Studies on heterophile antibodies in rheumatoid arthritis

Sera and synovial fluids of patients with rheumatoid arthritis were studied for the presence of heterophile antibodies to sheep and bovine erythrocytes by means of hemolysis in agar gel. It was demonstrated that 18 of 146 sera had hemolytic antibody titers of 160 or more; all 18 (12%) against sheep and 8 (6%) against bovine erythrocytes. Of 31 synovial fluids examined, 5 showed hemolysin titers of 40 or more; all 5 (16%) against sheep and 3 (10%) against bovine erythrocytes. These heterophile antibodies were shown to belong to IgM and/or IgG class. Absorption and inhibition studies revealed that antibodies of 10 positive sera and 2 synovial fluids were of Forssman specificity and antibodies of 6 sera and 3 synovial fluids were of Hanganutziu-Deicher specificity. Two remaining sera were shown to contain a mixture of Forssman antibodies and immune anti-B antibodies.     

Hidden and classical 19S IgM rheumatoid factor in a juvenile rheumatoid arthritis patient

Hidden 19S IgM rheumatoid factors (RF), i.e., 19S IgM RF which can be detected in the IgM containing fraction after acid gel filtration of serum, are found in 59-68% of patients with juvenile rheumatoid arthritis (JRA). Their presence generally correlates with disease activity. We describe a 12 year-old female with a polyarticular onset of JRA who, during her first 15 months of disease, was seronegative but had hidden RF titers of 1:128----1:256. Inhibition studies on her hidden RF showed specificity for HIgG greater than RIgG and equal specificity for the human IgG subclasses (IgG1 = IgG3). In the second and third year of disease, she became seropositive with RF titers varying from 1:40 to 1:320 while her hemolytic titers on her IgM fractions were decreased from 1:128 to 1:32. Inhibition studies now demonstrated a higher avidity for RIgG; and HIgG3 inhibited more than HIgG1. These studies documented for the first time a JRA patient who early in the disease was negative for RF and positive for hidden RF, and who later became seropositive.     

Antibodies to histones H1 and H5 in sera of patients with juvenile rheumatoid arthritis

The specificity of juvenile rheumatoid arthritis (JRA) sera for histone subclasses was examined by immunoblotting. Antibodies to H1 alone were found in 4 of 21 pauciarticular-onset JRA sera, 4 of 19 polyarticular-onset JRA sera, and 2 of 11 systemic-onset JRA sera. Antibodies to H5 alone were found in 1 of 21 pauciarticular JRA sera, 1 of 19 polyarticular JRA sera, and 3 of 11 systemic JRA sera. Antibodies to both H1 and H5 were found in 4 of 21 pauciarticular JRA sera, 4 of 19 polyarticular JRA sera, and 1 of 11 systemic JRA sera. Antibodies to the core histones (H2A and H2B) were found in 1 of 21 pauciarticular JRA sera, 1 of 19 polyarticular JRA sera, and no systemic JRA sera. No reactivity to histones was observed in 30 sera from age-matched children with nonrheumatic diseases. The presence of H1 and H5 antibodies did not correlate with antinuclear antibody titers or with a homogeneous pattern of immunofluorescence. The predominance of H1 and H5 antibodies and relative absence of antibodies binding to core histones in JRA contrast with findings in adult systemic lupus erythematosus. Further, the presence of antibodies to H5 alone in some of the JRA patients indicates that the immune response in these patients is directed to determinants that are not shared by sequences of mammalian proteins.     

Complement-fixing hidden rheumatoid factor in juvenile rheumatoid arthritis

Fifteen to twenty percent of patients with juvenile rheumatoid arthritis (JRA) have positive latex fixation tests (LFT), whereas approximately 46% have previously been demonstrated to have hidden rheumatoid factors (RF), i.e., 19S IgM RF which can be detected by the LFT after acid separation of the IgM-containing fraction from serum. In this study, hidden RF were found in 59% of patients with seronegative JRA by use of a complement-dependent hemolytic assay. The median titer of JRA patients was 1:42, and in healthy and disease controls it was 1:7. The difference was significant at P < 0.001. When data from patients with active disease were analyzed separately, the median titer for polyarticular JRA was 1:97 and for pauciarticular JRA, 1:91. The differences due to active disease were significant at P < 0.001 and P < 0.005, respectively. The results demonstrate that the hemolytic assay is more sensitive than the LFT in determining the presence of hidden RF, and activity of disease correlates well with high hemolytic RF titers.     

