RESUMO
OBJECTIVE: Higher self-reported disability (high Health Assessment Questionnaire [HAQ] score) has been associated with hospitalizations and mortality in established rheumatoid arthritis (RA), but associations in early RA are unknown. METHODS: Patients with early RA (symptom duration <1 year) enrolled in the Canadian Early Arthritis Cohort who initiated disease-modifying antirheumatic drugs and had completed HAQ data at baseline and 1 year were included in the study. Discrete-time proportional hazards models were used to estimate crude and multi-adjusted associations of baseline HAQ and HAQ at 1 year with all-cause mortality in each year of follow-up. RESULTS: A total of 1,724 patients with early RA were included. The mean age was 55 years, and 72% were women. Over 10 years, 62 deaths (3.6%) were recorded. Deceased patients had higher HAQ scores at baseline (mean ± SD 1.2 ± 0.7) and at 1 year (0.9 ± 0.7) than living patients (1.0 ± 0.7 and 0.5 ± 0.6, respectively; P < 0.001). Disease Activity Score in 28 joints (DAS28) was higher in deceased versus living patients at baseline (mean ± SD 5.4 ± 1.3 versus 4.9 ± 1.4) and at 1 year (mean ± SD 3.6 ± 1.4 versus 2.8 ± 1.4) (P < 0.001). Older age, male sex, lower education level, smoking, more comorbidities, higher baseline DAS28, and glucocorticoid use were associated with mortality. Contrary to HAQ score at baseline, the association between all-cause mortality and HAQ score at 1 year remained significant even after adjustment for confounders. For baseline HAQ score, the unadjusted hazard ratio (HR) was 1.46 (95% confidence interval [95% CI] 1.02-2.09), and the adjusted HR was 1.25 (95% CI 0.81-1.94). For HAQ score at 1 year, the unadjusted HR was 2.58 (95% CI 1.78-3.72), and the adjusted HR was 1.75 (95% CI 1.10-2.77). CONCLUSION: Our findings indicate that higher HAQ score and DAS28 at 1 year are significantly associated with all-cause mortality in a large early RA cohort.
Assuntos
Artrite Reumatoide/fisiopatologia , Estado Funcional , Mortalidade , Autorrelato , Atividades Cotidianas , Adulto , Fatores Etários , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Canadá , Causas de Morte , Escolaridade , Feminino , Glucocorticoides/uso terapêutico , Humanos , Canadenses Indígenas , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Fatores Sexuais , Fumar/epidemiologia , Inquéritos e Questionários , População BrancaRESUMO
Patients with rheumatoid arthritis (RA) have very different outcomes, particularly with regard to bone erosions. Since osteoclasts are responsible for bone destruction adjacent to rheumatoid synovium, profiling osteoclasts from circulating precursors in RA could help identify patients at risk for bone destruction. In this study, we sought to determine whether the functional characteristics of osteoclasts generated from their blood precursors were modified by RA activity or were intrinsic to osteoclasts and associated with the RA phenotype (erosive or not). Osteoclasts were generated in vitro from peripheral blood mononuclear cells (PBMCs) of subjects with RA (n = 140), as well as sex- and age-matched healthy controls (n = 101). Osteoclastic parameters were analyzed at baseline and during the follow-up for up to 4 years, with regular assessment of RA activity, bone erosions, and bone mineral density (BMD). As a validation cohort, we examined RA patients from the Early Undifferentiated PolyArthritis (EUPA) study (n = 163). The proportion of CD14+ PBMC was higher in RA than in control subjects, but inversely correlated with the 28-joint disease activity score (DAS28). Also surprisingly, in osteoclast cultures from PBMCs, active RA was associated with lower osteoclastogenic capacity, while in vitro bone resorption per osteoclast and resistance to apoptosis were similar in both active and quiescent RA. In a small subgroup analysis, osteoclasts from subjects with recent RA that had progressed at four years to an erosive RA exhibited at baseline greater resistance to apoptosis than those from patients remaining non-erosive. Our findings establish that when RA is active, circulating monocytes have a reduced potential to generate osteoclasts from PBMCs in vitro. In addition, osteoclasts associated with erosive disease had resistance to apoptosis from the start of RA.
