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Objective: Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone., Methods: Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED., Results: Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (r s ) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide., Conclusion: Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Objective: Many patients with systemic lupus erythematosus (SLE) and fibromyalgia (FM) may spend less time exposed to the sun than healthy individuals and thus might have low vitamin D levels. It is known that hydroxychloroquine (HCQ) inhibits conversion of 25(OH)- to 1,25(OH)2-vitamin D both in vitro and in patients with sarcoidosis. We assessed winter serum 25(OH)- and 1,25(OH)2-vitamin D levels in patients with SLE and FM., Methods: We recruited 25 consecutive female SLE and 25 female FM patients in London, Ontario, between January and March 2000. Subjects completed a brief questionnaire. Serum levels of 25(OH)-, 1,25(OH)2-vitamin D, and parathyroid hormone (PTH) were measured., Results: In SLE patients mean 25(OH)-vitamin D was 46.5 nmol/l and mean 1,25(OH)2-vitamin D was 74.4 pmol/l. In FM patients these means were 51.5 nmol/l and 90.1 pmol/l, respectively. Serum 25(OH)-vitamin D levels did not significantly differ between SLE and FM patients, nor after adjusting for age and vitamin D, milk consumption, and sun block use. In 14 of the SLE patients and 12 of the FM patients 25(OH)-vitamin D levels < 50 nmol/l were found. SLE patients not using vitamin D supplements had lower 25(OH)-vitamin D levels than those who did. 1,25(OH)2-vitamin D tended to be lower in the SLE compared to the FM patients. This difference could be attributed to HCQ use: HCQ users (n = 17) had lower 1,25(OH)2-vitamin D levels than nonusers (n = 33); the mean adjusted difference was 24.4 pmol/l (95% CI 2.8-49.9)., Conclusion: Half the SLE and FM patients had 25(OH)-vitamin D levels < 50 nmol/l, a level at which PTH stimulation occurs. Our data suggest that in SLE patients HCQ might inhibit conversion of 25(OH)-vitamin D to 1,25(OH)2-vitamin D.

A collaborative study was performed to compare the expression of a series of idiotypes defined on human anti-DNA and other autoantibodies. Three panels of human monoclonal antibodies were tested: eight derived from patients with systemic lupus erythematosus (SLE); 13 from an individual with lepromatous leprosy; and 38 from normal subjects. The following rabbit anti-idiotype sera were used: one (RId16/6) raised against the lupus-derived monoclonal anti-DNA antibody 16/6, four (RId8E7, RId4G7, RId4D5 and RIdTH9) against leprosy-derived monoclonal antibodies of various specificities, and one (anti-4.6.3) against a normal-derived anti-DNA monoclonal (KIM 4.6). In addition, two other anti-idiotypes were used--one a murine monoclonal (3I), the other a rabbit polyclonal (RIdD)--which had been raised against polyclonal anti-DNA antibodies from lupus serum. Further experiments were performed with immunoabsorbed fractions of RId8E7. Direct-binding and competition assays were used. All of the anti-idiotypes produced different patterns of positivity among the three panels of human monoclonal antibodies, with the exception of RId8E7 and RId4G7, which showed considerable concordance. There was a tendency towards anti-idiotypes being disease- or group-specific: thus anti-4.6.3 failed to bind to any of the lupus or leprosy-derived monoclonals, while RId16/6 and RId8E7 bound most strongly to the lupus- and leprosy-derived antibodies respectively. KIM 4.6 itself was bound only weakly by RId16/6, while 16/6 was not recognized by anti-4.6.3; 16/6 was, however, bound by 3I, while KIM 4.6 was not. 3I bound to several other monoclonals but RIdD, which has been shown to be specific for the anti-DNA fraction of lupus serum, did not bind to any of them. These results indicate that the majority of these anti-idiotype preparations recognize largely separate sets of determinants. The monoclonal antibodies which bind to DNA may be only partly representative of anti-DNA antibodies in the serum of lupus patients.

