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OBJECTIVE: To characterize clinical features of polymyositis/dermatomyositis (PM/DM) patients with different anti-aminoacyl transfer RNA synthetase (ARS) antibodies and their association with anti-Ro52. METHODS: Autoantibodies in sera from 97 Japanese patients (36 PM, 56 DM, and 5 clinically amyopathic DM), who satisfied Bohan and Peter or modified Sontheimer's criteria, were characterized by immunoprecipitation and enzyme-linked immunosorbent assay. Clinical information was from medical records. Features associated with different anti-ARS and anti-Ro52 antibodies were analyzed. RESULTS: The prevalence of anti-ARS was similar to other studies (Jo-1, 22%; EJ, 4%; OJ, 1%; PL-12, 1%), except for a high prevalence of anti-PL-7 (12%), which allowed us to characterize patients carrying this specificity. Serum creatine kinase >3000 IU/l was less common in anti-PL-7-positive patients (57%) than anti-Jo-1-positive patients (18%) (p = 0.0328) and was not found in anti-EJ-positive individuals. Interstitial lung disease was common in anti-ARS-positive patients (97%) (p < 0.0001 vs. 48% in anti-ARS-negative). Anti-Ro52 antibodies were frequently detected with anti-ARS (59%) (57% in anti-Jo-1, 67% in anti-PL-7) (vs. 21% in anti-ARS-negative, p < 0.0002). Anti-Ro52 was associated with overlap syndrome (26%) (vs. 7% in anti-Ro52-negative, p = 0.0119). CONCLUSIONS: Patients with different anti-ARS in combination with anti-Ro52 appear to be associated with distinctive clinical subsets.
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Aminoacil-ARNt Sintetasas/inmunología , Autoanticuerpos/sangre , Enfermedades Autoinmunes/inmunología , Dermatomiositis/inmunología , Ribonucleoproteínas/inmunología , Adulto , Anciano , Enfermedades Autoinmunes/sangre , Dermatomiositis/sangre , Dermatomiositis/complicaciones , Femenino , Humanos , Enfermedades Pulmonares Intersticiales/complicaciones , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVES: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease characterised by skin and muscle inflammation, internal organ involvement and serum disease-specific autoantibodies. The recently identified anti-MDA5 (melanoma differentiation-associated gene 5) antibodies are associated with clinically amyopathic DM (CADM), rapidly progressive interstitial lung disease, severe skin manifestations, and poor prognosis. Our objective was to examine the clinical significance of anti-MDA5 antibodies in a cohort of European Caucasian patients with PM/DM, considering that data on anti-MDA5 serology are limited to Asian and US cohorts. METHODS: Sera from 76 consecutive adult Italian patients with PM/DM were analysed by immunoprecipitation (IP) of 35S-methionine radiolabelled HeLa and K562 cell extracts, ELISA using recombinant MDA5 protein and IP-Western Blot using rabbit anti-MDA5 antibodies. Clinical associations of anti-MDA5 antibody positive patients were analysed. RESULTS: Anti-MDA5 antibodies were identified in 5/76 (7%) PM/DM cases and all 5 cases were CADM; anti-MDA5 was the second most common autoantibody in DM after anti-MJ/NXP-2, found in 24% of cases. Compared to 29 anti-MDA5 (-) DM, anti-MDA5 (+) patients have more typical DM skin disease (digit pulp/periungual lesions, Gottron's papules, heliotrope rash) (p=ns). Interstitial lung disease was observed in 3/5 anti-MDA5 (+) patients but only 14% of anti-MDA5 (-) cases (p=0.048). CONCLUSIONS: Our study on European patients with PM/DM confirms that anti-MDA5 antibodies are not uncommon. All anti-MDA5 (+) cases are affected by CADM with typical skin disease, while rapidly progressive pulmonary involvement was diagnosed only in one case. Further studies in larger cohorts are necessary to define the clinical significance of anti-MDA5 antibodies in European PM/DM.
