Anti-PM/Scl-100 and anti-RNA-polymerase III antibodies in scleroderma

BackgroundSystemic sclerosis (SSc) is a rare autoimmune disease characterized by the presence of various autoantibodies, including anti-centromere, anti-topoisomerase (Scl-70), anti-PM/Scl-100, and anti-RNA-polymerase III (RNA Pol-III) antibodies. Recently, new ELISA based immunoassays have become available for the detection of anti-PM/Scl and anti-RNA Pol-lII antibodies.ObjectiveWe studied the prevalence and clinical association of anti-PM/Scl-100 (PM1-Alpha) and anti-RNA Pol-III antibodies.MethodsAntibodies to PM1-Alpha and RNA Pol-III were measured by ELISA (DR. Fooke Laboratories and Inova Diagnostics, respectively) in 242 patients with various connective tissue diseases (CTD) (including 70 SSc patients) and in 36 non-CTD controls.ResultsLow levels of PM1-Alpha antibodies were found in various CTDs, whereas high levels were exclusively found in SSc, dermatomyositis and polymyositis, albeit at low frequency (4.7%). Anti-RNA Pol-III antibodies were found in 7% of SSc and in 1% of non-CTD and CTD controls. Anti-centromere and anti-Scl-70 antibodies were found in 37% and 21% of SSc patients, respectively. Anti-centromere antibodies were associated with limited cutaneous SSc and anti-Scl-70 antibodies with diffuse cutaneous SSc and interstitial lung disease. Because of the low number of samples positive for anti-PM/Scl-100 or RNA Pol-III antibodies, no clinical feature was statistically correlated with the presence of either reactivity, but taken together the presence of either antibody was correlated with interstitial lung disease. Anti-PM1-Alpha and anti-RNA Pol-III antibodies were mutually exclusive with anti-Scl-70 antibodies.ConclusionsAt high levels, anti-PM/Scl-100 antibodies were associated with SSc, PM, and DM, albeit at low frequency. Anti-RNA Pol-III antibodies were associated with SSc (in 7%) with high specificity.     

Antimyenteric neuronal antibodies in scleroderma.

The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.     

Antinuclear, anticentromere and anti-ScL-70 antibodies in rheumatic diseases]

Methods are described that are used for the titration of antinuclear, anticentromere, and anti-Scl-70 antibodies in systemic scleroderma, systemic lupus erythematosus, and rheumatoid arthritis: indirect immunofluorescence with various antigenic substrates (sections of fresh-frozen rat liver and Hep-2 cell culture), counter-current immunoelectrophoresis, isolation of Scl-70 antigen. Use of Hep-2 cells as a substrate for indirect immunofluorescence was found clinically and diagnostically more effective since it permitted the detection of anticentromere antibodies and anti-Scl-70. Nucleolar, mottled, homogeneous, marginal immunofluorescence types were observed when rat liver sections and Hep-2 cells were used for substrates. Anticentromere antibodies and anti-Scl-70 were isolated significantly more frequently in systemic lupus erythematosus or rheumatoid arthritis.     

Ku抗原的提取及其抗体的检测

目的 测定本组自身免疫性结缔组织疾病（ＣＴＤｓ）患者中抗Ｋｕ抗体的阳性率。方法 制备兔腺丙酮粉,进而提取Ｋｕ抗原,采用免疫双扩散法检测４３８份ＣＴＤｓ和５０份正常对照血清中的抗Ｋｕ抗体。结果 自制的Ｋｕ抗原与标准Ｋｕ抗血清免疫双扩散出现清晰的沉淀线。４８８份血清中,抗Ｋｕ抗体阳性率２．５％（１２／４８８）,其中皮肌炎为２．２％（１／４５）,弥漫型系统性硬化症为３．４％（２／５８）,系统性硬化症并多     

Diagnostic assays for Anti-PM/Scl IgG antibodies: Heterogeneity in antibody response or lack of standardization?

