Autoantibodies against alanyl-tRNA synthetase and tRNAAla coexist and are associated with myositis.

The sera of six patients with autoimmune disease, predominantly myositis with pulmonary fibrosis, contain antibodies of the PL-12 specificity. These autoantibodies react with both protein and RNA components of human cells. The protein has a subunit molecular mass of 110 kD, and the RNA comprises a group of bands in the tRNA size class. Aminoacylation experiments identify the antigens as alanyl-tRNA synthetase and its corresponding tRNAs, tRNAAla. Anti-tRNA antibody can be absorbed out without depleting antisynthetase activity, showing that the antigens are recognized independently by separable antibodies that coexist in these sera. The concurrence of separate antibodies to the two components suggests that the autoimmune response may be mounted against the charging enzyme-tRNA complex. However, the antisynthetase antibody fails to coprecipitate tRNA with the enzyme, suggesting that the antibody reacts with its target only when it is not complexed with tRNA.

Autoreactive epitope defined as the anticodon region of alanine transfer RNA.

Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.

Anti-RNA polymerase III antibodies in patients with systemic sclerosis detected by indirect immunofluorescence and ELISA.

OBJECTIVES: To evaluate the analytical performance of an ELISA for the detection of anti-RNA polymerase III antibody (ARA) and to assess IIF as a method for identifying this antibody. METHODS: A commercially available ELISA was used to assess the presence of ARA in sera from 1018 SSc patients. The sera had been divided into sub-populations based on the presence of specific autoantibodies, ANA pattern or the absence of both. Patients with ARA (n = 209) had been identified by characteristic ANA pattern by IIF on HEp-2 cell substrate [and additionally by radio-immunoprecipitation (IP) in 157/209 cases]. The remaining 809 SSc patients acted as a control group. RESULTS: Of 157 patients in whom ARA had been confirmed by IP, 150 were positive by ELISA providing a sensitivity of 96%. In the group where ARA had only been assessed by IIF, 100% (52/52) were ELISA positive. The ANA patterns indicating the presence of ARA were a fine-speckled nucleoplasmic stain with additional occasional bright dots, with or without concurrent punctate nucleolar staining. In the SSc control group, the ELISA attained a specificity of 98%, ARA being detected in 17/809 patients. CONCLUSIONS: We report the outcome of a study on a large population of SSc patients that shows the ARA ELISA to be of high analytical sensitivity and specificity. We confirm that there is minimal overlap between ARA and other SSc-specific autoantibodies. Additionally, it is demonstrated that the presence of ARA correlates with identifiable patterns by IIF on HEp-2 cell substrate.

Scleroderma renal crisis: patient characteristics and long-term outcomes.

BACKGROUND: Scleroderma renal crisis (SRC) is an important complication of systemic sclerosis, causing acute renal failure, and usually hypertension. AIMS: To review the clinical and pathological features of SRC, and correlate them with renal outcomes and mortality. DESIGN: Retrospective case series. METHODS: We identified 110 cases of SRC managed at a single centre between 1990 and 2005. RESULTS: SRC occurred in 5% of scleroderma cases under follow-up. Cases were predominantly female (81%), with diffuse cutaneous disease (78%). RNA polymerase antibodies were found in 59% of cases tested. Almost all (108/110) received treatment with ACE inhibitors (ACEIs). Dialysis was not required in 36%, was required temporarily (for up to 3 years) in 23%, was required permanently in 41%. Patients not on dialysis showed improvement in estimated glomerular filtration rate after SRC (mean change +23 ml/min over 3 years). Poor renal outcome was associated with lower blood pressure at presentation, and with higher age in those requiring dialysis. Steroid use, microangiopathic haemolytic anaemia, and antibody profile were not related to renal outcome. In the 58 renal biopsies available for clinical correlation, acute changes of mucoid intimal thickening in arteries and fibrinoid necrosis in arterioles were associated with a poorer renal outcome. Mortality was high (59% survival at 5 years), and was higher in men. DISCUSSION: Despite the efficacy of ACEIs in managing SRC, the poor long-term outcome warrants evaluation for additional treatments for this devastating complication of systemic sclerosis.

