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| Lisa Christopher-Stine, M.D., MPH
Pathogenesis Epidemiology Treatment | |||||||||||||||||||||
| Pathogenesis | |||||||||||||||||||||
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Abstract 643: The N-Terminal Domain of Jo-1 is Confirmationally Mobile: Implications for Immunogenicity in Myositis Purpose: To determine if the charged residues at positions 30-31 in the N-terminal regulatory domain of HRS are involved in autoantibody and Granzyme B cleavage binding. Histidyl tRNA synthetase (HRS, Jo-1) autoantibodies are found in up to 70% of myositis patients with ILD. Autoantibody binding to HRS inhibits enzymatic function and abolishes cleavage by Granzyme B; however the mechanism by which autoantibody binding to HRS mediates these effects is not known. Methods: A mutant HRS species containing positively charged amino acid subsytitutions in the region of interest was created by site-directed mutagenesis. These mutations preserved the coil-coil region of interest at the N-terminus. Radiolabeled proteins were generated and autoantibody binding and susceptibility to Granzyme B cleavage was assessed. Results: Autoantibody binding to the mutant HRS was unaffected in immunoprecipitation assays. Cleavage of mutant HRS in the presence of antibody was very efficient. In contrast, no cleavage of wild type HRS at this site was noted. Conclusions: the major epitope of HRS recognized by autoantibodies is located in a confirmationally functional region of the molecule delineated by its Granzyme B cleavage site. The cleavage site become inaccessible when autoantibodies are bound to it. This inhibition is abolished by mutations that do not affect autoantibody binding, suggesting that autoantibody binding to the wild type protein induces a conformational change which obscures the Granzyme B cleavage site. Editorial Comment: Dr. Levine and colleagues conclude that the HRS immune response in myositis may be a marker of conformational changes that occur during disease initiation, and thus lead to novel antigen processing and presentation of previously cryptic epitopes. This work draws on previous work by this group which first identified the N-terminal regulatory domain as the epitope target of HRS autoantibodies and provides further elucidates the generation and propagation of the immune response in anti-Jo-1 positive myositis. Abstract 644: In Adult Onset Myositis, the Presence of interstitial Lung Disease and Myositis Specific/associated antibodies are Governed by HLA Class II haplotypes Rather than By Myositis Subtype Purpose: to examine the primary HLA Class II associations in PM and DM as they relate to phenotypic differences. Methods: DNA samples were collected from 222 UK patients with probable or definite myositis by Bohan and Peter Criteria. The presence of clinical ILD as well as the type of detectable circulating myositis specific or myositis associated antibodies was recorded. A randomly selected control group was used for genetic comparisons. All patients in both groups were Caucasian. Patients and controls were genotyped at HLA DRB1, DQA1, and DQB1. Results: HLA-DRB1*03, DQA1*05, and DQB1*02 were all risk factors for PM and DM with the primary risk factor for PM being HLADQA1*05. Patients with ILD with or withourt antisynthetase antibodies also showed a strong association wit this haplotypes, regardless of whether they had PM or DM>. The HLA-DRB1*07-DQA1*02-DQB1*02 haplotype was a risk factor in anti-Mi-2 antibody positive subjects versus controls but protective in PM versus controls. Conclusions: HLA-DRB1*03-DQA1*05-DQB1*02 haplotype governs disease susceptibility in Caucasian DM/PM as well as phenotypic features common to the disease subtypes. In contrast HLA-DRB1*07-DQA1*02-DQB1*02 discriminates between PM/DM disease susceptibilities. These differences draw attention to the concept that myositis patients with different serologies have different immunogenetic profiles which may further define myositis subtypes. Editorial Comment: These differences draw attention to the concept that myositis patients with different serologies have different immunogenetic profiles which may be responsible for defining their particular myositis ssubtype. Abstract 772 A New Murine Model of Polymyositis Revealed Differential Requirement of Inflammatory Cytokines for Autoimmune Myositis Purpose: To establish a new mouse model to elucidate the pathology of PM. Current treatment polymyositis (PM) depends on non-specific immune suppression. Studies of its pathology and development of new treatment have been hampered in part by limited animal models. One available model (generated by immunization of myosin in SJL/J mice) requires repeated immunization, and is not a pure model because SJL/J mice also have a dysferlin gene mutation that causes spontaneous muscle necrosis and secondary inflammation. Methods: Recombinant human skeletal C-protein was prepared for immunization to provoke C-protein induced myositis (CIM). Muscle tissue from the immunized mice was examined with H & E staining and