红斑狼疮诊断价值的探讨

目的：探讨抗核小体抗体对系统性红斑狼疮的诊断和判断疾病活动度的价值。方法：采用酶联免疫吸附试验（ELISA）检测45例系统性红斑狼疮、25例类风湿关节炎、16例原发性干燥综合征、8例多发性肌炎／皮肌炎、5例混合性结缔组织病、18例骨性关节炎和30例健康对照组血清中的抗核小体抗体，将抗核小体抗体与疾病活动度（以SLEDAI评分）、抗双链DNA抗体、抗Sm抗体等指标进行比较。结果：抗核小体抗体在45例系统性红斑狼疮患者34例（75．5％）阳性，其敏感度和特异度分别为75．5％，93，1％；抗核小体抗体与SLEDAI评分、抗双链DNA抗体、肾损害有相关性。结论：抗核小体抗体在系统性红斑狼疮中敏感性较高，是诊断系统性红斑狼疮和了解狼疮活动度的良好指标。     

52例抗着丝点抗体阳性患者临床分析

目的　进一步探讨抗着丝点抗体 (ACA)检测的临床意义。方法　对 5 2例ACA阳性患者进行临床回顾性分析。结果　抗着丝点抗体阳性可在多种疾病中出现 ,其中包括系统性硬化征 ,局限性硬度病 ,CREST综合征 ,系统性红斑狼疮 ,类风湿性关节炎 ,干燥综合征 ,雷诺现象 ,重症肌无力 ,重叠综合征 ,原发性胆汁性肝硬化 ,慢性丙型肝炎和自身免疫性肝炎。ACA阳性患者可同时出现类风湿因子 ,多种核型的抗核抗体、抗线粒体抗体 ,抗心磷脂抗体和多种类型的抗ENA抗体。雷诺现象为ACA阳性患者主要临床表现。结论　ACA并非系统性硬化征局限型CREST亚型的特异性抗体 ,ACA阳性时 ,必须结合患者临床症状进行诊断。     

Clinical utility of common serum rheumatologic tests

Serum rheumatologic tests are generally most useful for confirming a clinically suspected diagnosis. Testing for rheumatoid factor is appropriate when rheumatoid arthritis, Sj?gren's syndrome or cryoglobulinemia is suspected. Antinuclear antibody testing is highly sensitive for systemic lupus erythematosus and drug-induced lupus. Anti-double-stranded DNA antibodies correlate with lupus nephritis; the titer often corresponds with disease activity in systemic lupus erythematosus. Testing for anti-Ro (anti-SS-A) or anti-La (anti-SS-B) may help confirm the diagnosis of Sj?gren's syndrome or systemic lupus erythematosus; these antibodies are associated with the extraglandular manifestations of Sj?gren's syndrome. Cytoplasmic antineutrophil cytoplasmic antibody testing is highly sensitive and specific for Wegener's granulomatosis. Human leukocyte antigen-B27 is frequently present in ankylosing spondylitis and Reiter's syndrome, but the background presence of this antibody in white populations limits the value of testing. An elevated erythrocyte sedimentation rate (ESR) is a diagnostic criterion for polymyalgia rheumatica and temporal arteritis; however, specificity is quite low. ESR values tend to correlate with disease activity in rheumatoid arthritis and may be useful for monitoring therapeutic response.     

Analyses of Lymphocytes from Patients with Rheumatoid Arthritis and Systemic Lupus Erythematosus OCCURRENCE OF INTERFERING COLD-REACTIVE ANTILYMPHOCYTE ANTIBODIES

Large percentages of the lymphocytes from some patients with rheumatoid arthritis and systemic lupus erythematosus were densely covered with Ig demonstrable by immunofluorescence, which was occasionally present in the form of caps. The amount and character of the Ig staining depended largely on the procedures used in the isolation and washing of the lymphocytes. Cold-reactive antilymphocyte antibodies present in many sera wre primarily responsible for these variations. Overnight culture of the lymphocytes proved to be an efficient procedure for the removal of adsorbed antibody. Some evidence was also obtained for the presence of circulating immune complexes and exogenous rheumatoid factor molecules on the lymphocyte surface. Thus on freshly isolated cells the demonstration of surface Ig proved to an unreliable marker of bone marrow-derived (B) cells in these disease: the actual percent of B cells with intrinsic surface Ig was often markedly decreased. In patients with systemic lupus erythematosus, this reduction was in agreement with the low numbers of cells that had a receptor for aggregated IgG. The mean percentage of thymus-derived (T) cells in both diseases was slightly greater than the normal level.The concentrations of lymphocytes in joint fluids from patients with rheumatoid arthritis were often greater than levels found in blood. T cells primarily accounted for this increase. The T cells typically formed unusually dense rosettes with sheep erythrocytes. B lymphocytes were proportionally much diminished. Evidence was obtained for the existence of a major joint fluid lymphocyte population that lacked all assayed surface markers.     

