Describing and expanding the clinical phenotype of anti-MDA5-associated rapidly progressive interstitial lung disease: case series of nine Canadian patients and literature review.

OBJECTIVE: To describe and expand the phenotype of anti-MDA5-associated rapidly progressive interstitial lung disease (MDA5-RPILD) in Canadian patients. METHOD: All proven cases of MDA5-RPILD hospitalized in the University of Montreal's affiliated centres from 2004 to 2015 were selected for inclusion. RESULTS: Of nine consecutive patients, RPILD was the presenting manifestation in seven, whereas two patients developed RPILD 2 years after the onset of arthritis and of chronic interstitial lung disease. In the case with arthritis, RPILD was probably triggered by initiation of tumour necrosis factor-α-inhibitor therapy. In most patients (89%), RPILD was accompanied by concomitant onset of palmar/lateral finger papules, skin ulcerations, and/or mechanic's hands. All patients experienced profound weight loss over 1-2 months (mean ± SD 10.2 ± 4.8 kg). All had arthralgias and/or arthritis. Six patients were clinically amyopathic; only one patient had creatine kinase (CK) levels > 500 U/L. Initial ferritin and transaminase levels were elevated in 86% and 67% of patients, respectively. The antinuclear antibody (ANA) test was negative for nuclear and cytoplasmic staining; antisynthetase autoantibodies were negative. Three patients died; time from initial symptoms to death ranged from 7 to 15 weeks. All six survivors received mycophenolate mofetil and/or tacrolimus as part of induction and/or maintenance therapy. CONCLUSION: In an inpatient setting, RPILD associated with characteristic skin rashes, profound weight loss, articular symptoms, normal or low CK with elevated ferritin, and absent fluorescence on ANA testing should alert the clinician to the possibility of MDA5-RPILD. T-cell-mediated therapies may play a role in this highly lethal condition.

Update on myositis-specific and myositis-associated autoantibodies.

Myositis-specific autoantibodies or myositis-associated autoantibodies can often be found in serum of patients with polymyositis and dermatomyositis. The presence of these autoantibodies can be significant in patient diagnosis and classification. Recent studies have provided new information about many of these specific autoantibodies. Among the more important developments were identification of a new antisynthetase, reacting with asparaginyl-tRNA synthetase; the detection of antibodies to the tRNA(his) in a over a third of anti-Jo-1 sera; and the description of distinctive features of the histopathology of patients with anti-Jo-1. New information about the cellular role of the antigens was discovered, including a role for Mi-2 antigen in chromosomally-mediated regulation of transcription as part of a nucleosome remodeling complex, and a potential role for PM-Scl antigen in ribosomal RNA processing as part of an exosome. The reason for the production of the autoantibodies, and the reason particular antigens are targeted, are key questions. Recent studies have suggested that antigen cleavage during apoptosis, particularly by granzyme B, may be an important factor. Whether the antibodies play a role in tissue injury remains unknown.

Antiendothelial cell antibodies in inflammatory myopathies: distribution among clinical and serologic groups and association with interstitial lung disease.

OBJECTIVE: To determine the prevalence and associations of antiendothelial cell antibodies (AECA) in a well characterized cohort of patients with idiopathic inflammatory myopathies (IIM). METHODS: Clinical characteristics, AECA, and myositis-specific autoantibodies were assessed by standard methods in 56 subjects with IIM. RESULTS: AECA were found in 20/56 patients with IIM, were seen in all the major clinical and serologic IIM groups, and were found in 10/15 patients with interstitial lung disease (ILD) (chi squared 6.5, p<0.01 with Yates' correction, relative risk 2.7, specificity 86% and sensitivity 50%). Antisynthetase antibodies, also associated with ILD as described (chi squared = 26.5, p<0.001 with Yates' correction, relative risk 8.7, specificity 95%, sensitivity 77%), did not correlate with the presence of AECA. CONCLUSION: AECA appear to be present in all forms of IIM and are markers for ILD that are independent of anti-synthetase autoantibodies. AECA may be a useful serologic marker for ILD in IIM.

