in 2005 , sato et al .   identified a novel autoantibody recognizing a 140-kda protein in patients with dermatomyositis ( dm ) , particularly in those with clinically amyopathic dermatomyositis ( cadm ) . the 140-kda autoantigen , which was identified as melanoma differentiation - associated protein 5 ( mda5 ) , is detected in 19% to 35% of the patients with dm . in the asian population , this autoantibody seems to be associated with rapidly progressive interstitial lung disease and with severe cutaneous vasculopathy ( skin ulceration , tender palmar papules , or both ) [ 16 ] . recently , the presence of anti - mda5 antibody - associated dermatopulmonary syndrome was described in the white population [ 79 ] . mda5 , also known as interferon - induced helicase-1 ( ifih1 ) , is a member of the retinoic acid - inducible gene i - like helicase ( rig - i or rlh ) family of proteins ,   which function by recognizing single - stranded rna viruses and are involved in the innate immune response , including type i ifn production . the main drawback to routine use of this antibody for clinical purposes is that its determination is limited to techniques that are only available in research laboratories , such as immunoprecipitation of radioactive - labeled protein   or enzyme - linked immunoassay ( elisa ) using in - house fabricated recombinant proteins [ 12 , 13 ] . our objective was to evaluate the prevalence and clinical manifestations of anti - mda5-positive patients in a large cohort of dm patients from a single center in barcelona , and to determine the feasibility of detecting this autoantibody with the use of more widely available techniques ( elisa and immunoblotting ) with commercially available recombinant mda5 as the antigen . this study was performed in 117 adult patients ( 92 women ) with dm ( 15 with clinically amyopathic dm ) . in addition , 45 patients with polymyositis ( pm ) , 30 with systemic sclerosis ( ssc ) , and 25 with systemic lupus erythematosus ( sle ) were included as controls . twenty - five healthy controls were also included to determine the cut - off value for establishing the positive status by elisa . the patients studied belong to a historical cohort diagnosed with idiopathic inflammatory myopathy at vall d'hebron general hospital in barcelona ( spain ) , between 1983 and 2012 . our center is a single teaching hospital with approximately 700 acute care beds , attending a population of nearly 450,000 inhabitants . all myositis patients in this population are referred to our hospital for diagnosis and therapy , regardless of the severity of the disease . serum samples from these patients are routinely collected at diagnosis and during follow - up in our outpatient clinic , and stored at 80c . patients and controls included in the study gave informed consent for the use of their serum for research purposes . the diagnosis of dm and pm was based on the criteria of bohan and peter [ 14 , 15 ] . the sontheimer criteria were used to diagnose amyopathic dm . interstitial lung disease ( ild ) was diagnosed according to the consensus classification of idiopathic interstitial pneumonias . the diagnosis of ild was established by high - resolution ct findings , and rapidly progressive - interstitial lung disease ( rp - ild ) was defined as a worsening of radiologic interstitial changes with progressive dyspnea and hypoxemia within 1 month after the onset of respiratory symptoms . cancer - associated myositis ( cam ) was defined as cancer occurring within 3 years of the myositis diagnosis . patients received treatment with corticosteroids and immunosuppressive drugs ( methotrexate , azathioprine , calcineurin inhibitors [ cyclosporine a or tacrolimus ] , or cyclophosphamide pulses ) were added when needed ; intravenous immunoglobulin was used as an adjuvant therapy , and biological therapy ( rituximab ) was also instituted when possible in refractory cases . a drug trial was defined as a single course from the beginning of the administration of a given drug to the time at which the drug was discontinued , or in the case of prednisone , as the time at which the dose was reduced to one quarter of the initial dose . serum samples from each patient were screened by indirect immunofluorescence for antinuclear antibodies ( ana ) using hep-2 cells , and by a commercial elisa used in our routine laboratory setting for antibodies against extractable nuclear antigens ( ro , la , rnp , sm ) and anti - histidyl - trna synthetase ( anti - jo-1 ) . anti - tif1 antibodies were detected by an in - house elisa and confirmed by immunoblot . in addition , all samples were tested by protein and rna immunoprecipitation , which enabled detection of other synthetases and myositis - specific and myositis - associated antibodies ( anti - mi-2 , anti - srp , anti - ro52 , anti - ro60 , anti - la , anti - pm / scl , anti - p155 , and anti - u1rnp ) that may have been overlooked by elisa , and confirmed the elisa results . briefly , 96-well elisa plates ( nunc , kamstrup , denmark ) were coated with 100  ng of purified recombinant mda5 ( origene , rockville , md ) , diluted in phosphate buffered saline ( pbs ) , and left to stand overnight at 4c . wells were incubated for 1 hour at room temperature ( rt ) with blocking buffer ( 10% nonfat dry milk in pbs ) . plates were then washed ( hrp wash , inova diagnostic inc . , san diego , ca ) ; human serum samples diluted 1  :  100 in blocking buffer were added in triplicate : two to mda5-coated wells and one to a pbs - coated well ( without antigen ) to determine the background absorbance . after washing , hrp - labeled goat anti - human igg antibody ( inova diagnostic inc . , san diego , ca ) was added to each well , and plates were incubated for 1  h at rt and washed again . color development was performed with peroxidase reagent tmb chromogen ( inova diagnostic inc . , san diego , ca ) and absorbances at 450  nm were determined . for each sample , the background absorbance from the pbs - coated well was subtracted from that of the corresponding mda5-coated wells ( the average of the two results ) . the same positive serum ( from patient 11 , confirmed by ip by casciola rosen , from baltimore , usa ) was used as the reference in each assay . briefly , 5  g of purified recombinant mda5 ( origene , rockville , md ) was run on 4% to 12% polyacrylamide - sds minigels with mops running buffer , and western blot was performed on a nitrocellulose membrane using the invitrogen nupage ( carlsbad , ca ) electrophoresis system . mda5-transferred nitrocellulose was vertically cut into several strips and incubated for 1 hour at rt in pbs with 0.05% tween ( pbs - t ) containing 3% nonfat dry milk ( blocking buffer ) . each strip was then incubated with the corresponding human serum sample diluted 1  :  100 in blocking buffer for 1 hour at rt . after washing , phosphatase alkaline - labeled goat anti - human igg antibody ( dako , glostrup , denmark 1  :  2000 ) was added to each strip and strips were incubated for 1 hour at rt . color development was performed by phosphatase reagent ( bcip / nbt , sigma - aldrich , st . , the results were classified into negative , weak positive ( + ) , or positive ( + + , + + + ) ( figure 1 ) . associations between anti - mda5 antibodies and qualitative variables were evaluated with the chi - square and fisher exact test . the strength of the associations between variables was measured using odds ratios ( ors ) with 95% confidence intervals ( cis ) . the corresponding area under the curve ( auc ) of the roc analysis of anti - mda5 antibody for detection of rp - ild , cadm , and total dm was analyzed with 95% of cis . all tests were two - sided , and probability ( p ) values of < 0.05 were considered statistically significant . all analyses were performed with spss , version 19.0 ( spss , chicago , il ) . one - hundred and seventeen adult dm patients , 15 of whom had cadm , were included in the study . anti - mda5 was determined by our in - house elisa and immunoblot techniques using a commercially available recombinant mda5 . the cut - off value for a positive result on elisa was established at 0.188 absorbance units , which corresponded to 2 standard deviations above the mean value obtained for the 25 healthy controls . the other control subjects included 45 patients with polymyositis ( pm ) , 30 with systemic sclerosis ( ssc ) , and 25 with systemic lupus erythematosus ( sle ) ( figure 2 ) . only two patients diagnosed with dm , 2 with pm , and 1 with ssc showed weak anti - mda5 reactivity by elisa , which was not confirmed by immunoblot ; these results were considered false positives . anti - mda5 antibodies detected by elisa and confirmed by immunoblot were only found in dm patients . roc curve analysis for the positivity of anti - mda5 of all patients with dm against controls and cadm patients versus remaining dm disclosed an auc of 0.56 , 95% ci 0.490.63 and 0.74 , patients with the highest absorbance unit values on elisa also showed the strongest anti - mda5 positivity on immunoblotting . fourteen patients , 8 with cadm , tested positive for anti - mda5 , which represents a prevalence of 12% of the dm patients from our cohort . median ( range ) age at diagnosis of anti - mda5 positive patients was 47 ( 2860 ) years , which did not differ significantly from the remainder of the cohort . seven patients also tested positive to anti - ro52 , but none of them was positive for any antisynthetase antibody . interstitial lung