The serum from 112 patients with systemic lupus erythematosus (SLE) was examined to compare serologic reactivity among anti-Ro positive and anti-Ro negative patients. While hypergammaglobulinemia, rheumatoid factor (RF) and elevated Clq binding were significantly more frequent among the anti-Ro positive group there was no increase in the frequency of anti-dsDNA antibody measured by the Farr assay or antibodies to ssDNA, dsDNA, poly dG . poly dC, poly (dA-dT) and cardiolipin measured by ELISA. Patients with the highest levels of anti-DNA antibody by the Farr assay did not have any increased frequency of anti-Ro antibodies. We concluded that anti-Ro and anti-DNA antibodies are independently regulated. The frequent occurrence of RF in anti-Ro positive SLE patients may provide a useful screening assay for this autoantibody among pregnant SLE patients.

A search for anti-Ro or anti-nRNP antibodies by precipitin analysis among a population of 64 patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease was undertaken. The 25% of SLE patients with anti-Ro did not have any distinct clinical features, compared to patients without anti-Ro or to anti-nRNP patients with SLE or mixed connective tissue disease. However, these patients did have a significantly greater frequency of rheumatoid factor (80%). Most importantly however, patients with anti-Ro had a significantly increased frequency of HLA-B8 (81%) and HLA-DRw3 (100%) compared to patients with anti-nRNP (B8--29%; DRw3--33%) or SLE patients without such antibodies (B8--41%; DRw3--25%). These data suggest the existence of a specific immune response gene for Ro close to the D region of the major histocompatibility complex.

Sixty-two Caucasian patients with systemic lupus erythematosus (SLE) were studied to determine whether any significant clinical correlations existed with various HLA antigens. While HLA-B8 and DR3 were significantly increased among the SLE population in general, these or other HLA antigens did not correlate with the severity of renal disease. HLA-B8 was significantly higher only in SLE males compared to male controls. The age of onset of disease in females was clustered into 2 groups, one with early age of onset (mean = 21.6 years, range 12-32 years) and another with later age of onset (mean = 48 years; range 38-60 years). SLE females with later age of onset showed an increased frequency of HLA-DR3 compared to sex and age matched controls, while those with early onset SLE showed no significant increase in any HLA antigens. Therefore any risk conferred by the presence of genes associated with HLA-B8 or DR3 is significant only in SLE males and SLE females whose disease onset was over the age of 35. Despite the greater frequency of HLA-B8, DR3 in SLE females with later onset of disease, this group did not have a greater frequency of anti-Ro antibodies compared to females with early age onset of SLE.

HLA profiles in 103 members of 4 separate kindred with multiple occurrence of SLE were compatible with the presence of a disease susceptibility factor linked to HLA or a direct effect of antigens B8, A11, and B35 on disease expression. Serum ANA was found in 52.9% of consanguineous, 56.5% of nonconsanguineous relatives of SLE probands, and 5% of controls, a finding that suggests the presence of a transmissible agent in the families. By contrast, lymphocytotoxic antibodies were not increased in relatives compared to controls.

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.