RESUMO
BACKGROUND: This multicenter incident cohort aimed to characterize how often early rheumatoid arthritis (ERA) patients self-report episodic joint inflammation (palindromic rheumatism) preceding ERA diagnosis and which characteristics differentiate these patients from those without prior episodic symptoms. METHODS: Data were from patients with early confirmed or suspected RA (more than 6 weeks and less than 12 months) enrolled in the Canadian Early ArThritis CoHort (CATCH) between April 2017 to March 2018 who completed study case report forms assessing joint pain and swelling prior to ERA diagnosis. Chi-square and t tests were used to compare characteristics of patients with and without self-reported episodic joint inflammation prior to ERA diagnosis. Multivariable logistic regression was used to identify sociodemographic and clinical measures associated with past episodic joint inflammation around the time of ERA diagnosis. RESULTS: A total of 154 ERA patients were included; 66% were female, and mean (SD) age and RA symptom duration were 54 (15) years and 141 (118) days. Sixty-five (42%) ERA patients reported a history of episodic joint pain and swelling, half of whom reported that these symptoms preceded ERA diagnosis by over 6 months. ERA patients with past episodic joint inflammation were more often female, had higher income, were seropositive, had more comorbidities, fewer swollen joints, and lower Clinical Disease Activity Index (CDAI) around the time of ERA diagnosis (P < 0.05). These associations remained significant in multivariable regression adjusting for other sociodemographic and RA clinical measures. CONCLUSION: Almost half of ERA patients experienced episodic joint inflammation prior to ERA diagnosis. These patients were more often female, had higher income, and presented with milder disease activity at ERA diagnosis.
RESUMO
Objective: Metabolic syndrome (MetS) prevalence in early rheumatoid arthritis (ERA) is conflicting. The impact of sex, including menopause, has not been described. We estimated the prevalence and factors associated with MetS in men and women with ERA. Methods: A cross-sectional study of the Canadian Early Arthritis Cohort (CATCH) was performed. Participants with baseline data to estimate key MetS components were included. Sex-stratified logistic regression identified baseline variables associated with MetS. Results: The sample included 1543 participants; 71% were female and the mean age was 54 (SD 15) years. MetS prevalence was higher in men 188 (42%) than women 288 (26%, P < 0.0001) and increased with age. Frequent MetS components in men were hypertension (62%), impaired glucose tolerance (IGT, 40%), obesity (36%), and low high-density lipoprotein cholesterol (36%). Postmenopausal women had greater frequency of hypertension (65%), IGT (32%), and high triglycerides (21%) compared with premenopausal women (P < 0.001). In multivariate analysis, MetS was negatively associated with seropositivity and pulmonary disease in men. Increasing age was associated with MetS in women. In postmenopausal women, corticosteroid use was associated with MetS. Psychiatric comorbidity was associated with MetS in premenopausal women. MetS status was not explained by disease activity or core RA measures. Conclusion: The characteristics and associations of MetS differed in men and women with ERA. Sex differences, including postmenopausal status, should be considered in comorbidity screening. With this knowledge, the interplay of MetS, sex, and RA therapeutic response on cardiovascular outcomes should be investigated.