The clinical and immunogenetic features of patients with autoantibodies to the nucleolar antigen PM-Scl.

The clinical and laboratory features of 32 patients with anti-PM-Scl were studied. Patients with this rare autoantibody suffered from a homogenous overlap connective tissue disease defined by Raynaud phenomenon (32/32), features of scleroderma (31/32), arthritis (31/32, erosive in 9/32), myositis (28/32), lung restriction (25/32), calcinosis (15/32), and sicca (11/32). Significant renal and neurologic involvement was uncommon. All patients examined (22/22) had HLA-DR3, and 50% of these patients were homozygous. Our patients responded favorably to moderate immunosuppression and, with therapy, the disease generally has a good prognosis; over 50% of our series (17/32) remained well on minimal or no immunosuppression after a median follow-up of 8 years.

Anti-PL 4 in patients with systemic lupus erythematosus with severe renal and haematological disease.

Anti-PL 4 is an autoantibody which binds to a 150 kDa polypeptide and is found in approximately 1% of SLE sera. The clinical and laboratory features of 16 patients who have had anti-PL 4 detected in their serum are presented. Anti-PL 4 is highly specific for SLE (100%) and identifies a population of patients who typically develop severe renal (75%) and haematological disease (100%).

Clinical features and prognosis of primary biliary cirrhosis associated with systemic sclerosis.

BACKGROUNDS AND AIMS: To evaluate the prognosis of primary biliary cirrhosis (PBC) together with systemic sclerosis (SSc), as this is unknown. METHODS AND RESULTS: A PBC database of 580 patients identified 43 with PBC and SSc: two patients with PBC alone were matched to each PBC-SSc patient for serum bilirubin concentration at the initial visit. Forty (93%) patients had limited cutaneous SSc. At diagnosis of PBC, median values were: 49.7 years, bilirubin 17 micromol/l, and albumin 40.5 g/l. Liver diagnosis occurred a median 4.9 years after SSc in 24 (56%) patients. In matched patients, median values at diagnosis were: 53.2 years, bilirubin 12 micromol/l, and albumin 41 g/l. Median follow up was similar: 3.16 years (PBC-SSc) and 4.8 years (PBC alone). The risk of transplantation or death from diagnosis, adjusting for sex, age, log bilirubin, and alkaline phosphatase was significantly lower in PBC-SSc (hazard ratio 0.116, p=0.01) due to less transplantation (hazard ratio 0.068, p=0.006). The rate of bilirubin increase was less in PBC-SSc (p=0.04). Overall survival was similar (hazard ratio 1.11, p=0.948); there were nine deaths (21%) in PBC-SSc (six SSc related and two liver related) and nine (11%) in PBC alone (six liver related). CONCLUSIONS: Liver disease has a slower progression in PBC-SSc compared with matched patients with PBC alone.

Two human tRNA(Ala) families are recognized by autoantibodies in polymyositis sera.

Autoantibodies to aminoacyl-tRNA synthetases are common in myositis. Sera of one particular class, the PL-12 specificity, contain separate antibodies reacting with alanyl-tRNA synthetase and tRNA(Ala). We show here that the anti-RNA antibodies recognize at least six distinguishable human tRNA(Ala) species, grouped in two sequence families. We have elucidated the complete nucleotide sequence of two tRNA(Ala) species from HeLa cells that are closely related to silkworm moth tRNA(Ala), as well as the partial sequence of a third species. All three contain the anticodon IGC. No tRNAs with pyrimidine in the "wobble" position were found in the immunoprecipitate, and such species may fail to interact with the antibody.

Anti-M4 antibodies measured by a sulphite oxidase ELISA in patients with both anti-centromere and anti-M2 antibodies.