Correlation of antiperinuclear factor with antibodies to streptococcal cell-wall peptidoglycan-polysaccharide polymers and rheumatoid factor.

Antiperinuclear factor (APF) has been noted in most seropositive rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) patients. The nature of the antigen is unknown; however, there are some suggestions that it might be a glycoprotein or proteoglycan. We studied the correlation of APF with antiproteoglycan antibodies and the reactivity of IgM-rheumatoid factor (RF) with the perinuclear antigen. Ten serum samples were separated to IgG, IgM, and IgM-RF enriched fractions. In seven samples, APF was found in the IgG fraction. Only 4 had APF in their IgM rheumatoid factor (RF)-containing fraction. In two of these, APF activity was present solely in the IgM RF fraction and was inhibited by pre-incubation with IgG. Fifty-five JRA patients' sera were also tested for the presence of antibodies to Streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-PSP). 76% of the APF-positive sera were anti-PG-PSP positive and 59% of the APF-negative sera were also anti-PG-PSP negative. Furthermore, 75% of the APF-positive sera lost their APF activity following adsorption to Streptococcal cell wall PG-PSP. Our results show that in JRA sera APF are polyclonal antibodies of both the IgG and IgM classes. Although the presence of APF correlates with RF positivity, and they sometimes may cross-react, many IgM RF-containing fractions do not show APF activity. However, the presence of APF does correlate with anti-PG-PSP positivity and the data suggest cross-reactivity between these two antibodies. This implies antigenic similarity between Streptococcal cell wall PG-PSP and the perinuclear antigen.     

Cross-reactive antiidiotypic antibodies against human rheumatoid factors from patients with juvenile rheumatoid arthritis

We prepared antiidiotypic (anti-Id) antibody to 2 polyclonal IgM rheumatoid factors (IgM-RF) and 2 polyclonal "hidden" IgM-RF. The anti-Id antibodies were isolated by chromatography on Sepharose 4B, to which was bound rabbit anti-human IgG Fc fragments. F(ab')2 fragments from the anti-Id antibodies were generated by pepsin digestion and isolated by gel filtration. The anti-Id antibodies directed against RF from 4 patients with juvenile rheumatoid arthritis (JRA) were tested by an inhibition hemolytic assay for cross-reactivity with IgM-RF from 4 adult patients with rheumatoid arthritis, 6 patients with JRA, and 13 JRA patients with hidden RF. The 4 anti-Id antibodies had variable cross-reactivity with the isolated adult RA RF, JRA RF, and JRA hidden RF. Similar results were obtained by a direct-binding enzyme-linked immunosorbent assay for the anti-Id antibodies. The broad pattern of cross-reactivity was apparently unrelated to a particular amino acid sequence, but was associated with the antigen-binding site of IgM-RF. These results suggest the possibility that the anti-Id antibodies prepared against isolated RF obtained from JRA patients bear the "internal image" of antigen; that is, the Fc region of human IgG. These anti-Id antibodies may be generated in JRA patients and may possess specific immunomodulatory properties.     

Antibody to streptococcal cell wall peptidoglycan-polysaccharide polymers in sera of patients with juvenile rheumatoid arthritis but absent in isolated immune complexes

Sera of 88 children with juvenile rheumatoid arthritis (JRA) (10 seropositive, polyarticular onset, 29 seronegative, polyarticular onset, 32 pauciarticular onset, and 17 systemic onset) were evaluated for the presence of serum antibodies to streptococcal cell wall peptidoglycan-polysaccharide polymers (PG-PSP). Immune complexes (IC) isolated by the antihuman IgM (HIgM) affinity column method were also evaluated for the presence of antibodies to PG-PSP. Forty-one of 88 patients with JRA (7 of 10 seropositive, polyarticular onset, 11 of 29 seronegative, polyarticular onset, 16 of 32 pauciarticular onset, and 7 of 17 systemic onset) showed elevated levels of antibodies to PG-PSP in their sera. IgM rheumatoid factors (RF) were demonstrated in 70/88 isolated IC fractions of patients with JRA and IgG RF in 7; however, none of the patients demonstrated the presence of antibodies to PG-PSP in their isolated IC fractions from the anti-HIgM affinity column. These data indicate that antibodies are produced to PG-PSP in all JRA onset types, but they are not constituents of isolated IC by the anti-HIgM affinity column method.     