RESUMO
Interventions targeting patients with recent fragility fracture and their physician were most successful at initiating osteoporosis treatment during the first 12 months. This window of opportunity had already closed after 1 year. The reasons for declining or accepting the intensive intervention were explored in patients still untreated at 12 months. INTRODUCTION: A fragility fracture (FF) event identifies patients most likely to benefit from osteoporosis treatment. Nonetheless, most FF patients go untreated. Our objective was to determine how long an incident FF remains a strong incentive to initiate osteoporosis treatment. METHODS: A total of 1086 men and women over age 50 with a recent FF event were assigned to either standard care (SC), to minimal (MIN), or intensive (INT) interventions targeting patients and their family physician to initiate osteoporosis treatment. Inpatients with FF (mainly hip) evaluated by rheumatologists were also included in a specialized group (SPE; n = 324). At 1 year, untreated patients in both the SC and the MIN groups were offered an INT intervention. The cohort was followed through 48 months. A qualitative analysis of patient-centered decision-making associated with initiation of treatment was conducted. RESULTS: In MIN and INT groups, osteoporosis treatment was initiated in 41.0 and 54.3% of untreated patients by 12 months, respectively, compared to 68.4% in SPE and 18.9% in SC groups; initiation rates drastically dropped thereafter. Over 4863 patient-years of follow-up, the rates of new FF were 3.4 per 100 patient-years, without significant differences between patients with initial major or minor FF, nor between control or intervention groups. Failure by patients and physicians to recognize FF as a sign of underlying bone disease contributed the most to lack of treatment. CONCLUSION: While incident FFs are an ideal opportunity for starting osteoporosis treatment, 1 year later, the therapeutic window of opportunity has already closed.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Osteoporose/tratamento farmacológico , Fraturas por Osteoporose/prevenção & controle , Idoso , Canadá , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Adesão à Medicação/estatística & dados numéricos , Pessoa de Meia-Idade , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Assistência Centrada no Paciente/organização & administração , Padrões de Prática Médica/estatística & dados numéricos , Medição de Risco/métodos , Fatores de TempoRESUMO
While fragility fractures (FFs) are one of the strongest predictors of subsequent osteoporotic fractures, it remains unclear whether low-trauma ankle fractures have this ability. The aim of the study was to identify the characteristics of patients with low-trauma ankle FFs who develop subsequent FFs. The OPTIMUS initiative is a strategy to improve osteoporosis treatment post-FF in orthopedic clinics. FRAX scores without BMD (FRAX-BMI) were calculated at time of inclusion. Recurrent FFs were recorded over a 48-month follow-up. All FFs were X-ray-confirmed. A total of 265 patients with initial ankle FF were included (190 women; mean age 62.8 ± 9.6 years), of whom 15 developed new FFs. Patients with ankle FFs had longer time until recurrence and lower 2-year incidence of recurrent FFs (3.2%) compared with those having wrist FFs (9.0%) or other initial FFs (9.6%), and 4-year incidence rates of 6.2, 13.4, and 15.3%, respectively (log-rank test, p = 0.001). With an ankle FF at inclusion, recurrent FFs were more frequent in patients with previous FF (6.2 per 100 patient-years; p < 0.01) or high-risk FRAX-BMI scores pre- or post-FF (2.4 or 2.0 per 100 patient-years, respectively; ns), compared to patients without any of these conditions (0.7 per 100 patient-years). Ankle FFs represent a clinical opportunity for identifying at-risk patients who should be targeted for treatment (previous FFs and/or high-risk FRAX). Because of mechanical factors and other contributors involved, ankle FFs themselves do not predict subsequent FFs overall, and their inclusion in FRAX-BMI risk calculation may thus not be warranted.