Characterization of an autoantigen associated with chronic ulcerative stomatitis: the CUSP autoantigen is a member of the p53 family.

A unique clinical syndrome has been described in which patients have chronic oral ulceration and autoantibodies to nuclei of stratified squamous epithelium. We have characterized the autoantibodies from patients sera and found that the major autoantigen is a 70 kDa epithelial nuclear protein. Sequencing of the cDNA for this protein, chronic ulcerative stomatitis protein, revealed it to be homologous to the p53 tumor suppressor and to the p73 putative tumor suppressor, and to be a splicing variant of the KET gene. The p53-like genes, p73 and the several KET splicing variants, are recently described genes of uncertain biologic and pathologic significance. This study provides the first clear association of a p53-like protein with a disease process.

Genetic risk and protective factors for idiopathic inflammatory myopathy in Koreans and American whites: a tale of two loci.

OBJECTIVE: To better understand genetic contributions to autoimmunity, immunogenetic markers were studied in two racially discrete and geographically isolated populations of patients with idiopathic inflammatory myopathy (IIM). METHODS: Clinical characteristics, as well as clinical and autoantibody subsets, were defined in 151 American white patients and 50 Korean patients with IIM. HLA-DRB1 and DQA1 genotyping was performed on patients and racially matched controls by standard molecular techniques. Gm allotypes and phenotypes were determined by the hemagglutination-inhibition method. RESULTS: HLA-DRB1*0301, the linked allele DQA1*0501, and DRB1 alleles sharing the first hypervariable region motif 9EYSTS13 were major genetic risk factors for the development of myositis in whites (corrected P [Pcorr] < 0.0004, odds ratio [OR] 11.2, 4.5, and 3.1, respectively, for each factor versus controls). Although both the white and Korean patients had a similar distribution of clinical characteristics, autoantibody profiles, and clinical groups, no HLA-DRB1 nor DQA1 allele or motif was found to be a risk factor for IIM in the Korean patients. However, DRB1*14 was a protective factor in Korean patients without myositis-specific autoantibodies (Pcorr = 0.004, OR 0.046). In addition, although no Gm phenotype or allotype was identified as a risk factor in whites, Gm 21 was a protective factor for the development of IIM in Koreans (Pcorr = 0.024, OR 0.3). CONCLUSION: Although myositis patients in the US and Korea share similar clinical and serologic features, the immune response genes predisposing to and protecting from myositis in each of these ethnic groups differ at two chromosomal loci. These data suggest that multiple genetic loci should be studied to identify risk and protective factors for some autoimmune diseases in various ethnic populations.

Anti-KS: identification of autoantibodies to asparaginyl-transfer RNA synthetase associated with interstitial lung disease.

Autoantibodies to five of the aminoacyl-transfer RNA (tRNA) synthetases have been described, and each is associated with a syndrome of inflammatory myopathy with interstitial lung disease (ILD) and arthritis. Serum KS, from a patient with ILD and inflammatory arthritis without evidence of myositis, immunoprecipitated a tRNA that was distinct from that precipitated by any described anti-synthetase or other reported tRNA-related Abs, along with a protein of 65 kDa. KS serum and IgG fraction each showed significant (88%) inhibition of asparaginyl-tRNA synthetase (AsnRS) activity, but not of any of the other 19 aminoacyl-tRNA synthetase activities. Among 884 patients with connective tissue diseases tested, only two other sera were found to immunoprecipitate tRNAs and proteins of identical gel mobility. These two and KS showed identical immunodiffusion lines using HeLa cell extract. The new sera significantly inhibited AsnRS without significant effects on other synthetases tested. Both patients had ILD but neither had evidence of myositis. These data strongly suggest that these three sera have autoantibodies to AsnRS, representing a sixth anti-synthetase. Anti-KS was more closely associated with ILD than with myositis. Further study of this Abs might prove useful in dissecting the stimuli responsible for the genesis of anti-synthetase autoantibodies.

