The association of anti-Ro52 autoantibodies with myositis and scleroderma autoantibodies

The presence of autoantibodies to the Ro52 protein in sera from patients with idiopathic inflammatory myopathies has recently been reported. These antibodies were found predominately in sera with the myositis-specific autoantibody anti-histidyl-tRNA synthetase (anti-Jo-1). In this report, we analysed sera from 216 patients to determine whether anti-Ro52 antibodies are associated with myositis autoantibodies other than anti-Jo-1. These included sera containing antibodies that recognize threonyl- or alanyl-tRNA synthetases, Mi-2, PM-Scl, signal recognition particle (SRP), as well as the systemic sclerosis-related antibodies anti-topoisomerase I (Scl-70) and anti-centromere. A high proportion of sera that contain anti-aminoacyl-tRNA synthetase antibodies, anti-SRP, or anti-PM-Scl antibodies were found to contain antibodies to the Ro52 protein. In contrast, in sera containing anti-Mi-2, anti-Scl-70 or anti-centromere antibodies, anti-Ro52 antibodies were absent or occurred infrequently. In addition, only one serum from 41 rheumatoid arthritis patients was positive for anti-Ro52 autoantibodies. These data indicate that anti-Ro52 antibodies are produced in particular subsets of myositis patients, and are not limited to sera with anti-Jo-1 antibodies.     

Relapsing annular erythema and myeloma successfully treated with cyclophosphamide

We report the history of a patient, who had recurrent patches of erythema on the trunk and extremities for some years. The diagnosis of erythema elevatum diutinum or annulare centrifugum was discussed. The patient developed a monoclonal IgA lambda fraction and clinical signs of myeloma. Treatment with cyclophosphamide stopped the development of erythema and also caused the M-component to disappear. This improvement has persisted even without cytostatic treatment. Another patient with prostatic carcinoma and relapsing annular erythema was cured of his skin lesions, when his carcinoma was treated with estrogens. The literature is reviewed.     

Anti-p80 coilin autoantibodies react with a conserved epitope and are associated with anti-DFS70/LEDGF autoantibodies

p80 coilin is a nuclear autoantigen that strongly accumulates in Cajal bodies (CB) and is considered a marker for CBs. No clear clinical features have been associated with anti-p80 autoantibodies, and the epitopes recognized by these antibodies are entirely unknown. We determined the epitope specificity for 34 anti-p80 coilin-positive sera from Japanese patients to better understand its clinical significance. Western blotting and immunoprecipitation analysis revealed that the autoepitope of p80 coilin was restricted to a single region encompassing amino acid residues 421-576 from the C-terminus of p80 coilin. Additionally, sera from 30 of 34 (88%) patients with anti-p80 coilin antibody titers were also positive for anti-DFS70/LEDGF antibody. This autoantibody is sometimes found in patients with some allergic diseases but rarely in patients with connective tissue disease (CTD). Other similar characteristics between these two autoantibodies were also noted. In contrast, few anti-p80 coilin positive patients were positive for CTD-marker antibodies, such as anti-Sm, anti-dsDNA, and anti-SS-A. We conclude that the presence of anti-p80 coilin autoantibodies is not associated with any particular clinical syndromes nor is it diagnostic of any conditions.     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.     

Modulation of natural IgM autoantibodies to oxidative stress-related neo-epitopes on apoptotic cells in newborns of mothers with anti-Ro autoimmunity

At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus.     

Pathogenesis of diseases associated with antineutrophil cytoplasm autoantibodies

Little is known about the etiologies of diseases associated with circulating antineutrophil cytoplasm autoantibodies (ANCA), such as primary vasculitides and inflammatory bowel diseases. However, the understanding of immune mechanisms supposedly involved in the pathogenesis of these diseases is still growing. In the present review, we first focus on the mechanisms triggering the development of ANCA, including the potential role of microbial superantigens and the possible defect(s) in the progression of apoptosis or in the removal of apoptotic cells. We next concentrate on the contribution of ANCA to the clinical symptoms and on the pathogenic role of ANCA, including the accessibility of ANCA antigens as targets for circulating antibodies and the mode of action of ANCA. Mechanisms of neutrophil activation by ANCA include the engagement of Fcgamma receptors, the possible mechanisms of neutrophil-mediated tissue damage, and the neutrophil-endothelial interaction.     