disease was present in 9 of the 14 ( 64.3% ) patients with anti - mda5 autoantibodies , and the condition was rapidly progressive in 8 patients . rp - ild was more frequent in patients with cadm , both in the anti - mda5 positive group ( 7 of 8 cadm versus 1 of 6 dm ; p < 0.05 ) and in the overall cohort ( 8 of 15 cadm patients versus 3 of 102 dm patients ; p < 0.05 ) . when rp - ild was evaluated in relation to anti - mda5 positivity , a highly significant association was observed between the two parameters . thus , 8 of the 14 anti - mda5-positive patients presented rp - ild versus 3 of the 103 anti - mda5-negative patients ( p < 0.05 ; or : 44.4 , 95% ci 9.3212 ; auc 0.84 , 95% ci 0.681 ) . nevertheless , no association was found between anti - mda5 elisa titers at diagnosis of dm and development of a rp - ild . moreover , 6 of the 8 ( 75% ) patients with anti - mda5 and rp - ild were ro52-positive in comparison to only 1 of the 6 ( 16% ) patients without rp - ild . the cadm diagnosis was significantly associated with anti - mda5 positive status ( 8 of 14 mda5-positive patients versus 7 of 103 mda5-negative ( p < 0.05 ; or : 18.3 , 95% ci 4.967.6 ) ) . no differences were observed in the frequency of gottron 's papules , heliotrope rash , photosensitivity , shawl or v  sign , cuticular overgrowth , calcinosis , or the presence of mechanic 's hands when the anti - mda5-positive and anti - mda5-negative groups were compared . panniculitis was the only manifestation significantly associated with the presence of anti - mda5 ( 5 out of 14 anti - mda5-positive versus 13 out of 103 anti - mda5-negative ; p < 0.05 ; or : 3.85 , 95% ci 1.1113.27 ) ; nevertheless , a multivariate analysis was not possible to be performed due to methodological reasons . cancer was diagnosed in 4 out of 14 ( 28.6% ) patients with anti - mda5 autoantibodies , and 3 ( 21% ) of them fulfilled criteria of cam . however , no association was found between anti - mda5 autoantibodies and cam ( p > 0.05 ) . a similar result was obtained when we repeated the analysis after excluding from the cohort the anti - tif1-positive patients ( 28 patients ) in order to avoid a possible confounding effect of the presence of these patients in the control group . the cumulative 70-month survival rate was significantly lower ( 38% ) in the group of patients with anti - mda5 than in the remainder of the cohort ( 62% ) ( log - rank test , p < 0.05 ) ( figure 3(a ) ) . comparison of cumulative 70-month survival between anti - mda5-associated ild and antisynthetase - associated ild also showed a statistical difference ( log - rank test , p < 0.05 ) ( figure 3(b ) ) . no differences in 70-month survival were observed between the cadm group and the classic dm group in anti - mda5- positive patients . all groups were comparable in age , gender , and number of immunosuppressive agents added to the corticosteroid treatment . differences in survival could not be attributed to a higher proportion of  deaths directly related to cancer  between groups . the results of this study prove the feasibility of detecting antibodies against mda5 in adult patients with dm by in - house elisa and immunoblot techniques using commercially available recombinant mda5 as the antigen . in addition , the findings in our patients contribute to support the previously reported association of anti - mda5 antibody with rp - ild and cadm . [ 16 ] , and the only studies performed in a white population have come from the united states [ 79 ] . our results in a large series of mediterranean patients from a single reference center , together with those from other articles published on this topic , [ 18 ] indicate that anti - mda5 antibodies may be a hallmark of adult cadm patients with rp - ild regardless of their origin . furthermore , some authors have suggested an association between this autoantibody and a specific , severe skin vasculopathy in adult dm , characterized by vascular fibrin deposition with variable perivascular inflammation [ 3 , 7 , 8 ] . we found an association only with panniculitis , one of the mucocutaneous findings previously described by fiorentino et al .   