In this study the clinical features of patients with a serological overlap between scleroderma and primary biliary cirrhosis (PBC) were analysed. The entity was defined by the presence of both anti-centromere antibody (ACA) and anti-mitochondrial antibodies to the M2 antigen, pyruvate dehydrogenase. In addition the sera were assayed for anti-mitochondrial antibodies to the M4 antigen measured by an ELISA to sulphite oxidase (SO). First, anti-M2 was detected, not only in 58 out of 60 patients with PBC but also in eight out of 61 patients with ACA. These sera, together with sera from 53 normals and 99 from patients with various connective tissue diseases were then evaluated for anti-SO, which has been proposed by Klein and Berg to be a marker of progressive liver disease. Again, a high proportion (62%) of sera from patients with PBC were positive for anti-SO, and three of the eight patients who had ACA and anti-M2 also reacted with SO. We subsequently identified and included for study a further 10 patients positive for ACA and anti-M2, making a total of 18 patients with this profile. Features of limited cutaneous scleroderma were present in 94% and evidence of liver disease in 56%. Eight out of the 18 patients had anti-SO, and of these four had PBC, two had abnormal biochemical liver function tests but two had no evidence of liver disease. These data confirm that detection of anti-SO is limited to an anti-M2 subpopulation, and may be a marker for liver involvement with prognostic significance in scleroderma patients with ACA.

Antibodies to extractable nuclear antigens in 173 patients with DNA-binding positive SLE: an association between antibodies to ribonucleoprotein and Sm antigens observed by counterimmunoelectrophoresis.

Sera from 173 patients with systemic lupus erythematosus (SLE) were examined for antibodies to extractable nuclear antigens (ENA) by counterimmunoelectrophoresis (CIE). Antibodies to four major constituents were identified and their frequencies noted as follows: anti-RNP (28%), anti-Sm (10%), anti-Ro (24%) and anti-La (9%). In addition to these a number of uncharacterised antigens were also noted. Using the CIE method we found that in cases where anti-Sm was present it was always accompanied by anti-RNP. Associations between these two antibodies have been noted previously, and many theories have been put forward in explanation. In this study a simple interpretation of the results is offered based on current knowledge of the molecular structure of Sm and RNP antigens.

Anti-RNA polymerases and other autoantibody specificities in systemic sclerosis.

Sera from 735 patients with systemic sclerosis were classified according to antinuclear antibody (ANA) pattern as follows: centromere (25%), homogeneous (26%), fine speckled (21%), fine speckled with nucleolar (14%), coarse speckled (7%), nucleolar only (3%) and cytoplasmic only (3%). Immunoprecipitations using 35S-labelled HeLa cell antigen extract were performed using sera from 374 of these patients to detect the systemic sclerosis-specific antibodies to RNA polymerases I and III. The sera were selected to represent each ANA group, but focused on those giving fine speckled nucleoplasmic staining (with or without nucleolar staining) where all 86 sera positive for these antibodies were concentrated. Immunoprecipitates from a further 93 sera from patients with ANA-positive autoimmune diseases other than systemic sclerosis did not precipitate RNA polymerases. In addition, all sera were tested for antibodies to the extractable nuclear antigens topoisomerase I, nRNP, Ro, La and PM-Scl. Sera positive for antibodies to these antigens gave clear correlations with ANA patterns but, of the examples tested, none contained antibodies precipitating RNA polymerase I or III. Thus, sera containing antibodies to RNA polymerases I and III were exclusive of both anticentromere and anti-topoisomerase I, and formed a major serological subgroup (11.7%). Clinically, 77% were patients with diffuse cutaneous disease reflected by higher skin scores and a significantly higher incidence of renal involvement (33%) than patients with antibodies to topoisomerase I (3%).

Anti-fibrillarin antibodies in systemic sclerosis.