immunohistochemistry. Scoring was based on number of muscle fibers with mononuclear cell infiltration. Muscle weakness was assessed with the Rotarod task test. Serum anti-C protein antibodies were detected with Western blotting. Results: Single immunization of the recombinant C-protein induced muscular inflammation and myocyte necrosis in several mouse strains including the black 6 mouse. While T cell epitopes were dispersed among the protein, the second fourth from the N end was determined to be the most immunogenic. Histological score was maximal after 2 to 3 weeks after the immunization. Shortened running time on the Rotarod device showed apparent muscle weakness of the affected mice. T cells expressing CD8 and perforin were abundant in the endomysial non-necrotic muscle fibers, the site of muscle damage. CD4 T cells and macrophages were found in perivascular areas as well as in the endomysium. B cells were limited. Interluekin (IL)-1 and tumor necrosis factor (TNF) alpha were predominantly expressed by infiltrating cells in the non-necrotic fibers. These observations closely resemble that of human PM. When IL-1 alpha/beta null mutant mice were immunized, CIM developed with a significantly lower incidence and lower severity. In contrast, TNF-alpha null mutant mice developed comparable myositis to those in wild type mice. Conclusions: This group has established a new murine myositis model that mimics human PM. The disease is readily induced with simple immunization and inducible in B6 mice. The results demonstrated that B cells are not essential to the disease development and that IL-1, but not TNF-alpha, is crucial in the pathogenesis. Editorial Comment: This new murine model sheds some light on the pathology of autoimmune myositis, and is potentially valuable in developing new treatments. It is of great interest that IL-1, and not TNF-alpha was essential for the development of myositis. This finding has implications for therapy. It could be inferred that anti-TNF alpha agents, currently commercially available and helpful in the treatment of rheumatoid arthritis and other related arthritis syndromes, may be weak agents in the treatment of human polymyositis. Abstract 778: Identification of Autoantibodies to Tyrosyl-tRNA Synthetase in Dermatomyositis with Features Consistent with Anti-synthetase Syndrome Purpose: To define an unidentified antibody found in a patient with myositis. The patients clinical picture suggested the antisynthetase syndrome, and immunoprecipitation raised the possibility of an antisynthetase antibody to Tyrosyl-tRNA Synthetase that had previously never been associated with a clinical syndrome. Methods: The patients phenotype included skin rash, ILD, arthritis, and muscle weakness with elevated muscle enzymes. IPP with HeLa cell extract was analyzed by PAGE. Aminoacylation reactions were tested for inhibition from the patients serum, normal control, and anti-Jo-1 and anti-KS reference sera. Antigen that was immuno-affinity purified was analyzed by PAGE and subjected to in-gel trypsin digestion. A peptide mass fingerprint was obtained by mass spectroscopy and compared to the NCBI database. Results: IPP showed a strong band in the tRNA region and a wide band between 61 and 63 kd. Only tyrosyl-tRNA synthetase was significantly inhibited by the patients serum, and not by control sera. The two bands were able to be separated and analyzed by mass spec. MASCOT search of the peptide fingerprint of band 2 suggested that it was tyrosyl-tRNA synthetase. Conclusions: This study confirms the presence of anti-tyrosyl-tRNA synthetase in conjunction with a clinical phenotype consistent with the antisynthetase syndrome. Editorial Comment: This exciting work demonstrates for the first time the presence of anti-tyrosyl tRNA antibodies in a patient with the anti-synthetase syndrome phenotype . For some synthetases, no antibody has been described. This study suggests that other synthetases (including cysteinyl-, Phenylalanyl-, Seryl-, tryptophanyl-, and Valyl-tRNA synthetases) may be targeted autoantigens. Future work should investigate the presence of autoantibodies to all synthetases when the clinical phenotype suggests the antisynthetase syndrome, especially in the absence of currently known anti-synthetase antibodies. | |||||||||||||||||||||
| Epidemiology | |||||||||||||||||||||