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Autoanticuerpos/sangre , ARN Helicasas DEAD-box/inmunología , Dermatomiositis/inmunología , Adulto , Anciano , Biomarcadores/sangre , Western Blotting , Dermatomiositis/sangre , Dermatomiositis/diagnóstico , Dermatomiositis/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HeLa , Humanos , Inmunoprecipitación , Helicasa Inducida por Interferón IFIH1 , Italia/epidemiología , Células K562 , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Prevalencia , Estudios Retrospectivos , Población BlancaRESUMEN
OBJECTIVE: Antinuclear antibodies (ANA) occur in up to 95% of patients with systemic sclerosis (SSc). In most, SSc-associated antibodies are detected (i.e., centromere, topoisomerase I, RNA polymerase III, PM/Scl, Ro52/TRIM21, and U1RNP). Ribonuclease P protein subunit p25, (Rpp25) is an autoantigenic component of the Th/To complex. The contribution of anti-Th/To and anti-Rpp25 antibodies to ANA positivity in patients with SSc remains unknown. METHODS: Sera from 873 patients with SSc were tested for ANA, and SSc-associated antibodies were measured. Samples without antibodies to extractable nuclear antigens (ENA; n = 53, ANA+/ENA-), were analyzed by immunoprecipitation (IP) and metabolically labeled proteins and for anti-Rpp25 antibodies (n = 50) by a chemiluminescent immunoassay (CLIA) and Rpp25 ELISA. RESULTS: Anti-Th/To antibodies occurred in 19/53 (36%), as determined by IP, and were the most common autoantibody in ANA+/ENA- SSc. Of those samples, 50/53 were available for additional testing by CLIA and ELISA. Anti-Rpp25 antibodies were detected in 12 (24% CLIA) or 10 (20% ELISA) of 50 patients. Receiver-operating characteristic curve analysis showed similar discrimination between Th/To IP-positive (n = 19) and -negative samples (n = 31) by CLIA and ELISA (area under the curve 0.90 vs 0.87; p = 0.6691). The positive percent agreement between IP and CLIA or ELISA was 12/19 (63.2%, 95% CI 38.4-83.7%) or 10/19 (52.6%, 95% CI 73.3-94.2%), respectively. Negative percent agreement was 100% for both assays. CONCLUSION: Autoantibodies to the Th/To autoantigen are important in patients with SSc who have been considered negative for SSc-specific or SSc-associated antibodies by widely available commercial assays. Rpp25 can be considered a major target of anti-Th/To antibodies. Assays detecting anti-Th/To and anti-Rpp25 antibodies may be important in SSc.
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Autoanticuerpos/análisis , Subunidades de Proteína/inmunología , Ribonucleasa P/inmunología , Esclerodermia Sistémica/inmunología , Adulto , Anciano , Homólogo de la Proteína Chromobox 5 , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
INTRODUCTION: Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico. METHODS: Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using 35S-methionine labeled K562 cell extract. Clinical information was obtained from medical records. RESULTS: DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy. CONCLUSIONS: Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.
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Autoanticuerpos/inmunología , Dermatomiositis/inmunología , Adulto , Autoanticuerpos/sangre , Autoantígenos/inmunología , Dermatomiositis/sangre , Femenino , Humanos , Inmunoprecipitación , Masculino , México/epidemiología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/inmunología , Persona de Mediana Edad , PrevalenciaRESUMEN
Like many other classical autoantibodies in systemic rheumatic diseases, anti-Su antibodies were originally defined by the double immunodiffusion assay in the early 80s. However, despite its high prevalence, only a few reports on anti-Su were published in the following years and the progress in characterizing the target antigens and clinical significance was slow, probably due to its inconsistent or poor reactivity in other standard immunoassays. In 2006 the target antigen was identified as the microRNA (miRNA)-binding protein Argonaute 2 (Ago2). Ago2 is a key component of the RNA-induced silencing complex enriched in cytoplasmic foci called GW bodies. Due to preferential reactivity of human autoantibodies with native antigens, immunoprecipitation is the only method to reliably detect anti-Su/Ago2 antibodies. Anti-Su/Ago2 does not appear to have disease specificity since it is found in 10-20% of patients with various rheumatic diseases, including systemic lupus erythematosus, scleroderma, polymyositis/dermatomyositis, and Sjögren's syndrome, as well as apparently healthy individuals at lower prevalence. The clinical significance and the mechanism of production of anti-Su/Ago2 remains to be clarified.