OBJECTIVES: To investigate the nature and extent of organ involvement in anti-fibrillarin antibody (AFA)-positive patients within a UK systemic sclerosis (SSc) population. METHODS: We investigated 1026 consecutive patients with SSc. AFA was identified by the characteristic clumpy nucleolar and coilin body pattern of staining in interphase cells and staining of fibrillarin in metaphase cells by indirect immunofluorescence using HEp-2 cells. Identity of the 34-kDa fibrillarin protein was confirmed by immunoprecipitation from [(35)S]methionine-labelled HeLa cell extract. RESULTS: AFA was detected in 42 patients (4.1%) with early disease onset (mean age 36 yr). Sixteen (38%) patients had limited cutaneous (lcSSc) and 26 (62%) diffuse cutaneous SSc (dcSSc). All eight Afro-Caribbean patients with AFA had dcSSc whereas the Caucasians were equally divided between dcSSc and lcSSc. Within the dcSSc subgroup, 54% had myositis, 35% had pulmonary hypertension, 15% had cardiac involvement and 23% had renal involvement. CONCLUSIONS: AFA identifies young SSc patients with frequent internal organ involvement, especially pulmonary hypertension, myositis and renal disease. In contrast to previous reports, AFA was not restricted to dcSSc patients in Caucasians.

Immunoglobulin classes in skin basement membrane in systemic lupus erythematosus: clinical significance and comparison with classes of serum anti-DNA antibodies.

Biopsies of apparently normal skin were obtained from 30 unselected patients with systemic lupus erythematosus. The immunoglobulin class distribution of the immune deposits at the dermal-epidermal junction was determined in order to assess associated disease patterns and to investigate the possibility that the immunoglobulin classes in the skin were an indication of the classes of serum anti-native DNA antibodies. Biopsy specimens containing IgG deposits were obtained from 10 patients with more active disease and a greater incidence of glomerulonephritis than those patients with only IgM deposits or negative biopsies. However, in this unselected group of patients the immunoglobulin class of the immune deposits did not necessarily indicate the class of serum anti-native DNA antibodies. Therefore biopsy of clinically uninvolved skin will not always identify SLE patients with an immunological restriction to IgM antibody production.

The SL autoantibody-antigen system: clinical and biochemical studies.

A recently described autoantibody, SL, was found in serum from 27 patients with autoimmune disease, including 20 with systemic lupus erythematosus (SLE) where the frequently was 7%. Analysis of clinical, serological, and HLA data from 119 SLE patients showed no positive associations with anti-SL antibody apart from a higher frequency of non-infective fever. Most SL positive sera contained other precipitins, notably antibodies to Ro(SS-A) and the proliferating cell nuclear antigen, PCNA. Anti-SL IgG recognised a protein of 32 000 daltons without associated RNA. This polypeptide was distinguished from a similarly sized component of the Sm and RNP ribonucleoprotein particles by demonstrating different products of partial proteolysis. Although anti-SL antibody is of limited clinical importance, it occurs with twice the frequency of anti-SM antibody in white patients with SLE. Preliminary studies indicate that SL and the Japanese Ki system are identical.

Complement fixation by anti-dsDNA antibodies in SLE: measurement by radioimmunoassay and relationship with disease activity.

We have developed a sensitive, solid phase radioimmunoassay (RIA) to quantify the amount of complement (C') fixation by anti-double-stranded DNA (anti-dsDNA) antibodies and have studied sera from 48 patients with systemic lupus erythematosus (SLE). 46% of the patients were positive in this assay. There was no correlation with serum C' levels, and only weak correlation with anti-dsDNA activity as measured by a solid phase RIA. An association was shown between positive values for C' fixation by anti-dsDNA antibodies and the presence of active lupus (p less than 0.01); there was a similar association with the presence of active renal disease in those patients with raised DNA binding (p less than 0.01). Our results suggest that quantitative measurement of C' fixation by anti-dsDNA antibodies may provide information about the pathogenicity of such antibodies in patients with SLE and be a better guide to potential end organ damage than the conventional measurement of DNA binding.

Autoimmune disease and HTLV-1 infection.

We describe two patients who presented with mixed connective tissue disease. Both had antinuclear antibodies and antibodies to the extractable nuclear antigen U1RNP, and both were found to be seropositive for HTLV-1 infection. We found no evidence of HTLV-1 infection in 20 other patients known to have antibodies to U1RNP or in 36 British and West German patients with idiopathic adult polymyositis. In addition the serum from 20 anti-HTLV-1 positive patients did not contain antinuclear antibodies or antibodies to U1RNP. We conclude that rheumatological disease is associated with HTLV-1 through geographical rather than aetiological means.