in white adults with dm and anti - mda5 antibody . although we routinely test all serum samples from patients with inflammatory myopathies by immunoprecipitation assays using s - protein - labeled hela cells , until the development of our proposed method , we had not been able to clearly identify patients with anti-140  kda antibodies ( anti - cadm-140 ; i.e , anti - mda5 ) . in our analyses , this polypeptide migrated to an area in which almost 70% of sera , including those from normal subjects , immunoprecipitated a weak line around 140  kda . [ 1 , 6 ] , and we tested these patients with a commercially available recombinant mda5 by our in - house elisa and immunoblot techniques , we were unable to recognize this autoantibody . our first patient with cadm and rp - ild died nearly 20 years ago , and anti - mda5 was detected in a stored frozen serum sample by our in - house techniques . thus , these methods could represent a significant advancement in identification of this autoantibody , even in laboratories with standard equipment . anti - ro52 antibodies were present in most of our patients with rp - ild and anti - mda5 , an association that has only recently been reported in anti - mda5-positive ild patients . this fact confers relevant significance on anti - ro52 as a costimulatory autoantibody , a concept reported in patients with antisynthetase syndrome [ 21 , 22 ] . one patient was positive to both anti - mda5 and anti - tif1. to our knowledge , this is the first description of this association . coexistence of two different myositis - specific antibodies has been rarely reported [ 19 , 23 ] . the clinical course of anti - mda5-positive dm patients can be divided into three groups . first ( and most important from the prognostic perspective ) , is the group of patients with cadm and rp - ild , who usually have a poor prognosis and a mortality rate of nearly 50% despite aggressive immunosuppressive therapy and even lung transplantation . second , the group with cadm and little lung involvement , who show skin manifestations , such as ulcerations , palmar pustules , and perhaps panniculitis , as was reported here . the prognosis does not seem to be unfavorable in this group . and finally , the third group of patients , who have ild that is not rapidly progressive and shows a disease pattern similar to that of classic antisynthetase syndrome . differences in the prognosis between the first and the third group may be due to genetic background , and either early immunosuppressive therapy or use of certain drugs , such as calcineurin inhibitors . the existence of a clinical syndrome of rapidly progressive ild ( usually in antisynthetase - negative patients ) was recognized some years ago , but the absence of useful biomarkers made it difficult to characterize these patients . the discovery of anti - mda5 antibodies will help to better define this population , facilitate an early diagnosis , establish the prognosis , and ultimately enable the development of randomized clinical trials to determine the optimal therapy in anti - mda5-positive patients with a poor prognosis . moreover , as it has been recently described , anti - mda5 antibody measurement seems to be useful for monitoring disease activity [ 13 , 26 ] . as our results show , elisa confirmed by immunoblot with commercially available recombinant mda5 antigen are useful techniques for anti - mda5 detection that can be reliably performed in a standard laboratory setting , with potential application in clinical practice .
a new myositis - specific autoantibody directed against melanoma differentiation - associated gene 5 ( anti - mda5 ) has been described in patients with dermatomyositis ( dm ) . 
 we report the clinical characteristics of patients with anti - mda5 in a large mediterranean cohort of dm patients from a single center , and analyze the feasibility of detecting this autoantibody in patient sera using new assays with commercially available recombinant mda5 . 
 the study included 117 white adult patients with dm , 15 ( 13% ) of them classified as clinically amyopathic dermatomyositis ( cadm ) . 
 clinical manifestations were analyzed , with special focus on interstitial lung disease and its severity . 
 determination of anti - mda5 antibodies was performed by a new elisa and immunoblot technique . in sera , from 14 ( 12% ) dm patients ( 8 cadm ) , mda5 was recognized by elisa , and confirmed by immunoblot . 
 eight of the 14 anti - mda5-positive patients ( 57.14% ) presented rapidly - progressive interstitial lung disease ( rp - ild ) versus 3 of 103 anti - mda5-negative patients ( 2.91% ) ( p < 0.05 ; or : 44.4 , 95% ci 9.3212 ) . 
 the cumulative survival rate was significantly lower in anti - mda5-positive patients than in the remainder of the series ( p < 0.05 ) . 
 patients with anti - mda5-associated ild presented significantly lower 70-month cumulative survival than antisynthetase - associated ild patients . among the cutaneous manifestations , 
 only panniculitis was significantly associated with the presence of anti - mda5 antibodies ( p < 0.05 ; or : 3.85 , 95% ci 1.1113.27 ) . 
 these findings support the reliability of using commercially available recombinant mda5 for detecting anti - mda5 antibodies and confirm the association of these antibodies with rp - ild in a large series of mediterranean patients with dm .