Enzyme linked (ELISA) immunoabsorbent assay for the detection of hidden 19S IgM rheumatoid factors in juvenile rheumatoid arthritis

Determination of hidden IgM rheumatoid factors (RF) in juvenile rheumatoid arthritis (JRA) offers advantages for diagnosis and in following disease activity. Sera from 30 patients with JRA were assayed for RF by latex fixation test (LFT), sensitized sheep cell agglutination test (SCAT), nephelometry, and by ELISA. IgM containing fractions were prepared by chromatography and assayed for hidden RF by the hemolytic method and ELISA. Ten patients were seropositive by LFT. All of them gave positive tests on the serum for RF with the SCAT, nephelometry, and ELISA, and on the IgM containing fractions by the hemolytic assay and ELISA. Seventeen patients seronegative by LFT were positive for hidden RF by the hemolytic test and ELISA on the IgM containing fraction. When unfractionated serum was used, 15 were positive by ELISA. Three patients were seronegative and also negative for hidden RF by the hemolytic assay and ELISA. Thus, only 2 of 30 patients had discordant results between the hemolytic assay on the IgM containing fraction and the ELISA on the serum. Our results indicate the ELISA on the serum in conjunction with the LFT offers a simple, rapid, alternative test for hidden 19S IgM RF in JRA patients.     

Autoantibodies to nonhistone chromosomal proteins HMG-1 and HMG-2 in sera of patients with juvenile rheumatoid arthritis

IgG antibodies against the high mobility group (HMG) nonhistone chromosomal proteins HMG-1 and/or HMG-2 were detected in the sera of 49 (39%) of 126 antinuclear antibody (ANA)-positive patients with juvenile rheumatoid arthritis (JRA), by immunoblotting. Clinical diagnosis classified these patients in 2 major groups, 105 with pauciarticular-onset JRA and 21 with polyarticular-onset JRA. Anti-HMG-1 and/or anti-HMG-2 antibodies were found in 8 (25%) of 32 pauciarticular-onset JRA patients with uveitis and in 34 (47%) of 73 patients without uveitis, whereas anti-HMG-1 and/or anti-HMG-2 antibodies were found in 4 (24%) of 17 children with polyarticular-onset JRA without uveitis. Among 53 sera from ANA-negative JRA patients, 3 (6%) were positive for anti-HMG-1 and/or anti-HMG-2 antibodies, whereas no reactivity to HMG-1 or HMG-2 proteins was observed in 48 sera from age-matched children with nonrheumatic diseases.     

Cytotoxic anti-T cell antibodies in children with juvenile rheumatoid arthritis

The object of this investigation was to determine the prevalence of anti-T cell antibodies in 66 children with various connective tissue diseases. Anti-T cell antibodies were found in 43/44 juvenile rheumatoid arthritis (JRA) patients (mean cytotoxicity 15.0%) and in 10/10 children with systemic lupus erythematosus (mean cytotoxicity 20.0%), but in only 1/15 normal controls and in none of 12 children with other arthritides. There was no significant difference in mean percent cytotoxicity among the JRA subclasses. In the JRA patients, the percent cytotoxicity was positively correlated with the erythrocyte sedimentation rate (P = 0.01), but not with the presence or absence of rheumatoid factor, antinuclear antibodies, or immune complexes. The sera of 3 JRA patients repeatedly inhibited the stimulation of normal lymphocytes by mitogens and antigens by 47-99% (measured by the incorporation of 3H-thymidine into DNA) when added to the culture system in the first 24 hours; normal sera did not. Sera from patients with JRA have increased reactivity with mitogen-activated lymphocytes and T cells compared with unstimulated cells as determined by flow cytometry. The expression of the "JRA antigen" requires protein synthesis but not DNA synthesis or cell division. We conclude that the majority of patients with active JRA have cytotoxic anti-T cell antibodies and that in selected patients, these antibodies may play a role in regulation of the immune response.     