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Complejo Antígeno-Anticuerpo/genética , Proteínas Argonautas/inmunología , Autoanticuerpos/inmunología , MicroARNs/metabolismo , Proteínas/inmunología , Enfermedades Reumáticas/inmunología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Autoanticuerpos/genética , Autoanticuerpos/metabolismo , Autoinmunidad , Técnica del Anticuerpo Fluorescente , Humanos , Inmunodifusión , Ratones , MicroARNs/genética , MicroARNs/inmunología , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Interferencia de ARN/inmunología , Enfermedades Reumáticas/genética , Enfermedades Reumáticas/metabolismoRESUMEN
INTRODUCTION: Autoantibodies in patients with polymyositis/dermatomyositis (PM/DM) are associated with unique subsets, clinical course and outcome. Anti-MJ antibodies, which recognize the nuclear protein NXP-2/MORC3, are reported in ~25% of juvenile DM. Prevalence and clinical significance of anti-MJ antibodies in adult Italian PM/DM patients were studied. METHODS: Sera from 58 consecutive adult Italian PM/DM patients were analyzed by immunoprecipitation of 35S-labeled K562 cells extract, ELISA (anti-MJ, Jo-1), Western blot and indirect immunofluorescence. Clinical associations were analyzed using information from medical charts. RESULTS: Anti-MJ antibodies were the most prevalent specificity (17%) found mainly in DM (30%, 8 cases) vs 8% of PM (2 cases, P = 0.02). Comparing 10 anti-MJ (+) vs 48 anti-MJ (-) cases, DM was more common (P = 0.03), and age at onset was younger in anti-MJ (+) (P = 0.0006). In anti-MJ (+), heliotrope rash (P = 0.01) and calcinosis (P = 0.09) were more frequent. None of them had heart or lung involvement, or malignancy. Myopathy in anti-MJ (+) patients responded well to therapy and none of them had elevated CPK at last visit (0% vs 25% in anti-MJ (-)). Only 60% of anti-MJ (+) showed immunofluorescent nuclear dots staining, despite PML localization of NXP-2/MORC3. CONCLUSIONS: Anti-MJ antibodies are the most frequent specificity in our cohort of adult Italian PM/DM. Anti-MJ (+) were associated with young onset DM, calcinosis, no internal organ involvement and good response of myopathy to therapy. Anti-MJ reported in juvenile DM is also found in adult PM/DM, and could be a new useful biomarker.
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Adenosina Trifosfatasas/inmunología , Especificidad de Anticuerpos , Autoanticuerpos/biosíntesis , Proteínas de Unión al ADN/inmunología , Dermatomiositis/epidemiología , Dermatomiositis/inmunología , Adulto , Anciano , Biomarcadores/metabolismo , Estudios de Cohortes , Dermatomiositis/diagnóstico , Femenino , Humanos , Italia/epidemiología , Células K562 , Masculino , Persona de Mediana EdadRESUMEN
INTRODUCTION: Myositis specific autoantibodies are associated with unique clinical subsets and are useful biomarkers in polymyositis/dermatomyositis (PM/DM). A 120 kD protein recognized by certain patients with DM was identified and clinical features of patients with this specificity were characterized. METHODS: The 120 kD protein recognized by a prototype serum was purified and identified by mass spectrometry and immunological methods. Autoantibody to this 120 kD protein was screened in sera from 2,356 patients with various diagnoses from four countries, including 254 PM/DM, by immunoprecipitation of 35S-methionine labeled K562 cell extracts. Clinical information of patients with this specificity was collected. RESULTS: The 120 kD protein, which exactly comigrated with PL-12, was identified as transcription intermediary factor TIF1ß (TRIM28) by mass spectrometry and validated by immunoassays. By immunofluorescence, anti-TIF1ß positivity showed a fine-speckled nuclear staining pattern. Four cases of anti-TIF1ß were identified; all are women, one each in a Japanese, African American, Caucasian, and Mexican individual. Three had a diagnosis of DM and one case was classified as having an undifferentiated connective tissue disease with an elevated CPK but without significant muscle symptoms. This individual also had a history of colon cancer, cervical squamous metaplasia and fibroid tumors of the uterus. Myopathy was mild in all cases and resolved without treatment in one case. The anti-TIF1ß specificity was not found in other conditions. CONCLUSIONS: Anti-TIF1ß is a new DM autoantibody associated with a mild form of myopathy. Whether it has an association with malignancy, as in the case of anti-TIF1γ, or other unique features will need to be evaluated in future studies.