Prostaglandins in the pathogenesis of various systemic diseases of the connective tissue

A study was made of the content of prostaglandins E, A, F2 alpha and malonic dialdehyde (MDA) in the blood plasma and urine of patients with systemic lupus erythematosus, systemic scleroderma, rheumatoid arthritis and healthy persons. The levels of plasma and urine prostaglandins and MDA were significantly elevated in systemic lupus erythematosus and rheumatoid arthritis, and in systemic scleroderma they did not differ from those in normal. The ratio of pressor and depressor prostaglandins in systemic lupus erythematosus and rheumatoid arthritis did not change, and in systemic scleroderma there was a strong shift to pressor prostaglandins. Change in the content and ratio of prostaglandins and MDA in systemic diseases of connective tissue was suggestive of differences of pathogenetic effects of these mediators in some diseases of this group.     

Detection of serum catalase antibodies in patients with inflammatory rheumatic diseases by immobilized magnetic sorbents

Sixty patients with systemic lupus erythematosus, 48 patients with rheumatoid arthritis, and 30 healthy individuals (a control group) were examined. Modified procedures for enzyme immunoassay (EIA) and immunofluorescence (IF) assays with an immobilized magnetic sorbent (MS) based on catalase as an antigenic matrix were used to detect catalase antibodies (Ab). The application of immobilized MSs and a photoelectric digital display attachment to a fluorescence microscope permits measurements of Ab to soluble antigens (catalase) in the IF assay. Nevertheless, for detection of catalase Ab in laboratory practice, preference should be given to EIA using a MS as a more sensitive procedure.     

Serum glycylproline p-nitroanilidase activity in rheumatoid arthritis and systemic lupus erythematosus

Glycylproline p-nitroanilidase activity in serum of patients with advanced rheumatoid arthritis or with systemic lupus erythematosus but with normal hepatic function was found to be significantly lower than that of normal adult controls. Decrease of this enzyme's serum activity was more pronounced in systemic lupus erythematosus. A significant inverse correlation was observed between the enzyme activity and the duration of rheumatoid arthritis.     

DEMONSTRATION OF AN EPITHELIAL ANTIGEN IN COLON BY MEANS OF FLUORESCENT ANTIBODIES FROM CHILDREN WITH ULCERATIVE COLITIS

Thirteen sera from children with ulcerative colitis were examined for antibodies reacting with constituents of human colonic tissue by means of immunofluorescent methods. 3 out of 10 sera reacted positively when tested by the direct staining method while 6 out of 13 reacted positively when tested by the indirect method with conjugates of rabbit anti-human gamma globulin. The specificity of the reactions could be confirmed by inhibition tests. 16 sera from healthy children and adults yielded completely negative results. The staining capacity of various sera was correlated to their hemagglutinating titer when they were tested with sheep erythrocytes, coated with phenol-water extract of human colon. Absorption experiments indicated that the stainable antigen was also present in the extracts used for the hemagglutination experiments. In unfixed tissue sections, fluorescent antibodies were adsorbed onto the epithelial cells of the mucosa. Adsorption on epithelial basement membranes could not be demonstrated. Fluorescent H agglutinins, isolated from eel serum, were adsorbed onto the same mucosal structures of human colon (blood group O) as the antibodies in the sera of patients with ulcerative colitis. However, any immunological relationship between H substance and the colonic antigen of ulcerative colitis could be ruled out by cross-inhibition and hemagglutination inhibition experiments. Fluorescent serum from patients with rheumatoid arthritis also stained sections of human colon but the localization of the stainable antigens was different from that visualized with the ulcerative colitis sera. Inhibition experiments indicated that the rheumatoid arthritis serum contained antibodies staining colon antigens different from those reacting with antibodies in the ulcerative colitis sera. Sera from patients with systemic lupus erythematosus or with the nephrotic syndrome, which all hemagglutinated erythrocytes coated with colon extract, did not stain the sections of the colon tissue either because of suboptimal antibody concentration or because of a difference in type or localization of the antigen.     

Markers of collagen metabolism in sera of patients with various rheumatic diseases

The activity of galactosylhydroxylysyl glucosyltransferase (S-GGT) and concentration of the amino-terminal propeptide of type III procollagen, measured with two different radioimmunoassays, S-Pro(III)-N-P and S-Fab, were determined in the sera of 209 patients with various rheumatic diseases. The mean values for all these three assays were elevated in the patients with rheumatoid arthritis, whereas only the mean value for S-GGT was elevated in osteoarthrosis. The mean S-GGT but not S-Pro(III)-N-P or S-Fab was also elevated in psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus. S-GGT correlated significantly both with S-Pro(III)-N-P and S-Fab in the pooled group of all the patients and in the cases of rheumatoid arthritis and psoriatic arthritis, and also with S-Fab in the cases of osteoarthrosis. S-Pro(III)-N-P and S-Fab correlated with each other in every disease group. The ratio S-Fab:S-Pro(III)-N-P was significantly higher in the patients with osteoarthrosis, ankylosing spondylitis and systemic lupus erythematosus than in those with rheumatoid arthritis. The data indicate that definite changes can be seen in the values of serum markers of collagen metabolism in rheumatoid arthritis, psoriatic arthritis, osteoarthrosis, ankylosing spondylitis and systemic lupus erythematosus, the most sensitive indicator being S-GGT.     