Assuntos
Fraturas do Tornozelo/epidemiologia , Fraturas por Osteoporose/epidemiologia , Idoso , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de RiscoRESUMO
OBJECTIVE: Simplified measures to quantify rheumatoid arthritis (RA) disease activity are increasingly used. The minimum clinically important differences (MCID) for some measures, such as the Clinical Disease Activity Index (CDAI), have not been well-defined in real-world clinic settings, especially for early RA patients with low/moderate disease activity. METHODS: Data from Canadian Early Arthritis Cohort patients were used to examine absolute change in CDAI in the first year after enrollment, stratified by disease activity. MCID cut points were derived to optimize the sum of sensitivity and specificity versus the gold standard of patient self-reported improvement or worsening. Sensitivity, specificity, positive predictive values, and negative predictive values were calculated against patient self-reported improvement (gold standard) and for change in pain, Health Assessment Questionnaire (HAQ), and Disease Activity Score in 28 joints (DAS28) improvement. Discrimination was examined using the area under receiver operator curves. Similar methods were used to evaluate MCIDs for worsening for patients who achieved low disease activity. RESULTS: A total of 578 patients (mean ± SD age 54.1 ± 15.3 years, 75% women, median [interquartile range] disease duration 5.3 [3.3, 8.0] months) contributed 1,169 visit pairs to the improvement analysis. The MCID cut points for improvement were 12 (patients starting in high disease activity: CDAI >22), 6 (moderate: CDAI 10-22), and 1 (low disease activity: CDAI <10). Performance characteristics were acceptable using these cut points for pain, HAQ, and DAS28. The MCID for CDAI worsening among patients who achieved low disease activity was 2 units. CONCLUSION: These minimum important absolute differences in CDAI can be used to evaluate improvement and worsening and increase the utility of CDAI in clinical practice.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/fisiopatologia , Progressão da Doença , Amplitude de Movimento Articular/fisiologia , Adulto , Fatores Etários , Idoso , Artrite Reumatoide/epidemiologia , Canadá , Estudos de Coortes , Bases de Dados Factuais , Avaliação da Deficiência , Diagnóstico Precoce , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Exame Físico/métodos , Prognóstico , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Autorrelato , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Fatores Sexuais , Perfil de Impacto da Doença , Resultado do TratamentoRESUMO
OBJECTIVE: To investigate the relationship between function and disease activity in early inflammatory arthritis (EIA). METHODS: Canadian Early Arthritis Cohort (CATCH) (n=1143) is a multi-site EIA cohort. Correlations between the Health Assessment Questionnaire Disability Index (HAQ) and DAS28 were done at every 3 months for the first year and then at 18 and 24 months. We also investigated the relationship between HAQ and DAS28 by age (<65 versus ≥65) and RF (positive vs negative). RESULTS: Mean HAQ and DAS28 scores were highest at the initial visit with HAQ decreasing over 24 months from a baseline of 0.94 to 0.40 and DAS28 scores decreasing from 4.54 to 2.29. All correlations between HAQ and DAS28 were significant at all time points (p<0.01). The correlations between HAQ and DAS28 were variable over time. The strongest correlation between HAQ and DAS28 occurred at initial visit (most DMARD naive) (n=1,143) and 18 months (r=0.57, n=321) and 24 months (r=0.59, n=214). The baseline correlation between HAQ and DAS28 was significantly different than correlations obtained at 3, 6, and 12 months (p=0.02, 0.01, and 0.01, respectively). Age did not change the association between HAQ and DAS28 {<65 years old (r=0.50, n=868) versus ≥65 (r=0.48, n=254), p=0.49}. The correlation between HAQ and DAS28 was stronger with RF+ patients (r=0.63, n=636) vs RF negative (r=0.47, n=477), p=0.0043. CONCLUSION: Over 2 years in EIA, HAQ and DAS both improved; correlations at time points were different over 2 years and RF status affected the correlations.
RESUMO
Increases in local and systemic bone resorption are hallmarks of rheumatoid arthritis (RA). Osteoclasts are implicated in these processes and their enhanced differentiation may contribute to bone destruction. We observed that in vitro osteoclastogenesis varies among healthy individuals and hypothesized that increased osteoclastogenesis could be a marker for the presence of RA. Our objective in the present study was to determine if in vitro osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) was different in patients with RA compared to healthy controls and osteoarthritis (OA) patients. Expression of CD14 in PBMCs was quantified and PBMCs were incubated for 21 days in the presence of the osteoclastogenic cytokines M-CSF and RANKL. Differentiation on cortical bone slices permitted the analysis of bone resorption while apoptotic potential was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. In vitro osteoclastogenesis was higher in PBMCs from RA patients compared to controls, and a similar increase was observed in the percentage of osteoclast precursors in RA patients. Osteoclasts from RA patients showed lower apoptotic rates than osteoclasts from healthy controls. No difference was observed in bone resorption activity between RA patients and controls. Interestingly, the difference in osteoclast number and apoptosis rate allowed the implementation of an algorithm capable of distinguishing patients with RA from controls. In conclusion, our study shows that osteoclast differentiation from PBMCs is enhanced in patients with RA, and this difference can be explained by both a higher percentage of osteoclast precursors in the blood and by the reduced apoptotic potential of mature osteoclasts.