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Autoanticuerpos/biosíntesis , Dermatomiositis/diagnóstico , Dermatomiositis/inmunología , Proteínas Represoras/inmunología , Secuencia de Aminoácidos , Biomarcadores/sangre , Femenino , Humanos , Células K562 , Datos de Secuencia Molecular , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/inmunología , Sistema de Registros , Proteína 28 que Contiene Motivos TripartitoRESUMEN
OBJECTIVE: To estimate the prevalence, types, and sociodemographic and biobehavioral correlates of antinuclear antibodies (ANAs) in the US. METHODS: We conducted a cross-sectional analysis of 4,754 individuals from the National Health and Nutrition Examination Survey 1999-2004. ANAs were assessed by indirect immunofluorescence. In ANA-positive individuals, cellular staining patterns were determined, and specific autoantibody reactivities were assessed by immunoprecipitation. RESULTS: The ANA prevalence in the US population of individuals ages 12 years and older was 13.8% (95% confidence interval [95% CI] 12.2-15.5%). ANA prevalence increased with age (P=0.01), and ANAs were more prevalent among females than males (17.8% versus 9.6%; P<0.001), with the female-to-male ratio peaking at 40-49 years of age. ANA prevalence was modestly higher in African Americans compared with whites (age-adjusted prevalence odds ratio [POR] 1.30, 95% CI 1.00-1.70). Remarkably, ANAs were less common in overweight and obese individuals (age-adjusted POR 0.74) than in persons of normal weight. No significant associations of ANA with education, family income, alcohol use, smoking history, serum levels of cotinine, or C-reactive protein were observed. In ANA-positive individuals, nuclear patterns were seen in 84.6%, cytoplasmic patterns were seen in 21.8%, and nucleolar patterns were seen in 6.1%; the most common specific autoantibodies were anti-Ro (3.9%) and anti-Su (2.4%). CONCLUSION: These findings suggest that more than 32 million persons in the US have ANAs, and that the prevalence is higher among females, older individuals, African Americans, and those with a normal body weight. These data will serve as a useful baseline for future investigations of predictors and changes in ANA prevalence over time.
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Anticuerpos Antinucleares/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antinucleares/inmunología , Población Negra , Niño , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas Nutricionales , Prevalencia , Factores Sexuales , Estados Unidos/epidemiología , Población BlancaRESUMEN
INTRODUCTION: Anti-RNA polymerase III (RNAP III) antibodies are highly specific markers of scleroderma (systemic sclerosis, SSc) and associated with a rapidly progressing subset of SSc. The clinical presentation of anti-RNAP III positive patients, onset of Raynaud's phenomenon (RP) and SSc in unselected patients in a rheumatology clinic were evaluated. METHODS: Autoantibodies in sera from 1,966 unselected patients (including 434 systemic lupus erythematosus (SLE), 119 SSc, 85 polymyositis/dermatomyositis (PM/DM)) in a rheumatology clinic were screened by radioimmunoprecipitation. Anti-RNAP III positive sera were also tested by immunofluorescence antinuclear antibodies and anti-RNAP III ELISA. Medical records of anti-RNAP III positive patients were reviewed. RESULTS: Among 21 anti-RNAP III positive patients, 16 met the American College of Rheumatology (ACR) SSc criteria at the initial visit but 5 did not; diagnoses were vasculitis, early polyarthritis, renal failure with RP, interstitial lung disease, and Sjögren's syndrome. The first two patients developed rapidly progressive diffuse SSc. An additional case presented with diffuse scleroderma without RP and RP developed two years later. Anti-RNAP III antibodies in these 6 cases of atypical clinical presentation were compared with those in 15 cases of typical (SSc with RP) cases. Anti-RNAP III levels by ELISA were lower in the former group (P = 0.04 by Mann-Whitney test) and 3 of 6 were negative versus only 1 of 15 negative in the latter (P < 0.05 by Fisher's exact test). Three cases of non-SSc anti-RNAP III positive patients had predominant reactivity with RNAP I with weak RNAP III reactivity and had a strong nucleolar staining. Three anti-RNAP III patients, who did not have RP at the initial visit, developed RP months later. Scleroderma developed prior to RP in 5 out of 16 (31%) in the anti-RNAP III group, but this was rare in patients with other autoantibodies. The interval between the onset of RP to scleroderma was short in anti-RNAP III positive patients. CONCLUSIONS: Anti-RNAP III antibodies are highly specific for SSc; however, a subset of anti-RNAP III positive patients do not present as typical SSc. The interval between RP and scleroderma in this group is short, and 31% of patients developed scleroderma prior to RP in this group. Anti-RNAP III positive patients may not present as typical SSc and detecting anti-RNAP III may have predictive value.