Rabbit antiserum raised against a normal-derived monoclonal anti-DNA antibody KIM 4.6.3 (IgM lambda) was used for idiotype analyses. This anti-serum (anti-4.6.3 ID) was rendered specific for KIM 4.6.3 idiotype (4.6.3 ID) by absorption with normal human IgM and IgG. The specificity of anti-4.6.3 was shown by its ability to bind to KIM 4.6.3 antibody but not to normal human IgM and IgG, by inhibition of anti-4.6.3 ID reactivity with KIM 4.6.3 antibody by the homologous monoclonal antibody and by the ability of anti-4.6.3 ID to inhibit the binding of single stranded DNA with KIM 4.6.3 antibody. The 4.6.3 ID was found to be commonly expressed since it was detected among 33% (10/30) DNA and 32% (23/72) non-DNA-reactive monoclonal antibodies that were obtained from five different unrelated normal individuals. The binding to ssDNA of the majority of idiotype positive anti-DNA antibodies however was not blocked by anti-4.6.3 ID suggesting that among these other monoclonal antibodies its expression is outside of the antigen binding site. The 4.6.3 ID, which was present among some normal-derived monoclonal IgM molecules was also found at a high frequency (90%) in the sera of patients with systemic lupus erythematosus (SLE) but only at a low frequency (24%) and concentration in normal sera. The level of 4.6.3 ID in SLE did not correlate with serum IgM and IgG nor with anti-DNA antibody concentrations. Idiotypic relatedness between SLE serum antibodies and monoclonal anti-DNA antibodies of normals implies the existence of a cross-reactive idiotype family and implies that a conserved common gene or closely related genes exist in the germ line encoding these 4.6.3 ID positive antibodies some of which are not exclusively associated with nucleic acid reactivity. The expression of these germ line genes in vivo thus distinguishes SLE from normals.

Human lysosomes were isolated from normal peripheral blood leukoyctes and characterized by electron microscopy, enzyme analysis, and assays for DNA and RNA. Stored sera from 37 unselected patients with systemic lupus erythematosus (SLE), including active and inactive, treated and untreated cases, were tested in complement fixation (CF) reactions with these lysosome preparations. 23 SLE sera exhibited positive CR reactions, as did sera from two patients with "lupoid" hepatitis. The seven SLE sera with strongest CF reactivity also demonstrated gel precipitin reactions with lysosomes. Neither CF nor precipitin reactions with lysosomes were observed with normal sera or with sera of patients with drug-induced lupus syndrome, rheumatoid arthritis (RA), polymyositis, or autoimmune hemolytic anemia. By several criteria the antilysosome CF and precipitin reactions of SLE sera cound not be attributed to antibody to DNA, RNA, or other intracellular organelles. The lysosomal component reactive with SLE sera in CF assays was sedimentable at high speed and is presumably membrane associated. The CF activity of two representative SLE sera was associated with IgG globulins by Sephadex filtration. A search for lysosomal antigen in SLE and related disorders was also made. By employing rabbit antiserum to human lysosomes in immunodiffusion, a soluble lysosomal component, apparently distinct from the sedimentable (membrane-associated) antigen described above, was identified in serum, synovial fluid, or pleural fluid from patients with SLE, RA, ankylosing spondylitis, and leukemoid reaction. An antigenically identical soluble component reactive with the rabbit antiserum could be released in vitro from intact lysosomes by repeated freeze-thaw cycles..

The families of two patients with SLE and IgA deficiency were examined. A study of the first patient's family revealed that IgA deficiency was determined by an incompletely penetrant autosomal dominant gene not linked to the HLA locus. Antinuclear antibodies, found in consanguineous and nonconsanguineous relatives were not related to the presence of IgA deficiency or any HLA haplotype. The second patient and his father with antinuclear, anti RBC, and antithyroid auto-antibodies shared an HLA haplotype not present in other sibs. IgA deficiency was not inherited in this family but may have influenced the expression of disease in the propositus or could have resulted from the disease itself.

One idiotype family 4.6.3 derived from a human hybridoma IgML monoclonal anti-DNA antibody of normal origin is commonly expressed (approximately 85%) in the serum of patients with systemic lupus erythematosus but not in normal controls, where its level appears to fluctuate with clinical disease activity. In contrast, the IgM released by normal cultured lymphocytes in vitro expresses this idiotype family but does not bind DNA. Both idiotype 4.6.3 and anti-DNA antibodies are frequently detectable in the serum of patients with monoclonal IgM but not IgG. Therefore, it is likely that idiotype 4.6.3 reflects the expression of a common germ line immunoglobulin gene family. Some of the IgM molecules encoded by these germ line immunoglobulin genes also bind DNA.