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.

Detection of anti-PL-12 autoantibodies by ELISA using a recombinant antigen; study of the immunoreactive region.

Autoantibodies to aminoacyl-tRNA synthetases are highly associated with myositis and detection is important in clinical diagnosis; however, current methods of screening limit its clinical utility. In the present study, alanyl-tRNA synthetase (PL-12) recombinant protein was obtained by immunological screening of a HeLa expression library and used in an ELISA with 22 anti-PL-12 sera, 200 autoimmune sera negative for PL-12 and 100 healthy individual sera. Sensitivity of the method was 95% (21/22) and specificity 100%. Mapping of the immunoreactive region was carried out using three anti-PL-12 sera and different recombinant protein-derived peptides. Results show that the same conformational epitope located within amino acids 730-951 of the PL-12 antigen outside the catalytic region was recognized by the three anti-PL-12 sera tested. We conclude that ELISA using recombinant protein is an effective and useful method for routine screening for anti-PL-12 autoantibodies.

Clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy.

OBJECTIVE: To describe the clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy (IIM) and to compare these with the features of sporadic IIM. METHODS: Clinical signs and symptoms, autoantibodies, HLA-DRB1 and DQA1 alleles, and GM/KM phenotypes were compared among 36 affected and 28 unaffected members of 16 unrelated families in which 2 or more blood relatives developed an IIM. In addition, findings in patients with familial IIM were compared with those in 181 patients with sporadic IIM. The families included 3 pairs of monozygotic twins with juvenile dermatomyositis, 11 families with other siblings or relatives with polymyositis or dermatomyositis, and 2 families with inclusion body myositis. RESULTS: The clinical features of familial IIM were similar to those of sporadic IIM, although the frequency of myositis-specific autoantibodies was lower in familial than in sporadic IIM. DRB1*0301 was a common genetic risk factor for familial and sporadic IIM, but contributed less to the genetic risk of familial IIM (etiologic fraction 0.35 versus 0.51 in sporadic IIM). Homozygosity at the HLA-DQA1 locus was found to be a genetic risk factor unique to familial IIM (57% versus 24% of controls; odds ratio 4.2, corrected P = 0.002). CONCLUSION: These findings emphasize that 1) familial muscle weakness is not always due to inherited metabolic defects or dystrophies, but may be the result of the development of IIM in several members of the same family, and 2) multiple genetic factors are likely important in the etiology and disease expression of familial IIM, as is also the case for sporadic myositis, but DQA1 homozygosity is a distinct risk factor for familial IIM.

Anti-endothelial cell antibody, thrombomodulin, and von Willebrand factor in idiopathic inflammatory myopathies.

Sera from 19 patients with idiopathic inflammatory myopathy (IIM) were examined for the presence of anti-endothelial cell antibodies (AECA) by an immunoglobulin G-specific cellular enzyme-linked immunosorbent assay. The mean binding index of AECA was found to be 37.7% +/- 26.5% for the patients, compared with a mean of 7.2% +/- 2.7% for normal controls (P < 0.04). Levels of thrombomodulin, von Willebrand factor antigen, and serum creatine kinase were also shown to be augmented. Interestingly, positive correlations between AECA on the one hand and Raynaud's phenomenon and interstitial lung disease on the other were demonstrated. Given that the pathogenesis of IIM remains uncertain, these findings may be of importance.

Autoantibodies to ribosomal P proteins penetrate into live hepatocytes and cause cellular dysfunction in culture.