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Autoanticuerpos/sangre , ARN Polimerasa III/inmunología , ARN Polimerasa I/inmunología , Enfermedad de Raynaud/inmunología , Esclerodermia Sistémica/inmunología , Adulto , Anciano , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Nucléolo Celular/inmunología , Nucléolo Celular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Persona de Mediana Edad , Ensayo de Radioinmunoprecipitación , Enfermedad de Raynaud/diagnóstico , Enfermedad de Raynaud/metabolismo , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/metabolismoRESUMEN
OBJECTIVE: Autoantibodies in the systemic rheumatic diseases are clinically useful biomarkers of the diagnosis or of certain clinical characteristics. An unusual pattern of immunoprecipitation, in which the D, E, F, and G proteins of small nuclear RNPs (snRNP) but without other components of the snRNP, was noticed at the autoantibody screening. The purpose of this study was to examine the target antigens and clinical manifestations associated with this specificity. METHODS: Autoantibodies in sera from 1,966 American patients (including 434 with systemic lupus erythematosus, 121 with scleroderma, 86 with polymyositis/dermatomyositis [PM/DM]) and 248 Italian patients with autoimmune diseases were screened by immunoprecipitation of (35) S-methionine-labeled cell extracts. Sera with which D, E, F, and G proteins of snRNP was immunoprecipitated, but without the other snRNP proteins, were further examined by analysis of RNA components by immunoprecipitation (silver staining), Western blotting using survival of motor neuron (SMN) complex, and immunofluorescence. RESULTS: Three sera that immunoprecipitated D, E, F, and G proteins without other components (U1-70K, A, B'/B, C) of the snRNP were found. Four additional proteins (130 kd, 120 kd, 38 kd, and 33 kd) were also commonly immunoprecipitated. The target antigen was identified as SMN complex (Gemin 3, Gemin 4, SMN, and Gemin 2, respectively), which plays a critical role in the assembly of snRNP. In immunofluorescence analyses, all 3 sera showed nuclear dots (Cajal bodies) and cytoplasmic staining. Only 1 serum was weakly positive on Western blotting of SMN, suggesting that these sera mainly recognize native molecule or quaternary structure. All 3 patients were white women with PM, an interesting finding, since deletion or mutation of SMN is known to cause spinal muscular atrophy. CONCLUSION: SMN complex was identified as a new Cajal body autoantigen recognized by sera from white patients with PM. The biologic and clinical significance of anti-SMN autoantibodies will need to be clarified.
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Autoanticuerpos/inmunología , Polimiositis/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Proteínas del Complejo SMN/inmunología , Adulto , Enfermedades Autoinmunes/inmunología , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/inmunologíaRESUMEN
OBJECTIVES: Primary anti-phospholipid syndrome (PAPS) is an autoimmune condition defined by anti-phospholipid antibodies (aPL) and thrombotic or obstetric events. Some PAPS can evolve into systemic lupus erythematosus (SLE) during follow-up. Few studies systematically examined lupus autoantibodies and their clinical significance in PAPS. The aim of our study is to analyze the clinical and laboratory correlations with lupus-related autoantibodies, detected by immunoprecipitation (IP), a technique not yet systematically applied to investigate autoantibodies in this condition. METHODS: Sera from 52 PAPS patients were screened by indirect immunofluorescence (IIF) antinuclear antibodies (ANA), IP of ³5S-labeled K562 cell extract, and ELISA [anti-Argonaute2 (Ago2, Su), 60kRo, 52kRo, La, dsDNA)]. Anti-Ago2/Su positive sera were also tested for anti-GW bodies (GWBs) by IIF double staining, using rabbit anti-Rck/p54 serum. RESULTS: First, 56% of PAPS patients (29/52) were ANA positive, mainly with speckled pattern. Anti-Ago2/Su antibodies were found in 13% (7/52), anti-Ro/SSA in 10% (5/52), anti-La in one case. The clinical profile of patients did not seem to be related to the presence of these antibody specificities. However, levels of IgG anti-ß2 glycoprotein I antibodies were lower in anti-Ago2/Su positive patients (p = 0.02). None of anti-Ago2/Su or -Ro patients developed SLE during a 2-year follow-up. Ago2 is a key component of GWBs, however, only 1/7 anti-Ago2/Su serum showed a typical cytoplasmic GWBs staining. CONCLUSIONS: Anti-Ago2/Su and -Ro antibodies are the two autoantibodies detected by IP in our PAPS cohort. Clarifying why Ago2/Su and Ro are specific targets of autoimmunity may help to understand the mechanisms of autoantibody production.
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Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Factor 2 Eucariótico de Iniciación/inmunología , Inmunoprecipitación/métodos , Ribonucleoproteínas/inmunología , Adulto , Animales , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Especificidad de Anticuerpos , Síndrome Antifosfolípido/diagnóstico , Proteínas Argonautas , Autoanticuerpos/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Persona de Mediana Edad , Conejos , Adulto JovenRESUMEN
BACKGROUND: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. METHODOLOGY/PRINCIPAL FINDINGS: Distinct cytoplasmic rods (â¼3-10 µm in length) and rings (â¼2-5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin. CONCLUSIONS/SIGNIFICANCE: RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.