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Abstract 779: Myositis-Related Autoantibodies in Patients with Elevated CPK or Interstitial Lung Disease without Diagnosis of PM/DM Y Yamasaki, S Narain, L Hernandez, C Nichols, R Lyons, A Chin Loy, SK Erbay, P Hahn, E Sobel, H Richards, W Reeves, M Satoh Purpose: To investigate the clinical significance of myositis-related autoantibodies in patients with elevated CPK or ILD without clinically apparent myositis. Autoantibodies to histidyl (Jo-1) and other aminoacyl tRNA synthetases are a serological marker associated with a subset of PM/DM with a specific clinical phenotype that includes interstitial lung disease (ILD) and Raynauds phenomenon. Other autoantibodies (Anti-nRNP, -Ku, and -PM-Scl) are associated with PM/DM-overlap syndrome. Methods: 1119 subjects enrolled from 2000-2005 were studied. The specific connective tissue disease diagnosis was established by ACR criteria (SLE, SSc) or Bohan and Peters criteria (PM/DM). Autoantibodies were analyzed by immunoprecipitation (IP) and gel analysis of 35S-labeled proteins and small RNAs. Anti-Jo-1 was also tested by ELISA Clinical information was from a database. Results: Out of 1119 subjects, 57 had ILD and 99 had elevated CPK. In patients with ILD, anti-synthetase antibodies were found in 27% of SLE with ILD vs. 0/285 without ILD (p < 0.0001 by Fishers exact test) and in 18% of UCTD. Anti-nRNP was found in 60% of SLE with ILD vs. 39% without ILD (p = 0.0063). Among patients with elevated CPK, Anti-synthetase was found in 9% of SLE (vs. 1/256 in SLE without elevated CPK, p = 0.0057) and 8% of UCTD. Anti-nRNP was found in 78% of SLE with elevated CPK (vs. 27% with normal CPK, p < 0.0001) and 11% of UCTD. One case each of PL-12, PL-7, and Ku coexisted with anti-nRNP antibodies. In SSc with elevated CPK, anti-PM-Scl was found in 29%. Conclusions: Anti-synthetase and nRNP antibodies are frequently found in SLE or UCTD with ILD or elevated CPK. The authors conclude that patients with these autoantibodies should be monitored closely for myositis/ILD regardless of their diagnosis. Editorial Comment: This study examines several groups of patients labeled with a particular connective tissue disease (SLE, UCTD, etc). It underscores the question of whether myositis-specific antibodies are truly being observed in these diseases, or rather, the patients are labeled inappropriately based on available clinical data. It is believed that myositis-specific antibodies such as the anti-synthetase antibodies are rarely found in other connective tissue diseases. Perhaps a better conclusion is that when myositis-specific antibodies are found in patients suspected of having SLE or another CTD, the original clinical diagnosis should be re-thought. Abstract 780: Epidemiology of Sporadic Inclusion Body Myositis and Polymyositis in Olmsted County, Minnesota Purpose: To determine the incidence and prevalence of sporadic inclusion body myositis (sIBM) and polymyositis (PM), and compare patient demographic parameters. Methods: A retrospective chart review was conducted at the Mayo Clinic involving patients of Olmsted County, MN over 20 years between 1981 and 2000. Patients were classified as sIBM or PM according to established published criteria. Incidence and prevalence rates were determined assuming that individuals aged 30 and older in the population were at risk. Age-and-sex-specific person years used as denominators were estimated from decennial census data for the county. Only those subjects who resided in Olmsted County at the onset of disease were included in the incidence calculations for sIBM and PM. Incidence figures were directly age-and-sex-adjusted to the population structure of the US population in 1990. The 95% confidence intervals around the rates were estimated from the cumulative Poisson distribution. Results: The incidence rate per 100,000 persons with sIBM was 0.56 for males and 0.66 for females. The age-and-sex adjusted rate for males and females was 0.72 (95% CI = 0.18-1.26). The total incidence rate per 100,000 persons with PM was 0.37 for males and 0.66 for females. The age-and-sex adjusted rate for males and females was 0.52 (95% CI = 0.10-0.94). The age-and-sex adjusted prevalence rate of sIBM per 100,000 was 12.9 (95% CI = 4.0-21.9). The age-and-sex adjusted prevalence rate of PM per 100,000 was 8.3 (95% CI = 1.6-14.9). The incidence of sIBM compared to PM did not increase over 2 decades. The demographics of sIBM showed a 1.3:1 female-to-male ratio, and all patients were white. Conclusions: Sporadic IBM may be more common than once believed. This group reported the highest incidence and prevalence rates for sIBM recorded in the literature. More research is needed to explore the possibility that the prevalence of sIBM in males may vary according to population characteristics or environmental factors. Editorial Comment: The authors astutely conclude that sIBM may have been previously under-diagnosed. It is well-known that in the absence of characteristic biopsy findings of IBM, the Bohan and Peter criteria may label IBM patients as having polymyositis (PM). This group also found the incidence of PM to be lower than previous estimates, thus affirming the rarity of the disease that has been suggested by Dalakis and others. Of interest, this group is the first to report an unexpected female-to-male ratio of 1.3:1 despite previous reports of a male predominance. This may be a function of an identified population characteristic in the Olmstead County cohort and should be investigated in other populations including those with a more uniform racial distribution. Abstract 784: Sensitivity and Specificity of Magnetic Resonance Imaging in Discriminating Dermatomyositis and Polymyositis from Muscular Dystrophy and Normal Controls Purpose: This pilot study sought to discriminate between inflammatory