BackgroundThe aim of this study was to compare the correlation between new diagnostic methodologies for detecting anti-polymyositis/scleroderma (anti-PM/Scl) IgG antibodies associated with myositis and/or systemic scleroderma assays with existing platforms.MethodsSera from 164 samples previously tested for anti-PM/Scl IgG antibody by immunodiffusion, ID; 171 sera screened for anti-PM/Scl IgG by immunoprecipitation, IP; an additional group of 215 sera tested by ID and 46 healthy blood donor sera were retrospectively evaluated. Anti-PM/Scl IgG antibodies were measured using three PM/Scl-100 specific enzyme immunoassays (EIAs), PM1-alpha (PM1-α) EIA and a line immunoblot assay (LIA) for anti-PM/Scl-75 and ? 100 IgG antibodies. Selected samples were tested for the presence of antinuclear antibody (ANA) by indirect fluorescent antibody (IFA) assay.ResultsThe overall agreement between ID and all anti-PM/Scl IgG EIAs as determined by Crohnbach's alpha was unacceptable (α < 0.50). The concordance between the IP and either LIA or PM1-α EIA was greater than 90% however, the best agreement was seen between the IP and LIA PM/Scl-100 assays (98.3%). Compared to the LIA PM/Scl-75 and PM1-α tests, the LIA PM/Scl-100 IgG assay showed the best specificity in the healthy control group.ConclusionsOur results demonstrate considerable differences between assays for detecting anti-PM/Scl IgG antibodies which cannot be attributable to heterogeneity in antibody response alone. Further characterization and standardization of these assays are needed.     

ANTINUCLEAR ANTIBODIES IN RABBIT ANTISERA

Antinuclear antibodies were detected by immunofluorescence in most sera from rabbits immunized with whole human serum emulsified in Freund's complete adjuvant. Four of 14 sera from rabbits immunized with Cohn fractions III, IV-1, or IV-4 also gave positive nuclear fluorescence with human leukocyte nuclei. Other human and animal nuclei gave negative results with these rabbit antisera. Three rabbit anti-whole human sera had complement-fixing antibody against DNA in sufficient titer for study in quantitative complement fixation tests. Antibody with greatest reactivity with human single strand DNA was detected in the 3 rabbit antisera. Reactivity with rabbit, calf thymus, and B. natto DNA was also detected. In each case reactivity was greater with single strand than with native DNA. Antibodies against human histone and purine-6-oly BSA were also detected. No correlation was found between the titers of rheumatoidlike factors and antinuclear antibodies present in the rabbit antisera. The induction of antinuclear antibodies in these rabbits was not associated with disease attributable to the antibodies. The induction of antinuclear antibody by immunization with whole human serum was interpreted as indicating the presence of antigenic nuclear material in whole human serum.     

Elevated Levels of Antibodies to Epstein-Barr Virus Antigens in Sera and Synovial Fluids of Patients with Rheumatoid Arthritis

The frequencies and levels of antibodies to Epstein-Barr virus (EBV)-specific antigens were determined in paired sera and synovial fluids from patients with rheumatoid arthritis (RA) and in sera from patients with other connective tissue diseases; i.e., systemic lupus erythematosus, progressive systemic sclerosis, and osteoarthritis (OA). The specimens were also tested for the presence of antibodies to RA-associated nuclear antigen. Compared to healthy controls, the patients' sera showed increased frequencies of elevated antibody titers (≥320) to Epstein-Barr viral capsid antigen, a correspondingly enhanced (twofold to threefold) geometric mean titer, and an increased frequency of antibodies at elevated titers (≥10), usually to the restricted component and rarely the diffuse component of the early antigen complex. Levels of antibody to the EBV-associated nuclear antigen were within the normal range. Enhancement of antibody titers was more pronounced in seropositive RA patients (i.e., positive for rheumatoid factor) than in those who were not. Enhancement was also found in systemic lupus erythematosus and progressive systemic sclerosis. Antibody to RA-associated nuclear antigen was detected at an increased frequency only in the group of seropositive RA patients (90%), as compared to 8-15% in the other connective tissue diseases and 6-8% in healthy controls. The antibody titers in the synovial fluids equaled or were at most twofold higher or lower than those in the sera. In addition, levels of EBV-specific antibodies were studied serially over a period of 6-10 mo in patients with RA and OA. Parameters of disease activity were determined and compared to antibody levels. EBV-specific antibodies in sera of OA patients remained constant and within normal limits throughout the study. Although EBV-specific antibodies were often elevated in RA patients, they also remained constant, with the exception of three patients, who showed gradual increases in one of the four antibodies, which did not correlate with disease activity.     