Assuntos
Apoptose , Artrite Reumatoide/patologia , Diferenciação Celular , Osteoclastos/patologia , Osteogênese , Células-Tronco/patologia , Adulto , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Movimento Celular , Estudos de Coortes , Demografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Análise Multivariada , Osteoartrite/patologia , Estudos ProspectivosRESUMO
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Lúpus Eritematoso Sistêmico/genética , Doenças Autoimunes/genética , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Ro ribonucleoproteins (RNPs) are autoantigens that result from the association of a 60-kDa protein (Ro60) with a small RNA (hY1, hY3, hY4 or hY5 in humans, mY1 or mY3 in mice). Previous studies localized Ro RNPs to the cytoplasm. Because Ro RNPs containing hY5 RNA (Ro(hY5) RNPs) have unique biochemical and immunological properties, their intracellular localization was reassessed. Subcellular distribution of mouse and human Ro RNPs in intact and hY-RNA transfected cells was assessed by immunoprecipitation and Northern hybridization. Human Ro(hY1--4) RNPs as well as murine Ro(mY1, mY3) RNPs are exclusively cytoplasmic. Ro RNPs containing an intact hY5 RNA, but not those containing a mutated form of hY5 RNA, are found in the nuclear fractions of human and mouse cells. Ro(hY5) RNPs are stably associated with transcriptionally active La protein and are known to associate with RoBPI, a nuclear autoantigen. Our results demonstrate that Ro(hY5) RNPs are specifically present in the nucleus of cultured human and murine cells. The signal for nuclear localization of Ro(hY5) RNPs appears to reside within the hY5 sequence itself. In conclusion, we suggest that the unique localization and interactions of primate-specific Ro(hY5) RNPs reflect functions that are distinct from the predicted cytoplasmic function(s) of more conserved Ro RNPs.
Assuntos
Autoantígenos/metabolismo , Heterogeneidade Genética , RNA Citoplasmático Pequeno , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Células 3T3 , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expressão Gênica , Células HeLa , Humanos , Camundongos , Testes de PrecipitinaRESUMO
Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.
Assuntos
Proteínas de Transporte/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/metabolismo , Extratos Celulares , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Ligação Proteica , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Coelhos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras , Ribonucleoproteínas/metabolismo , LevedurasRESUMO
We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.
Assuntos
Técnicas de Sonda Molecular , Ribonucleoproteínas , Saccharomyces cerevisiae/genética , Sítios de Ligação , DNA Complementar/análise , Escherichia coli/genética , Biblioteca Gênica , Células HeLa , Humanos , Plasmídeos/genética , RNA/metabolismo , Proteínas Recombinantes , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ro and La RNP complexes were reassembled from in vitro labelled hY5 RNA and HeLa cell extracts. These complexes were then visualized through retardation of migration of labelled hY5 RNA in non-denaturing polyacrylamide gels. Three major complexes (named A, B, and C) were formed when crude cellular extracts (S100 fraction) were used. Using monospecific anti-60-kD Ro (Ro60) and anti-La antibodies to retard RNPs containing these antigens during migration in the gels, the three major complexes were shown to contain Ro60 (C), La (B), or both proteins (A). The specificity of RNA-protein interactions in the reassembled complexes was further demonstrated using two 3'-shortened hY5 RNA transcripts lacking the La-binding site (hY5-Alu I RNA) and both the Ro60 and La-binding sites (hY5-Hha I RNA). hY5-Hha I RNA still formed a single, minor complex when incubated with S100 extract, suggesting interaction with a yet undefined protein. In addition, we used the capacity of specific antibodies to retard the migration of the reassembled complexes to design a detection assay for anti-Ro and anti-La autoantibodies. Using 84 human sera, our assay was shown to approximate the specificity and sensitivity of an immunoprecipitation assay where 32P-labelled cell extracts are used as source of antigens. Our assay may be used to detect low levels of antibodies to conformational determinants on Ro60 and La proteins in human sera and antibody preparations.