Abs to ribosomal P protein have been shown to bind a membrane form of the P0 38-kDa ribosomal phosphoprotein. This study shows that after affinity-purified Abs to ribosomal P proteins bind living HepG2 cells, they then penetrate these live cells and cause cellular dysfunction. Binding and penetration of anti-P Abs is the property of F(ab')2 fragments as well as whole IgG molecules showing that neither binding nor penetration depends on Fc fragments or their cognate receptors. Confocal microscopy shows that internalized Ab concentrates in perinuclear vesicles (presumably lysosomes), but substantial quantities of Ab are also found in the cytosol. This intracellular Ab adversely affects the synthesis of apolipoprotein B resulting in a threefold increase in cellular cholesterol with lipid droplet accumulation as seen in some chronic liver diseases. It also has a profound inhibitory effect on global protein synthesis as measured by [35S]methionine incorporation. These studies therefore describe a model of cellular injury effected by specific Ab to ribosomal "P" protein that may underlie certain forms of autoimmune hepatic diseases.

Clinical, serologic, and immunogenetic features in Polish patients with idiopathic inflammatory myopathies.

OBJECTIVE: To determine the clinical, serologic, and immunogenetic correlations in patients with idiopathic inflammatory myopathies (IIM), and to evaluate the useful grouping of some diseases for practical clinical purposes. METHODS: Patients with IIM were categorized according to clinical presentation as compared with autoantibody specificity. Serum samples from 84 patients were screened for myositis-specific autoantibodies (MSAs) by indirect immunofluorescence and double immunodiffusion. All sera were also studied by protein A-assisted immunoprecipitation. Genomic DNA was isolated from peripheral blood mononuclear cells, and HLA-DQA1 and DRB1 alleles were determined. The patients were seen and followed up for many years in the same center. RESULTS: MSAs were present in 19% of patients. The most common MSAs were antisynthetases in 13% of patients (Jo-1 10.7%, PL-12 1.2%, and EJ 1.2%), associated with the antisynthetase syndrome. Anti-SRP was found in 1.2% of patients, associated with polymyositis, and anti-Mi-2 in 4.9%, found exclusively in patients with dermatomyositis. The most frequent MSA was PM-Scl in 23.8% of patients, associated with scleromyositis, and Ku was present in 9.6% of patients with overlap syndromes. The alleles that were found at a significantly increased frequency were HLA-DRB1*0301 (59.4%) and DQA1*0501 (71.6%), which are in linkage disequilibrium. DQA1*0501 was present in 85.7% of patients with antisynthetases, and in 100% of patients with PM-Scl and Ku. CONCLUSION: The HLA-DRB1*0301; DQA1*0501 haplotype was found to be significantly increased in this population overall and in those myositis patients with antisynthetase, anti-PM-Scl, and anti-Ku antibodies. The results of this study confirm that IIM are heterogeneous syndromes, but can be divided into more useful groups on the basis of clinical, serologic, and immunogenetic features.

Classification criteria for the idiopathic inflammatory myopathies.

Inasmuch as the clinical features of the idiopathic inflammatory myopathies are not easily differentiated from those of other similar rheumatic and neurologic conditions, diagnosis is often difficult. Various classification criteria for polymyositis and dermatomyositis have been suggested by a number of investigators. The most commonly accepted and used criteria include symmetric proximal muscle weakness, serum elevations of muscle enzymes, the classic electromyographic and muscle biopsy findings of inflammatory myopathy, and the typical skin rash of dermatomyositis. Although these criteria are clinically useful, they can result in misdiagnoses and inappropriate therapies. They also result in heterogeneous patient groups being selected for clinical and laboratory studies. Furthermore, they do not include recent findings related to the myositis-specific autoantibodies and magnetic resonance imaging of muscle that have been found to be important adjuncts in assessing patients with muscle weakness or elevations of muscle enzymes. A modification to the Bohan and Peter criteria is proposed to include myositis-specific autoantibodies and magnetic resonance imaging. This proposal could initiate productive discussions and investigations of the sensitivity and specificity of new classification criteria for myositis and could ultimately enhance our treatment capabilities.

Anti-PM/Scl autoantibodies in patients without clinical polymyositis or scleroderma.