A prospective evaluation of heterophile and Epstein-Barr virus-specific IgM antibody tests in clinical and subclinical infectious mononucleosis: Specificity and sensitivity of the tests and persistence of antibody.

Three heterophile antibody tests and a test specific for IgM antibody to Epstein-Barr virus were evaluated during prospective studies of infectious mononucleosis. Specificity was judged by the frequency of false-positive results in sera of known qualities taken before illness; except for two patients bled during early, unrecognized illnes,, titers of greater than or equal to 1:40 were detected in 12% by the absorbed sheep red cell test, in 6.7% by the absorbed horse red cell test, and in none by the beef cell hemolysin test. None had IgM antibody specific for Epstein-Barr virus in sera obtained before illness. In addition, no rises in titer of heterophile antibody were detected by the horse cell test in 38 patients with proved rubella and/or influenza infection. In terms of sensitivity (indicated by the percentage of cases with diagnostic titers during infectious mononucleosis), 97% were positive by the Epstein-Barr virus IgM test, 96% by the horse cell agglutination test, 85% by the beef hemolysin test, and 81% by the sheep cell agglutination test. Persistence of antibody was judged by serial bleedings up to three years after illness; titers of heterophile antibody by the sheep agglutination and beef hemolysin tests as well as titers of IgM antibody to Epstein-Barr virus returned to normal in two to three months, whereas the horse cell heterophile test remained positive for a year or more in 75%. Inapparent and mild infections with Epstein-Barr virus resulted in the production of horse cell heterophile antibody in 48.4% of 122 subjects.     

High diagnostic value of anticalpastatin autoantibodies in rheumatoid arthritis detected by ELISA using human erythrocyte calpastatin as antigen

OBJECTIVE: To develop a quantitative method of measuring autoantibodies against human calpastatin in rheumatoid arthritis (RA) and to determine their diagnostic value compared with other autoimmune and articular diseases. METHODS: We performed a highly sensitive ELISA for IgG and IgM anticalpastatin autoantibodies in human sera using human erythrocyte calpastatin as an antigen. Samples were diluted 1:2000 for the measurement of IgG and 1:400 for IgM. RESULTS: IgG anticalpastatin antibodies were found in the sera of 48 of 58 patients (82.8%) with RA. In contrast, IgG anticalpastatin antibodies were found in the sera of only 2 of 11 (8.3%) patients with osteoarthritis (OA). Compared to sera from patients with other autoimmune diseases, anticalpastatin antibody sensitivity for RA was better than that of systemic lupus erythematosus (5.6%), systemic sclerosis (0%), mixed connective tissue disease (0%), and Sj?gren's syndrome (20%). IgG anticalpastatin antibodies also showed high specificity (96.1%) for RA. Almost 90% of patients with RA were positive for IgG or IgM anticalpastatin antibodies. CONCLUSION: We have developed a simple, sensitive, specific, and quantitative ELISA for anticalpastatin antibodies that may have a high diagnostic value for RA.     

Antiperinuclear factor in juvenile rheumatoid arthritis.

The serological diagnosis of juvenile rheumatoid arthritis (JRA) is difficult, with only 7-10% of patients 19S IgM rheumatoid factor positive. About 60-70% of patients are positive for hidden 19S IgM rheumatoid factor, but this test requires serum separation and is not available in most laboratories. Antiperinuclear factor has been described in both seropositive and seronegative adult patients with rheumatoid arthritis, but has not been thoroughly evaluated in children with JRA. This study determined the diagnostic sensitivity and specificity of antiperinuclear factor in patients with JRA. Serum samples from 64 children with JRA, 24 with systemic lupus erythematosus (SLE), and 24 control subjects were tested for the presence of antiperinuclear factor. A total of 10 (83%) of seropositive, polyarticular onset and six (37%) of seronegative, polyarticular onset patients with JRA were positive for antiperinuclear factor. The occurrence of antiperinuclear factor in five (19%) with pauciarticular onset and one (10%) with systemic onset (JRA) as well as in four (17%) with SLE was not increased compared with the control subjects (1/24 (4%)). These data show an overall diagnostic sensitivity and specificity of 34 and 90% respectively in this group of patients. Although less sensitive than the hidden rheumatoid factor assay, the antiperinuclear factor assay is easier to perform and may contribute to the serological diagnosis of JRA.     