Anti-Scl-70 antibodies in autoimmune hypothyroidism

The relationship between autoimmune thyroiditis and systemic sclerosis is controversial. Data exist on the presence of thyroid autoantibodies in patients with systemic sclerosis but, as far as we could ascertain, anti-Scl-70 antibodies, which are highly specific for systemic sclerosis, have not been investigated in autoimmune hypothyroidism. This study compares the presence of anti-Scl-70 in females with autoimmune hypothyroidism (n = 24) and in healthy age-matched female controls (n = 26). Free thyroxine levels were similar in both groups. Thyroid stimulating hormone (TSH), antithyroid peroxidase (anti-TPO), antithyroglobulin (anti-Tg) and index values for anti-Scl-70 levels were significantly higher in patients with autoimmune hypothyroidism compared with controls, although the anti-Scl-70 test was negative in both groups. Anti-TPO, anti-Tg and TSH significantly correlated with anti-Scl-70. In conclusion, autoimmune hypothyroidism seems to be associated with a higher index level of anti-Scl-70, yet a negative anti-Scl-70 antibody test. This suggests that autoimmune hypothyroidism might have common aetiological factors with systemic sclerosis.     

The non immunogenicity of CALLA in leukemic children

The sera from 55 children with acute lymphoblastic leukemia (ALL) treated with active immunotherapy were examined for the presence of antibodies against common ALL antigen (CALLA). A solid phase indirect radioimmunometric assay (IRA) was developed which enabled the detection of anti-CALLA antibody in patients' sera, utilizing the ability of affinity-purified CALLA to bind Ricinus communis agglutinin and anti-CALLA antibody simultaneously. Using IRA, anti-CALLA antibody activity could not be detected in a majority of patients. We concluded that the patients did not raise comparable antibodies against CALLA, indicating this antigen is not immunogenic for ALL patients.     

Evaluation of adsorption selectivity dextran sulfate bound cellulose beads for the removal of anti-DNA antibodies.

The purpose of this study was to clarify the basic adsorption selectivity characteristics of dextran sulfate (DS) columns (Selesorb, Kaneka Corporation, Osaka, Japan). Recovery rates of blood chemical components, hormones, coagulation factors, and antinuclear antibodies (anti-SS-A, SS-B, Sm, Scl-70, and RNP antibody) in vitro were assessed by mixing normal volunteers' or patients' sera with DS bound cellulose beads. For tested blood chemical components other than triglyceride and total cholesterol, the recovery rate was not changed significantly by incubation. No significant changes in hormone levels resulted from incubation. Among coagulation factors, the activities of antithrombin III, plasminogen, and factors V, VIII, IX, XI, and XII were significantly reduced by incubation. Among antinuclear antibodies tested, anti-SS-A and anti-RNP were absorbed to some extent, but not anti-SS-B, Sm, or Scl-70 antibodies. Taking into account these characteristics, apheresis therapy using a DS column should be performed.     

Characterization of a distinct nuclear acidic protein antigen (MA) and clinical findings in systemic lupus erythematosus patients with MA antibodies.

Circulating antibodies against certain nuclear acidic protein antigens have been shown to have diagnostic and prognostic importance in connective tissue disease. We describe a new precipitin system found in the sera of patients with systemic lupus erythematosus. The antigen, called MA, was prepared from calf thymus nuclei, and was shown to be distinct from other nuclear acidic protein antigens by physicochemical and immunologic techniques. MA antibodies were detected in the serum of 12 of 66 lupus patients and in none of 554 sera from normal controls or patients with other rheumatic diseases. Lupus patients having MA antibodies had more severe disease than did lupus patients with Sm or native DNA antibodies, manifested by recalcitrant skin rashes and a significantly greater incidence of hypocomplementemia, serious renal disease, hypertension, hepatosplenomegaly, lymphadenopathy, and neurological disease (P values range from 0.025 to 0.005). The presence of circulating MA antigen was demonstrated in three lupus patients immediately before a flare of nephritis. These data suggest that MA is a nuclear acidic protein antigen that may identify a subset of lupus patients with very severe disease. The presence of the antigen in the circulation before clinical flares suggests a possible biologic role for the MA system in an immune complex nephritis.     