Cellular protein and RNA antigens in autoimmune disease.

Antibodies directed against soluble cellular antigens are a distinctive feature of systemic autoimmune disease. We have examined 22 autoantibodies in sera from 1111 patients and present the disease associations together with a biochemical analysis of the antigens. The data emphasize the clinical specificity of the antibodies and the restricted number of cellular components that commonly elicit an immune response. In several instances, serological relationships between antibodies mirror biochemical relationships between the corresponding antigens. The antigens are mainly proteins and are often present in complexes with additional protein or nucleic acid molecules. In myositis the antibodies react chiefly with cytoplasmic antigens such as aminoacyl-tRNA synthetases, in contrast to the mainly antinuclear response in SLE. It is argued that both environmental stimuli and genetic factors govern autoantibody specificity, and that molecular characterization of the cellular antigens may yield clues to the aetiology of the disease and of the concomitant, specific autoimmune response.

Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis.

The presence of autoantibodies in the serum of 110 patients with primary biliary cirrhosis (PBC), 50 with HBsAg negative chronic active hepatitis (HBsAg- CAH) and 30 with HBsAg positive chronic active hepatitis (HBsAg+ CAH) was assessed using two methods: indirect immunofluorescence on cells grown in tissue culture (HEp-2 cell line) or standard mouse tissue sections, and counter immunoelectrophoresis (CIE) with soluble tissue extracts. Anti-nuclear antibodies (ANA) were found in 38% of sera from patients with PBC using HEp-2 cells compared with 10% using mouse tissue. A variety of staining patterns were detected including a pattern of multiple nuclear dots. In contrast, ANA was detected in 70% of sera from patients with HBsAg- CAH and 27% with HBsAg+ CAH. Using CIE four distinct antibody antigen systems were detected: Ro (SS-A), La (SS-B) and two new systems, designated XH and XR, reacting with extracts of human spleen and rabbit thymus, respectively. Correlation of the presence of antibody with clinical conditions confirmed the close association between anti-centromere antibody and sclerodactyly in patients with PBC and indicated an association between 'multiple nuclear dot' staining and the sicca syndrome in PBC. No association was found between the presence of either Ro or La antibody and the sicca syndrome in patients with PBC.

Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes.

The clinical and laboratory features of 29 patients who had one of three anti-aminoacyl-tRNA synthetase autoantibodies, anti-Jo1 (histidyl-tRNA synthetase), anti-PL12 (alanyl-tRNA synthetase) or anti-PL7 (threonyl-tRNA synthetase) were analysed and compared with the findings of other published reports. These autoantibodies were found to be associated with a syndrome delineated by inflammatory myositis (24 patients) and pulmonary fibrosis (23 of 29), but also including inflammatory arthritis (26/29), keratoconjunctivitis sicca (17/29), sclerodactyly (21/29), Raynaud's phenomenon (27/29), hepatitis (8/29) and subcutaneous calcinosis (7/29). The most important clinical determinant of outcome in this group of patients was the severity of the interstitial pulmonary disease. No patient fulfilled the classification criteria for systemic lupus erythematosus, although 10 had autoantibodies to extractable nuclear antigens including Ro, La, RNP, and Sm, and two patients had anti-dsDNA antibodies. Although it seems unlikely that anti-aminoacyl-tRNA synthetase antibodies are directly responsible for causing disease, they may provide an important clue to the aetiology of this unusual syndrome.

Anti-Jo-1 antibody: a marker for myositis with interstitial lung disease.

An autoantibody known as anti-Jo-1 antibody is found in 25% of patients with myositis. Its prevalence in patients with both myositis and cryptogenic fibrosing alveolitis was 68% (13 out of 19 patients), compared with 7.5% in patients with myositis alone (four of 53) and 3% in patients with cryptogenic fibrosing alveolitis alone (two of 62). Anti-Jo-1 antibody may be useful in indicating patients with myositis and cryptogenic fibrosing alveolitis. Raynaud's phenomenon, the sicca syndrome, and mild arthritis are also often part of the syndrome.