Assessment of antibodies to double-stranded DNA induced in rheumatoid arthritis patients following treatment with infliximab, a monoclonal antibody to tumor necrosis factor alpha: findings in open-label and randomized placebo-controlled trials

OBJECTIVE: To compare the incidence of anti-double-stranded DNA (anti-dsDNA) antibodies in rheumatoid arthritis (RA) patients receiving either single or multiple doses of a chimeric anti-tumor necrosis factor alpha (anti-TNFalpha) antibody or placebo infusions, with or without methotrexate, in open-label, randomized, placebo-controlled trials. METHODS: Multiple sera obtained from 156 patients before and after treatment with infliximab and from 37 patients treated with placebo infusions were tested for anti-dsDNA antibodies by 3 methods: Crithidia luciliae indirect immunofluorescence test (CLIFT), a commercial Farr assay (Ortho Diagnostics radioimmunoassay [RIA]) in which the antigen source is mammalian DNA, and a Farr assay employing 125I-labeled circular plasmid DNA (Central Laboratory of The Netherlands Red Cross Blood Transfusion Service [CLB] RIA). Patients with positive findings on the CLIFT were also tested for antibodies to histones (H1-H5) and chromatin and for IgM rheumatoid factors (IgM-RFs). RESULTS: None of the RA patients had a serum sample that was positive for anti-dsDNA antibodies by the CLIFT prior to infliximab therapy. Of the 22 patients who developed a positive CLIFT result, 11 (7% of 156 exposed to infliximab) also had positive findings on the Ortho RIA at a concentration of >10 units/ml and another 8 (5%) were positive at a concentration of >25 units/ml. In all but 1 patient, the anti-dsDNA antibodies were solely of the IgM isotype. Only 1 patient had detectable anti-dsDNA antibodies by the CLB RIA. All sera containing anti-dsDNA by the CLIFT contained antibodies to chromatin, and sera from 2 patients also contained antibodies to histones. IgM-RF titers showed a significant reduction following infliximab therapy in these 22 patients. One patient developed anti-dsDNA antibodies of IgG, IgA, and IgM isotype and had positive results on both Farr assays (peaking at 22 weeks and resolving by 54 weeks); this was associated with a reversible lupus syndrome. CONCLUSION: Anti-dsDNA antibodies of IgM class are induced by infliximab therapy; the frequency is dependent on the assay method used. Only 1 of the 156 patients who were treated with infliximab developed a self-limiting clinical lupus syndrome; that patient developed high titers of anti-dsDNA antibodies of IgG, IgM, and IgA class, as detected by the CLIFT and by 2 different Farr assays.     

Enzyme immunoassays for the detection of IgG and IgM anti-dsDNA antibodies: clinical significance and specificity

A solid phase Enzyme-Linked Immunosorbent Assay (ELISA) was developed for the measurement of IgG and IgM antibodies to double-stranded DNA (anti-dsDNA). This method is sensitive, specific, relatively simple and suitable for routine use. Thus, we evaluated sera from 224 Greek patients with the following autoimmune rheumatic diseases: 54 patients with classical rheumatoid arthritis (RA), 50 patients with primary Sj?gren's syndrome (SS), 41 patients with systemic lupus erythematosus (SLE), 30 patients with scleroderma, 20 patients with idiopathic Raynaud's phenomenon (IRP) and 29 patients with juvenile rheumatoid arthritis (JRA). Sera from 119 age- and sex-matched healthy blood donors were tested as normal controls. The presence of both IgG and IgM anti-dsDNA highly correlated with SLE. However, IgM anti-dsDNA levels were significantly lower. Serum complement C3 and C4 levels correlated negatively with anti-dsDNA levels in the SLE group. Finally, in sequential sera from five SLE patients, the anti-dsDNA activity proved to be a relatively sensitive marker of SLE activity.     