OBJECTIVE: To characterize patients with anti-PM/Scl but no definite myositis-scleroderma (PM/SSc) overlap or either polymyositis/dermatomyositis (PM/DM) or systemic sclerosis (SSc) alone. METHODS: Review of all patients with anti-PM/Scl identified at a reference serologic clinical laboratory. RESULTS: We identified 5 patients with anti-PM/Scl not considered to have either PM, SSc, or PM/SSc. One had primary Sjögren's syndrome but the other 4 had some feature(s) of illness consistent with PM/DM or SSc, such as hypertensive crisis or interstitial lung disease. These 5 patients represented 10% of the total number of patients with anti-PM/Scl identified in our clinical laboratory. CONCLUSION: Anti-PM/Scl may be a marker for atypical or subclinical presentations of the usually associated disease, or this autoantibody may precede expression of the underlying disease. The antibody may also be present in patients who never express PM, SSc, or PM/SSc. Anti-PM/Scl without the usually described illnesses is not uncommon in our series of 55 patients with anti-PM/Scl.

Clinical significance of specific autoantibodies in juvenile dermatomyositis.

OBJECTIVE: To determine the prevalence and clinical association of myositis specific antibodies in an unselected group of patients with juvenile dermatomyositis (DM). METHODS: The sera of 42 subjects, representing an unselected group of patients from a single center, with juvenile DM and 7 others with idiopathic inflammatory myopathy (IIM) were examined for the presence of myositis specific antibodies by immunodiffusion against calf thymus extract and immunoprecipitation with HeLa extract. RESULTS: Of the subjects with juvenile DM, only 2 had evidence of antibodies specific to myositis (anti-Mi2). Three other patients with juvenile DM had defined autoantibodies not usually considered to be specific to myositis. Two of the 3 subjects had anti-PM-Scl; both developed features of scleroderma after the juvenile DM remitted. The 5 subjects with defined autoantibodies did not differ clinically from the remainder of the subjects with the exception of the late development of scleroderma features in 2. Fourteen other subjects with juvenile DM had unidentified bands on immunoprecipitation, which may represent as yet undiscovered myositis specific antibodies. No myositis specific antibodies were detected in any of the 7 subjects with other IIM syndromes. CONCLUSION: Based on our findings, we do not recommend routine clinical testing for these antibodies in children with typical juvenile DM. Further study of the unidentified bands seen in our subjects may lead to better understanding of the clinical groupings and etiopathogenesis of childhood myositis.

Anti-U5 small nuclear ribonucleoprotein (snRNP) antibodies: a rare anti-U snRNP specificity.

We surveyed for autoantibodies to the U small nuclear ribonucleoproteins (snRNPs) in sera from 1171 patients with various connective tissue diseases by immunoprecipitation assay. We found serum, termed LaJ, which principally recognized the U5 snRNP from one patient with systemic sclerosis-polymyositis overlap syndrome. Anti-LaJ serum immunoprecipitated predominantly U5 snRNA from a 32PO4-labeled HeLa cell extract and at least five U5-specific proteins as well as the Sm core proteins from a [35S]methionine-labeled extract. Anti-LaJ serum immunoprecipitated both U5 snRNA and these five proteins in identical fractions at 15-20S by parallel sucrose gradient, suggesting physical association of these two components. Thus, we concluded that anti-LaJ serum contained antibody specific to the U5 snRNP. Antibodies specific to U5 snRNP were found in a single serum; in contrast, the prevalence of anti-U1 snRNP and anti-Sm antibody was considerably higher. Thus, the specific proteins of the U5 snRNP are rare targets for autoantibodies in patients with connective tissue disease.

Interrelationship of major histocompatibility complex class II alleles and autoantibodies in four ethnic groups with various forms of myositis.