Microplate ELISA for detection of antibodies to DNA in patients with systemic lupus erythematosus: specificity and correlation with Farr radioimmunoassay

A 96-well microplate ELISA for the detection of antibodies to DNA is described. A number of buffers and precoating treatments were used to evaluate the optimal method for coating the plate with DNA. These included pretreatment of the plates with poly-L-lysine or protamine sulfate, and posttreatment with glutaraldehyde, none of which improved the performance of the assay. Whereas bicarbonate and borate coating buffers gave equivalent and satisfactory results, TRIS buffer resulted in very high binding of immunoglobulin to wells not coated with antigen. Sera from groups of patients with autoimmune disease as well as normal sera were tested against plates optimally coated with native E. coli DNA, calf thymus DNA, and heat-denatured DNA. Using native E. coli DNA, virtually none of 35 normal sera had any detectable antibody. With this antigen, as well as with native calf thymus DNA, significant levels of DNA antibody were found only in SLE patients. Most patients with SLE or drug-induced lupus, as well as some patients with rheumatoid arthritis and normal individuals had antibodies that bound to heat-denatured (single-stranded) DNA. Using either native E. coli or calf thymus DNA, a good correlation was found between the amount of DNA antibody detected by ELISA and the Farr-type radioimmunoassay.     

Evaluation of the antigen specific to the mycelial phase of Candida albicans in the serodiagnosis of candidiasis.

We demonstrated 2 different antibodies against Candida albicans in patients' sera, the detection rates were proportional to the severity of candidiasis. One antibody was directed toward antigen shared by Candida in both blastospore and mycelial phases and the other was directed against antigen found only in the mycelial phase of Candida. The presence of the latter may reflect the invasive form of candidiasis.     

IMMUNOLOGICAL STUDIES IN ULCERATIVE COLITIS : IV. ORIGIN OF AUTOANTIBODIES

The incidence and height of antibody titers to colon, assayed by indirect hemagglutination with a heat stable colon extract from germ free rats, is significantly higher in sera from patients with ulcerative colitis than in those from healthy controls or from patients with amebic liver abscess or dysentery. While sera from ulcerative colitis patients and controls are indistinguishable in regard to incidence and height of antibody titers to Forsman antigen, Staphylococcus aureus S 209, Clostridium difficile, and several common strains of E. coli, they have elevated titers and increased incidence of antibodies to a heat stable antigen of E. coli O14. Patients with amebic dysentery have normal titers of such antibodies. Absorption of patients' sera with E. coli O14 antigen inhibits the colon directed hemagglutination reaction in approximately 30% of the cases tested. Likewise, the anti-E. coli O14 reaction can sometimes be inhibited with the colon extract. Other E. coli strains and other bacteria are inactive or have only weak inhibitory activity. Hemagglutination inhibition experiments show that germ free rat colon and E. coli O14 contain common structures, depicted by antibodies in the patients' sera. This pattern of reactivity closely resembles that seen in rats made autoimmune to colon by injection of newborn rabbit colon. E. coli O14 is known to carry a heterogenetic antigen present in lower concentration (or activity) in most Enterobacteriaceae. Hemagglutination inhibition experiments with rabbit antisera to E. coli O14 suggest that the antigen common for E. coli O14 and colon is related to this heterogenetic antigen. The findings imply that this antigen, which is constantly present in low concentrations in the human colon, may give rise to anticolon antibody formation in ulcerative colitis through breakage of tolerance. Since this antigen is present in healthy individuals as well, additional factors are required to explain the induction of anti-colon autoimmunity in ulcerative colitis.     

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.     

抗核抗体谱在自身免疫性疾病中的临床应用

目的 探讨抗核抗体谱在自身免疫病中的应用价值.方法 ANA 检测采用间接免疫荧光法,ENA多肽抗体谱用免疫印迹法.结果 729例患者中,有58例自身免疫病患者,其中SLE 31例,混合性结缔组织病12例,干燥综合征13例,硬皮病和皮肌炎各1例.所有自身免疫病患者的ANA均为阳性,但SLE患者的ENA抗体谱中可显示抗nRNP、抗Sm、抗SSA、抗SSB、抗dsDNA等抗体阳性;而干燥综合征患者仅抗SSA或抗SSB抗体阳性;皮肌炎患者仅显示抗Jo-1抗体阳性;硬皮病患者仪显示抗Scl-70抗体阳性.结论 ENA抗体谱对种自身免疫病的诊断具有重要意义.     

Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy

Using a hemagglutination test which can detect antibodies to (a) native and denatured deoxyribonucleic acid (DNA) and (b) an extractable nuclear antigen (ENA), a comparative study of patterns of autoantibody formation has been done in systemic lupus erythematosus (SLE) and related rheumatic diseases. Antibody to native DNA was present in the serum in 96% of patients with active SLE and disappeared during remissions. Antibody to ENA was found in 86% of those patients with SLE nephritis who responded to treatment but in only 8% of those who did not. The highest titers of antibody to ENA were found in patients having a mixed connective tissue disease syndrome with features of SLE, scleroderma, and myositis. The latter syndrome was notable for the absence of renal disease and for a striking responsiveness to corticosteroid therapy. Hemagglutination testing of 277 sera from normal persons and patients with a wide variety of acute diseases other than SLE revealed the presence of antibody to native DNA in only 1.4% and antibody to ENA in only 0.4%.These results yield significant correlations among the pattern of autoimmune reactivity, the clinical form of the rheumatic disease, and responsiveness to treatment. They implicate the qualitative nature of the patient's immune response as a conditioning factor in the type of disease. Together with other correlations they may allow classification of rheumatic diseases into more biologically meaningful groups and lead to more selective methods of therapy.     

Evaluation of a line immunoblot assay for detection of antibodies recognizing extractable nuclear antigens

Sera (n = 90) giving positive results in a screening test for antibodies to extractable nuclear antigens (ENAs) were tested in a line immunoblot assay that measures antibody reactivity with individual ENAs in a single test field. Results were then compared to those obtained in monospecific ENA antibody enzyme immunoassays (EIAs). Discordant results were resolved by immunodiffusion. Of 540 result pairs (90 sera tested for 6 ENAs [Sm/RNP, Sm, SSA, SSB, Scl-70, Jo-1]), 509 (94%) showed concordance. Immunodiffusion resolved 28 of 31 discordant result pairs in favor of the immunoblot result. After resolution of discordant data, the immunoblot assay exhibited 100% sensitivity for all ENA antibodies except those recognizing Scl-70, for which the sensitivity was 89%; specificity was over 96% for all 6 ENA antibodies. These findings show that a line immunoblot assay for the characterization of ENA antibodies yields results comparable to those obtained using monospecific ENA antibody EIAs. The immunoblot assay is easier and less expensive to perform due to its utilization of a single test field. J. Clin. Lab. Anal. 12:320–324, 1998. © 1998 Wiley-Liss, Inc.     

Chemically humanized murine monoclonal antibody against a cell nuclear antigen: usefulness in autoimmune diagnostics.

The cellular nuclear antigen SS-B/La is known to be a major antigenic target to an autoantibody in patients with Sjogren's syndrome and systemic lupus erythematosus. It is useful to detect an anti-SS-B/La antibody from patients' sera in a clinical point of view. We purified SS-B/La from rabbit thymus acetone powder by affinity chromatography with a murine anti-SS-B/La monoclonal antibody (1C3-H7). An enzyme-linked immunosorbent assay method, in which SS-B/La was used to coat a plate, was also successfully established. It is difficult to obtain a large volume of patient's serum with high antibody titer and high specificity as a positive control. We investigated whether or not a positive control from human could be replaced by a murine monoclonal antibody to SS-B/La. The 1C3-H7 was conjugated with a human IgG Fc' fragment using N-gamma-maleimidobutyryloxysuccinimide as a cross-linker. The chemically humanized murine monoclonal antibody (1C3-Fc') was recognized by antiserum specific for human IgG Fc fragment. 1C3-Fc' reacted to SS-B/La but not to other antigens. Furthermore, the titration curve of this conjugate ran parallel with those of patients' sera specific for SS-B/La. It is concluded that a chemically humanized murine monoclonal antibody is useful as a positive control in place of a human patient's serum.