Serum antiimmunoglobulins reactive with human and rabbit IgG in rheumatoid arthritis and other conditions

IgG, IgA, and IgM antiimmunoglobulins reactive with human and rabbit IgG were measured in patients with rheumatoid arthritis (RA), patients with rheumatic fever or osteoarthritis, and normal individuals. Values of all antiimmunoglobulins were significantly evaluated in RA patients as compared with other groups and depended upon activity and stage of the disease. IgM antibodies with specificity for human IgG predominated quantitatively over others in sera of RA patients with high titers of RF, whereas most of those reactive with rabbit IgG in latex negative or positive RA patients belonged to IgG class. The reaction with human IgG included thermostable and thermolabile IgM antiimmunoglobulins but in that with rabbit IgG only thermostable antibodies were active.     

IgM anti-dsDNA antibodies in systemic lupus erythematosus: negative association with nephritis

Antibodies against dsDNA of the IgM class were measured in sera of 352 patients with systemic lupus erythematosus, 81 blood donors and 189 patients with rheumatoid arthritis using a new ELISA based on human recombinant dsDNA as antigen. IgM anti-dsDNA antibodies were found in 52.3% of the sera from patients with systemic lupus erythematosus, but in none of the sera from 81 normal controls and 189 patients with rheumatoid arthritis. The association of these autoantibodies with 31 clinical and 37 laboratory parameters was calculated. There was a highly significant negative correlation between IgM anti-dsDNA antibodies and nephritis as well as all the laboratory parameters indicating renal disease (elevated serum creatinine concentration, proteinuria, erythrocyte casts in the urine). IgM anti-dsDNA antibodies indicate protection of lupus patients against the development of lupus nephritis. Further experiments will show whether application of IgM anti-dsDNA antibodies is effective in treating lupus nephritis. Received: 10 August 1998 / Accepted: 11 September 1998     

Autoantibodies to the chromosomal protein HMG-17 in juvenile rheumatoid arthritis

Objective. To determine the antibody profiles in sera from patients with juvenile rheumatoid arthritis (JRA). Methods. Immunoblotting using nuclear extracts and recombinant high-mobility group (HMG) nonhistone chromosomal proteins. Results. Antibodies directed against HMG-17 were found in 47% of antinuclear antibody (ANA)–positive patients with pauciarticular-onset JRA and in 16% of ANA-positive patients with polyarticular-onset JRA. HMG-17 values of 6% and 8%, respectively, were detected in ANA-negative patients with JRA and in those with nonrheumatic diseases. Conclusion. There is evidence for a high prevalence of anti—HMG-17 antibodies in sera of patients with pauciarticular-onset JRA.     

Serum IgM rheumatoid factor by enzyme-linked immunosorbent assay (ELISA) delineates a subset of patients with deforming joint disease in seronegative juvenile rheumatoid arthritis

Using human IgG as an antigen in an enzymelinked immunosorbent assay (ELISA), we looked for the presence of IgM rheumatoid factor (RF) in the sera of 74 children with juvenile rheumatoid arthritis (JRA). Nine children had RF detectable by both latex agglutination and ELISA. Forty-five percent (26 of 65) of the children who were seronegative by latex agglutination were found to be positive for IgM RF by ELISA. The prevalence of IgM RF was higher in patients with polyarticular onset disease (57.4%) than in those with pauciarticular onset (38.5%) or systemic onset (27.2%) disease. The prevalence of RF was higher in sera from patients with deforming joint disease than those without deformities (P<0.01).     

A solid-phase radioimmunoassay for detection of IgM antibodies to hepatitis A virus.

The conditions for a sensitive and specific solid-phase radioimmunoassay (RIA) for the detection of IgM antibodies to hepatitis A virus (HAV) were optimized, and the RIA was used to assay sera from patients with hepatitis. IgM antibodies to HAV reached highest concentrations between one and three weeks after onset of icterus and were measurable in follow-up sera for at least 12 months after infection. To prove the specificity, the IgG antibodies were separated from patient sera by sucrose density-gradient centrifugation. The remaining IgM antibodies, after treatment with beta-mercaptoethanol, did not bind in the RIA, and, when the anti-IgM antibody bound to the solid phase was replaced with anti-IgG, a negative result was obtained with incubation of IgM antibody to HAV. Also, the presence of IgG was shown not to interfere with measurement of IgM antibody to HAV. Finally, as a further specificity control, 50 sera positive for rheumatoid factor or from patients infected with hepatitis B virus, cytomegalic inclusion disease, infectious mononucleosis, influenza A virus, rubella, or measles were tested, and all of these sera were negative for IgM antibody to HAV.