OBJECTIVE: To examine interrelationships among myositis subsets, autoantibodies, and major histocompatibility complex (MHC) class II alleles across ethnic lines, and to localize genetic susceptibility (presence of HLA-DR versus DQ) to myositis within the MHC class II region. METHODS: MHC class II alleles (HLA-DRB1, DQA1, and DQB1, detected by DNA oligotyping) and myositis-specific autoantibodies (MSA) were determined in 224 patients with various myositis syndromes, including 89 whites, 89 African-Americans, 25 Mexican-Americans, and 21 Japanese. RESULTS: Anti-Jo-1 (histidyl-transfer RNA [tRNA] synthetase) and other MSAs (anti-PL-12, anti-PL-7, anti-OJ, anti-EJ, anti-KJ, anti-tRNA, and anti-signal recognition particle) were equally distributed among the races, but occurred more often in patients with polymyositis (PM) than in those with dermatomyositis (DM) or other myositis syndromes. MSA frequencies were significantly positively associated with anti-Ro (SS-A) (P = 0.002), and significantly negatively associated with anti-U1 RNP (P = 0.003). Frequencies of the HLA-DRB1*0301 (DR3), DQA1*0501, and DQB1*0201 (DQ2) alleles (and haplotype) were each increased in white patients with myositis, especially those with PM, but most strikingly in those with MSAs. However, in the other ethnic groups, except the Japanese group, only frequencies of HLA-DQA1*0501 and the structurally similar DQA1*0401 alleles were significantly increased. The presence of HLA-DQA1*0501 or *0401 was most significantly associated with anti-Jo-1, anti-PL-12, and other MSAs, compared with myositis patients without MSAs (P = 0.0008, Pcorr = 0.01, odds ratio [OR] = 3.7), and with normal, ethnically matched controls (P = 3 x 10(-7), Pcorr = 1 x 10(-6), OR = 6.5). Among MSA-positive patients who were negative for HLA-DQA1*0501 and *0401, including Japanese patients, the HLA-DQA1*0102 and *0103 alleles predominated. In addition, there appeared to be a negative association of the HLA-DR2 alleles (DRB1*1501 and *1503) with PM (P = 0.007, Pcorr not significant, OR = 0.39), but not with DM or myositis overall. CONCLUSION: By transracial gene mapping, genetic susceptibility to anti-Jo-1 and other MSAs in patients with myositis can be localized within the MHC region to the HLA-DQA1 locus.

Epitope analysis of the major reactive region of the 100-kd protein of PM-Scl autoantigen.

OBJECTIVE: To localize the epitope(s) bound by anti-PM-Scl antibodies in the N-terminal half of the 100-kd protein, the major antigen of the PM-Scl complex. METHODS: Investigations were performed by immunoblotting 20 anti-PM-Scl positive sera against bacterially expressed, polymerase chain reaction-derived deletion mutants of the S1 fragment (amino acids 11-437), enzyme-linked immunosorbent assay (ELISA) screening against synthesized serial octapeptides, and ELISA screening, with anti-PM-Scl positive sera, against a synthesized 21-amino acid peptide covering the active region. RESULTS: Anti-PM-Scl positive sera retained full immunoblot activity with fragment 207-436 and most activity with fragment 11-241, but had markedly decreased activity against fragments 236-436 and 11-212, indicating a major epitope in the aa 207-241 region. Fusion proteins with smaller fragments localized this activity between aa 226 and aa 246. Of 42 anti-S1-positive, anti-PM-Scl positive sera tested by ELISA against a synthetic peptide of this region, 36 were definitely positive, 4 borderline, and 2 negative. Similar activity was seen with a peptide from which proline 228 was deleted. Three additional epitope areas were found in S1, but each reacted with only a few sera. Anti-PMScl positive sera did not react with any octapeptide spanning the major epitope area (aa 207-246). CONCLUSION: The main immunoblot epitope of the PM-Scl 100-kd protein is within a central area of 21 aa (aa 226-246), but is longer than the usual linear epitope. This peptide may be useful in patient testing. Three minor epitopes in S1 may also be recognized by some sera.