Subclass restriction of anti-SS-B (La) autoantibodies

Antibodies to the SS-B (La) nuclear ribonucleoprotein particle are relatively specific for the diagnoses of Sj?gren's syndrome or systemic lupus erythematosus. The formation of such autoantibodies is likely, then, to reflect the basic immunopathogenesis of these disorders. We have studied the isotype distribution of anti-SS-B antibodies as a clue to their immunoregulation. Using specific ELISA assays, we found that nearly all anti-SS-B antibodies in 39 patients were IgG, and, of these, only the IgG1 and, to a much lesser extent, IgG3 subclasses were represented. Both kappa and lambda light chain antibodies were found in most sera, and the overall kappa/lambda ratio approximated that of normal serum immunoglobulin. These results suggest that the formation of anti-SS-B antibodies is T-cell dependent and that the response is polyclonal in most patients.     

Characterization of human-human hybridoma monoclonal anti-Ro(SS-A) autoantibodies derived from normal tonsil lymphoid cells

Human-human hybridomas obtained from the separate fusion of tonsillar lymphoid cells from three different normal individuals to the lymphoblastoid cell line GM 4672 were screened by ELISA for the presence of autoantibody to Ro(SS-A). Those anti-Ro(SS-A) reactive hybridomas were then cloned by limiting dilution. Nineteen monoclonal IgM anti-Ro(SS-A) antibodies were obtained, which showed specificity to Ro(SS-A) by ELISA and Western blotting (60 kDa). Some of these monoclonal anti-Ro(SS-A) antibodies showed reactivity to DNA (2/19), cardiolipin (9/19), Sm/RNP (15/19) by ELISA, and to IgG (12/19) and La(SS-B) (19/19) by ELISA and Western blotting. None showed reactivity to the unrelated proteins casein and BSA, nor to RNA. Inhibition studies revealed that the binding to Ro(SS-A) of both IgM hybridoma monoclonal and SLE serum polyclonal IgM anti-Ro(SS-A) antibodies was inhibited with Ro(SS-A), La(SS-B) and Sm/RNP but not with IgG, DNA, RNA and BSA. These data indicate that (1) normal humans have the genetic potential to express antibodies to Ro(SS-A) and (2) the normally derived monoclonal and SLE serum IgM anti-Ro(SS-A) antibodies share similar antigen binding properties and therefore may possibly originate from a common pool of precursor B cells.     

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

The B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145–164, 289–308, 301–320 and 349–368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes:¹⁴⁷HKAFKGSI¹⁵⁴, ²⁹¹NGNLQLRNKEVT³⁰², ³⁰¹VTWEVLEGEVEKEALKKI³¹⁸ and ³⁴⁹GSGKGKVQFQGKKTKF³⁶⁴. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presented 83.3% similarity with the ¹³⁹HKGFKGVD¹⁴⁶ region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope ¹⁴⁷HKAFKGSI¹⁵⁴ to 100% to epitope ³⁴⁹GSGKGKVQGKKTKF³⁶⁴. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.     

Clinical and serological features of patients with autoantibodies to GW/P bodies

GW bodies (GWBs) are unique cytoplasmic structures involved in messenger RNA (mRNA) processing and RNA interference (RNAi). GWBs contain mRNA, components of the RNA-induced silencing complex (RISC), microRNA (miRNA), Argonaute proteins, the Ge-1/Hedls protein and other enzymes involving mRNA degradation. The objective of this study was to identify the target GWB autoantigens reactive with 55 sera from patients with anti-GWB autoantibodies and to identify clinical features associated with these antibodies. Analysis by addressable laser bead immunoassay (ALBIA) and immunoprecipitation of recombinant proteins indicated that autoantibodies in this cohort of anti-GWB sera were directed against Ge-1/Hedls (58%), GW182 (40%) and Ago2 (16%). GWB autoantibodies targeted epitopes that included the N-terminus of Ago2 and the nuclear localization signal (NLS) containing region of Ge-1/Hedls. Clinical data were available on 42 patients of which 39 were female and the mean age was 61 years. The most common clinical presentations were neurological symptoms (i.e. ataxia, motor and sensory neuropathy) (33%), Sj?gren's syndrome (SjS) (31%) and the remainder had a variety of other diagnoses that included systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC). Moreover, 44% of patients with anti-GWB antibodies had reactivity to Ro52. These studies indicate that Ge-1 is a common target of anti-GWB sera and the majority of patients in a GWB cohort had SjS and neurological disease.     