Antibody-dependent cell-mediated cytotoxicity in selected autoimmune diseases.

Antibody-dependent cell-mediated cytotoxicity mediated by peripheral blood lymphocytes was studied in patients with systemic lupus erythematosus, polyarteritis nodosa. Sjogren's syndrome, and rheumatoid arthritis. The target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody. Antibody-dependent cell-mediated cytotoxic activity was normal in Sjogren's syndrome and rheumatoid arthritis but significantly decreased (P is less than 0.001) in active systemic lupus erythematosus and in two patients with polyarteritis nodosa. A partial regeneration of antibody-dependent cell-mediated cytotoxic activity was obtained by treatment with pronase and DNase followed by overnight incubation. Sera from patients with systemic lupus erythematosus inhibited antibody-dependent cell-mediated cytotoxic activity of normal lymphocytes. The inhibitory activity was studied by specific immunoadsorption and sucrose density geadient ultracentrifugation. Removal of IgG but not IgM greatly reduced inhibition. Inhibitory factors were present in 7S and heavier fractions containing IgG. Five systemic lupus erythematosus patients were studied serially to determine if improvement in clinical status could be correlated with a decrease in serum inhibitory factors as studied by inhibition of normal antibody-dependent cell-mediated cytotoxicity. Indeed, a greater serum inhibitory capacity was found in each patient during periods of greater disease activity.     

Clinical significance of serum hydroxyproline-containing peptides with special reference to hyproprotein

Serum hydroxyproline-containing peptides were separated into three fractions, i.e. large protein, polypeptide and smaller peptides, by gel filtration on Sephadex G-200. The large protein was presumed to be the C1q subunit of the first component of complement by an immunological analysis. The polypeptide had an electrophoretic mobility corresponding to beta-globulin and its molecular weight was estimated between 35000 and 45000 by gel filtration analysis. The polypeptide might well be defined as hyproprotein. The molecular weight of smaller peptides was estimated between 1400 and 3000. As compared with the control value, hyproprotein levels were significantly increased in rheumatoid arthritis, systemic lupus erythematosus and chronic liver disease. C1q levels were slightly lowered in rheumatoid arthritis and systemic lupus erythematosus, and were slightly increased in chronic liver disease but were not significant. In all patient groups smaller peptides levels remained in the normal range. These results suggest that serum hyproprotein levels are raised in some fibroproliferative disorders, probably reflecting collagen metabolism.     

Antinuclear antibodies in patients with chronic fatigue syndrome

Significance of antinuclear antibodies (ANA) in the patients with chronic fatigue syndrome (CFS) was reviewed. When indirect immunofluorescence with the HEp-2 cells as the substrates was used, prevalence of the positive ANA was reportedly 15-25%. The ANA titers were low and the immunofluorescent staining patterns were heterogeneous. One group in the USA reported that 'nuclear envelope staining pattern' was found in more than 50% of the patients with CFS. This results, however, have not been confirmed by any other research groups. Clinical significance of the positive ANA in the CFS patients resides in differential diagnoses of systemic lupus erythematosus and other diffuse connective tissue diseases. Recently, several ANAs specific to CFS have been described. We reported anti-68/48kD protein antibodies utilizing SDS-PAGE/ immunoblot method. These autoantibodies were found in 13% of 114 CFS patients and 0% in healthy subjects (p < 0.05). Hypersomnia and difficulty in concentration were found more frequently in the CFS patients with this specific autoantibody.     

自身抗体联合检测在自身免疫性疾病诊断中的应用价值

目的运用间接免疫荧光法（IIF）检测抗核抗体（ANA）、抗双链脱氧核糖核酸（抗ds—DNA）及免疫印迹法（IBT）检测抗可提取性抗核抗体（抗ENA）。方法ANA、抗ds—DNA均采用IIF，其中ANA抗原片同时固定有Hep-2细胞和猴肝切片，抗ds-DNA检测抗原片采用绿蝇短膜虫。抗ENA检测采用IBT。共检测自身免疫性疾病患者170例。结果ANA在系统性红斑狼疮（SLE）、干燥综合征、肌炎／皮肌炎、混合性结缔组织病、系统性硬皮病、类风湿关节炎、强直性脊柱炎、过敏性紫癜的阳性率分别为91．6％、100．0%、100．0％、82．4％、65．4％、35．7％、16．7％、42．9％。抗ds—DNA对SLE阳性率为65．1％，特异性为96．6％。抗ENA对自身免疫性疾病（AID）鉴别诊断具有重要意义。结论运用Hep—Z／猴肝复合抗原片检测ANA有利于AID的初步筛选和判断。抗ENA抗体谱检测有利于自身免疫疾病的鉴别诊断，抗ds—DNA可作为SLE治疗和监测的指标。     

Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis

We examined the activities of peptidases in plasma and tissues of the New Zealand Black (NZB) mouse as an animal model of human systemic lupus erythematosus, and also in serum from patients with rheumatoid arthritis and systemic lupus erythematosus. Activities of dipeptidyl peptidase II (DAP II) and post-proline cleaving enzyme (PPCE) were increased, and dipeptidyl peptidase IV (DAP IV) activity was decreased in plasma and spleen of NZB mice, as compared with the control BALB/c mice. Likewise, the activity of DAP II was increased and that of DAP IV was decreased in serum of patients with rheumatoid arthritis and systemic lupus erythematosus. These results indicate the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.     

Importance of the determination of antibodies to n-DNA in systemic lupus erythematosus and other rheumatic diseases (comparative study of radioisotope and immunoenzyme technics)

Altogether 95 patients with systemic lupus erythematosus, 36 patients with rheumatoid arthritis, 10 with sclerodermia systematic, 10 with Bekhterev's disease, 9 with rheumatic fever, and 20 healthy persons were examined. An analysis of the results made it possible to establish that in SLE, sclerodermia systematic and Bekhterev's disease the frequency of detection of higher levels of antibodies to n-DNA using enzyme immunoassay and radionuclide methods was approximately the same. SLE patients were characterized by a 2-fold increase in the level of antibodies to n-DNA as compared to patients with sclerodermia systematic, Bekhterev's disease and rheumatic fever suggesting usefulness for differentiation of these diseases. In rheumatic fever and rheumatoid arthritis antibodies to n-DNA in minimal elevated levels were revealed more frequently under enzyme immunoassay than in radionuclide ones. In SLE high levels of antibodies to n-DNA under the method of radionuclide binding showed significant correlation with a decrease in complement hemolytic activity, high levels of circulating immune complexes, with a higher frequency of detection of the antinuclear factor, and reflected lupus nephritis severity. Enzyme immunoassay used for the detection of antibodies to n-DNA showed correlation of high levels of these antibodies with signs of developing cerebrovasculitis and pulmonary lesion.     

A soluble acidic protein of the cell nucleus which reacts with serum from patients with systemic lupus erythermatosus and Sjögren''s syndrome.

A soluble nuclear antigen that reacts with sera obtained from patients with systemic lupus erythematosus and Sj?gren's syndrome has been described. The antigen, tentatively named the Ha antigen after the prototype serum, was shown to react with specific antibodies by precipitin, complement fixation, and immunofluorescence techniques. The Ha antigen prepared from isolated nuclei of calf thymus glands, calf liver, and rat liver showed identical immunological reactivities; a wide distribution among different species and tissues is presumed. The Ha antigen was destroyed by trypsin and relatively mild heat or pH variation from neutrality, but was resistant to DNase or RNase. Many of these characteristics are similar to those of the "B" antigen to which antibodies have recently been described in Sj?gren's syndrome. The nuclear origin of the Ha antigen was confirmed by the speckled nuclear immunofluorescence staining pattern given by purified antibody to Ha obtained from a specific immune precipitate. Preliminary results showed approximately 13% of patients with systemic lupus erythematosus and 30% of patients with Sj?gren's syndrome had precipitating antibodies to the Ha antigen.     

C-reactive protein in inflammatory articular diseases: comparison of concentrations in blood and synovial fluid

We evaluated the diagnostic value of measuring C-Reactive Protein (CRP) in blood and in synovial fluid for the detection of inflammatory articular diseases in 154 patients. High concentrations of CRP in blood were found in microcrystalin arthritis, polymyalgia rheumatica and Horton's disease. Our results show a good correlation between CRP and erythrocyte sedimentation rate for ankylosing spondylitis (p less than 0.01), systemic lupus erythematosus (p less than 0.01), rheumatoid arthritis (p less than 0.05), polymyalgia rheumatica and Horton's disease (p less than 0.05). The CRP measurement in blood did not separate seropositive versus seronegative rheumatoid arthritis, systemic lupus erythematosus versus rheumatoid arthritis and treated versus non-treated rheumatoid arthritis. There was a good correlation between CRP concentration in blood and in synovial fluid but the concentration was lower in synovial fluid than in blood (p less than 0.01). Then, the CRP measurement in synovial fluid does not have a higher diagnostic value than in blood.