myopathies (IIM) from late-onset muscular dystrophies, as this represents a diagnostic challenge. The objective was to assess the sensitivity and specificity of MRI to discriminate among IIM and muscular dystrophy and normal controls. Because the T2-weighted STIR MRI sequence detects edema, one would predict that patients with IIM with active muscle inflammation would have abnormalities on this sequence. Likewise, as T1-weighted MRI imaging demonstrates atrophy and fatty replacement, characteristics of muscular dystrophy, one would predict that this sequence would be abnormal in patients with muscular dystrophy. The objective was to assess the sensitivity and specificity of MRI to discriminate among IIM and muscular dystrophy and normal controls. Methods: A pilot study was performed to examine MRI patterns on 14 patients with IIM [5 definite myositis, 5 probable myositis, 4 with possible myositis by Bohan and Peter criteria]. MRIs of 5 patients with late-onset muscular dystrophy and 5 normal controls were also examined. Each patient underwent MRI of the lower extremities with T1- and T2-weighted STIR imaging. All 14 myositis patients [polymyositis (n=10); dermatomyositis (n=4)] had active disease by clinical assessment. Average disease duration for the myositis patients was 25 months; the average age was 51 years; racial composition was: Caucasian 57%, African American 36%, Hispanic 7%. MRI abnormalities (see table below) were analyzed in a dichotomous fashion (absence vs. presence of any abnormal MR sequence). Both of the diagnostic groups (IIM, muscular dystrophy) were combined, compared with normal controls, and then compared with each other. Results: 17 of 19 patients (89%) with IIM or dystrophy had an abnormal MRI (T1, T2-STIR or both) compared to none of the normal controls (p=0.0005 by Fishers exact test). MRI abnormalities by Disease process and MRI Sequence
Abnormal T2-STIR only is a sensitive measure for discriminating IIM from dystrophy. Sensitivity is 71% (10/14) and specificity is 100% (5/5) (p=0.011 by Fishers exact test). However, MRI is inconclusive at differentiating IIM from dystrophy only when BOTH T1 and T2 are abnormal. Conclusion: This pilot study demonstrates that MRI may be both a sensitive and specific test for discriminating between IIM, muscular dystrophy, and normal muscle and may prove to be a valuable diagnostic tool in the diagnosis of IIM. Editorial Comment: Our group undertook this investigation because it is becoming increasingly clear that some patients labeled with polymyositis may indeed have a late-onset muscular dystrophy. Inflammation on biopsy may be present in both disease states. We sought to determine a non-invasive method to help discriminate between these two diseases, as the therapy and follow-up for each differs greatly. A larger study with a greater number of patients will be needed to confirm our findings. In addition, stratification by disease duration would also be helpful. ((top of page) | |||||||||||||||||||||
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Abstract 790: Preliminary Trial of Creatine and Magnesium Supplementation in Myositis Patients J Park, J Qi, S Kroop, N. Olsen Purpose: To determine whether supplemental creatine and magnesium would be tolerated and enhance muscle performance in patients with idiopathic inflammatory myopathy (IIM). Methods: Five IIM patients (1 DM, 4 PM) completed a 6-month open-label trial of creatine and magnesium as adjunctive treatment. Cr was given as a loading dose of 0.3 g/kg/day for one week and 0.75 g/kg/day thereafter. After 30 days, 400 or 800 mg/day of Mg was added. PCr, ATP, and Mg levels were determined by P-31 magnetic resonance spectroscopy (MRS) at baseline and 6 months. Strength testing was measured, and clinical status was monitored via questionnaires at baseline, and months 1,3, and 6. DEXA scans were also performed at baseline and 6 months. Results: All subjects tolerated the treatment without incident. P-31 MRS showed increased PCr levels for four patients (P=0.020. ATP increases were not statistically significant. Work/cost ratio increased significantly, indicating increased muscle contraction efficiency. MRI was completed on only 3 patients but did demonstrate decreased T2 signal, suggesting decreased edema/inflammation. Patients demonstrated functional improvement and body composition scans via DEXA showed increases in total muscle mass as well as lean tissue. Conclusions: This study demonstrated statistically significant improvement in strength, function, and muscle mass with supplemental creatine and magnesium. Editorial Comment: This study draws upon previous work by this group that demonstrated that muscles of IIM patients have decreased levels of phosphocreatine (pCr, ATP, and magnesium. It concurs with previous studies demonstrating benefit to supplemental creatine alone. The authors correctly indicate that this is a safe adjunctive therapy which should be further investigated in a larger, randomized double-blinded trial. | |||||||||||||||||||||
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