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen,enhancing recognition by anti-Ro60 kD autoantibodies

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide–calreticulin is recognized specifically by anti‐Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi‐step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175–184aa(10p) and 216–232aa(17p). The interaction of the peptide–calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty‐eight anti‐Ro60 KD positive and 23 anti‐Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti‐Ro60 kD positive sera bound the complex calreticulin‐17p, while 95% of the same sera had activity against the complex calreticulin ? 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti‐Ro60 kD positive sera. Anti‐Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10ρ and 17ρ or with the complexes calreticulin ? 10p and calreticulin‐17p (<5%). These results suggest that calreticulin can induce conformation‐dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.     

Paraneoplastic intestinal pseudo-obstruction associated with high titres of Hu autoantibodies

Anti-Hu autoantibodies in high titres, as revealed with immunocytochemistry and Western blot, were present in a patient with gastrointestinal pseudoobstruction and small-cell lung cancer (SCLC) bearing the Hu antigen. Marked neuron and nerve fibre loss were found in the myenteric plexus at postmortem. These findings show that neuronopathic Hu-associated gastrointestinal pseudo-obstruction can occur as the only paraneoplastic neurological symptom in patients with SCLC.     

Clinical implications of the presence of anti-Ro (SSA) antibodies in patients with rheumatoid arthritis

The clinical and serologic findings in 25 patients with rheumatoid arthritis (RA) of more than 5 years' duration, who had antibodies to Ro(SSA) cellular antigen, were analyzed and compared with those in 50 anti-Ro(SSA) negative RA patients with similar disease duration. Ninety-six percent of the anti-Ro(SSA) positive patients were female, compared to 74% in the negative group. The majority of antibody positive patients had symmetric erosive synovitis, similar to that observed in the negative group. Extra-articular manifestations were similar in both groups, except for features of Sj?gren's syndrome (keratoconjunctivitis sicca and positive labial salivary gland biopsy), which were significantly more common in patients with anti-Ro(SSA) antibodies. Anemia of chronic disease was also more common in these patients, as were rheumatoid factor (RF) and antinuclear antibodies (ANAs). No patient in either group had antibodies to ds-DNA or Sm and none developed clinical features suggestive of systemic lupus erythematosus (SLE). D-penicillamine, given in the majority of patients in both groups, produced adverse reactions in 72% of the anti-Ro(SSA) positive patients, but only in 27% of the negative ones. Thus, anti-Ro(SSA) antibodies seem to characterize a distinct group of RA patients who are almost exclusively female, express more activated B-cell function, have a high prevalence of Sj?gren's features and commonly develop side effects to D-penicillamine.     

Factors associated with parietal cell autoantibodies in the general population

The presence in serum of parietal cell autoantibodies (PCA) is a characteristic of autoimmune gastritis. We determined the prevalence of PCA in the general population and investigate their association with type 2 diabetes, insulin resistance and lifestyle factors related with autoimmune gastritis. A cross-sectional study was performed, involving 429 individuals enrolled in a cohort study of the general population of the Canary Islands. All participants underwent physical examination, provided a blood sample and responded to a questionnaire regarding health and lifestyle factors. Serum concentrations of PCA, soluble CD40 ligand (sCD40L), C-peptide and glucose (to determine insulin resistance) were measured. The association of PCA with the other factors was determined with bivariate analysis, and logistic regression models were used to adjust the associations for age and sex. The prevalence of PCA was 7.8% (95% CI=10.3-5.3). The factors associated with PCA were female sex (p=0.032), insulin resistance (p=0.016), menopause (p=0.029) and sCD40L (p=0.019). Alcohol consumption (p=0.006) and smoking (p=0.005) were associated with low prevalences of PCA. After adjustment for age and sex, the association with PCA was confirmed for smoking (OR=0.1 [0.0-0.9]), alcohol consumption (OR=0.3 [0.1-0.9]), insulin resistance (OR=2.4 [1.1-4.9]), female sex (OR=2.4 [1.1-5.3]), sCD40L (OR=3.7 [1.2-11.4]) and menopause (OR=5.3 [1.2-23.3]). In conclusion, smoking and alcohol consumption acted as protective factors against the appearance of PCA in the general population, whereas female sex, menopause, insulin resistance and elevated serum sCD40L were risk markers for PCA. In patients who smoke or drink alcohol, clinicians should be cautious when using PCA to rule out autoimmune gastritis.