Assuntos
Autoantígenos/metabolismo , RNA Citoplasmático Pequeno , Ribonucleoproteínas/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Microesferas , Ligação Proteica/genética , RNA/análise , Antígeno SS-BAssuntos
Autoantígenos/imunologia , Biomarcadores/análise , Lúpus Eritematoso Sistêmico/imunologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas/imunologia , Doenças Autoimunes/etiologia , Humanos , Sistema Imunitário/fisiopatologia , Lúpus Eritematoso Sistêmico/etiologia , Sensibilidade e EspecificidadeRESUMO
We recently reported the identification in human anti-Ro serum of Abs specifically immunoprecipitating deproteinized hY5 RNA. In the present report, we characterized the epitopes recognized by anti-hY5 RNA Abs. Using deletion and site-directed mutagenesis of hY5 cDNA and in vitro transcribed RNAs with intact and 3'-shortened ends, we have defined two conformational antigenic determinants distinct from the regions known to bind Ro and La proteins. One of these epitopes (epitope A) is present in the middle portion of hY5 RNA and is dependent on the presence of a four-nucleotide sequence (AACC at position 58-61) that may form a single-stranded loop. Deleting these four nucleotides or modifying the stem structures proximal or distal to this loop abolishes recognition of the mutated RNAs by Abs. The second epitope (epitope B) requires the presence of another four-nucleotide sequence (CUUG at position 74-77) in between the Ro and La binding sites. Deleting this CUUG sequence or modifying nucleotides on the 5' side of the stem structure below the Ro60 binding site severely compromises the interaction with Abs. Since Abs to deproteinized hY RNAs are restricted to anti-hY5 RNA and target determinants not involved in interactions with known hY5 RNA-binding proteins, human RohY5 particles may play a direct role in the immunization process, leading to the production of anti-hY5 RNA autoantibodies.
Assuntos
Autoantígenos/imunologia , Epitopos Imunodominantes/imunologia , RNA Citoplasmático Pequeno , RNA/imunologia , Ribonucleoproteínas/imunologia , Especificidade de Anticorpos , Autoanticorpos/imunologia , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Humanos , Epitopos Imunodominantes/química , Dados de Sequência Molecular , RNA/química , Antígeno SS-BRESUMO
Human T-cell leukemia virus type I (HTLV-I) is associated with a large spectrum of clinical manifestation including adult T-cell leukemia (ATL) and tropical spastic paraparesis or HTLV-I-associated myelopathy (TSP/HAM). In most cases, however, infected patients remain asymptomatic. The participation of the immune system in the pathogenesis of TSP/HAM has been suggested. In this study the IgG antibody response of HTLV-I-infected individuals has been investigated using both ELISA with a panel of nuclear and cytoplasmic proteins and peptides known to be recognized by antibodies from patients with various systemic autoimmune diseases, and immunoprecipitation of ribonucleoproteins from HeLa cell extracts. The results were compared with the reactivity of sera from individuals with non-HTLV-I-related neurological diseases and healthy blood donors. Raised levels of autoantibodies reacting with several nuclear and cytoplasmic antigens were found in TSP/HAM and ATL patients. In asymptomatic HTLV-I-seropositive individuals, both the prevalence and level of IgG antibodies were lower and directed only against a restricted set of antigens. The mechanism of induction of these antibodies still remains obscure. However, the results show that a significant autoimmune response exists in these patients and it may contribute to the pathogenesis of the disease.