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OBJECTIVE: To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. METHODS: Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. RESULTS: Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. CONCLUSION: We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.
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Autoanticuerpos , Lupus Eritematoso Sistémico , Humanos , Proteínas Ribosómicas , Complejo de la Endopetidasa Proteasomal , Proteínas Argonautas , Lupus Eritematoso Sistémico/diagnóstico , Autoantígenos , Inmunoglobulina G , Inmunoglobulina ARESUMEN
BACKGROUND: Patients with systemic lupus erythematosus (SLE) have autoantibodies against the L1-encoded open-reading frame 1 protein (ORF1p). Here, we report (i) which immune cells ORF1p emanates from, (ii) which L1 loci are transcriptionally active, (iii) whether the cells express L1-dependent interferon and interferon-stimulated genes, and (iv) the effect of inhibition of L1 ORF2p by reverse transcriptase inhibitors. RESULTS: L1 ORF1p was detected by flow cytometry primarily in SLE CD66b+CD15+ regular and low-density granulocytes, but much less in other immune cell lineages. The amount of ORF1p was higher in neutrophils from patients with SLE disease activity index (SLEDAI) > 6 (p = 0.011) compared to patients with inactive disease, SLEDAI < 4. Patient neutrophils transcribed seven to twelve human-specific L1 loci (L1Hs), but only 3 that are full-length and with an intact ORF1. Besides serving as a source of detectable ORF1p, the most abundant transcript encoded a truncated ORF2p reverse transcriptase predicted to remain cytosolic, while the two other encoded an intact full-length ORF2p. A number of genes encoding proteins that influence L1 transcription positively or negatively were altered in patients, particularly those with active disease, compared to healthy controls. Components of nucleic acid sensing and interferon induction were also altered. SLE neutrophils also expressed type I interferon-inducible genes and interferon ß, which were substantially reduced after treatment of the cells with drugs known to inhibit ORF2p reverse transcriptase activity. CONCLUSIONS: We identified L1Hs loci that are transcriptionally active in SLE neutrophils, and a reduction in the epigenetic silencing mechanisms that normally counteract L1 transcription. SLE neutrophils contained L1-encoded ORF1p protein, as well as activation of the type I interferon system, which was inhibited by treatment with reverse transcriptase inhibitors. Our findings will enable a deeper analysis of L1 dysregulation and its potential role in SLE pathogenesis.
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OBJECTIVE: Autoantibodies against proteins encoded by human endogenous retrovirus K (HERV-K) have been reported in patients with rheumatoid arthritis (RA), but their relevance, if any, has remained unresolved. We revisited this question and tested if such autoantibodies may react with citrullinated epitopes on the envelope (Env) protein of HERV-K. METHODS: Immunoblotting and ELISAs were conducted with unmodified Env protein and with Env citrullinated by protein arginine deiminase 4 (PAD4). Sera from 100 patients with RA, plasma from 32 patients with juvenile idiopathic arthritis (JIA), and healthy adult and pediatric controls were included. Antibody reactivity was evaluated for correlations with clinical and laboratory variables of the patients. RESULTS: We replicated and expanded upon published data suggesting that patients with RA or JIA have autoantibodies against HERV-K Env, some with high titers. Anti-HERV-K antibodies correlated with cigarette smoking and with circulating myeloperoxidase-DNA complexes indicative of nonapoptotic neutrophil cell death. Further, most of the patients with RA, but not those with JIA, had autoantibodies that reacted more strongly with Env that was citrullinated by PAD4. These anticitrullinated Env autoantibodies correlated with seropositivity and tended to be higher in patients with erosive disease. CONCLUSION: Our data suggest that anti-HERV-K immunity is elevated in RA and JIA and may have a connection with pathogenic protein citrullination in RA.
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Artritis Reumatoide , Retrovirus Endógenos , Autoanticuerpos , Niño , Productos del Gen env , Humanos , Arginina Deiminasa Proteína-Tipo 4RESUMEN
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. While its etiology remains elusive, current understanding suggests a multifactorial process with contributions by genetic, immunologic, hormonal, and environmental factors. A hypothesis that combines several of these factors proposes that genomic elements, the L1 retrotransposons, are instrumental in SLE pathogenesis. L1 retroelements are transcriptionally activated in SLE and produce two proteins, ORF1p and ORF2p, which are immunogenic and can drive type I interferon (IFN) production by producing DNA species that activate cytosolic DNA sensors. In addition, these two proteins reside in RNA-rich macromolecular assemblies that also contain well-known SLE autoantigens like Ro60. We surmise that cells expressing L1 will exhibit all the hallmarks of cells infected by a virus, resulting in a cellular and humoral immune response similar to those in chronic viral infections. However, unlike exogenous viruses, L1 retroelements cannot be eliminated from the host genome. Hence, dysregulated L1 will cause a chronic, but perhaps episodic, challenge for the immune system. The clinical and immunological features of SLE can be at least partly explained by this model. Here we review the support for, and the gaps in, this hypothesis of SLE and its potential for new diagnostic, prognostic, and therapeutic options in SLE.
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BACKGROUND: Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells. METHODS: Autoantibodies in the plasma of pSLE patients (n = 30), healthy children (n = 37), and disease controls juvenile idiopathic arthritis (JIA) (n = 32) and juvenile dermatomyositis (JDM) (n = 60), were measured by ELISA. Expression of p40 in immune cells was assessed by flow cytometry. Markers of neutrophil activation and death were quantitated by ELISA. RESULTS: IgG and IgA autoantibodies reactive with p40 were detected in the pSLE patients, but were low in healthy controls and in JIA or JDM. pSLE patients with active disease (13 of them newly diagnosed) had higher titers than the same patients after effective therapy (p = 0.0003). IgG titers correlated with SLEDAI (r = 0.65, p = 0.0001), ESR (r = 0.43, p = 0.02), and anti-dsDNA antibodies (r = 0.49, p < 0.03), and inversely with complement C3 (r = -0.55, p = 0.002) and C4 (r = -0.51, p = 0.006). p40 protein was detected in a subpopulation of CD66b+ granulocytes in pSLE, as well as in adult SLE patients. Myeloperoxidase and neutrophil elastase complexed with DNA and the neutrophil-derived S100A8/A9 were elevated in plasma from pSLE patients with active disease and correlated with anti-p40 autoantibodies and disease activity. CONCLUSIONS: Children with active SLE have elevated IgG and IgA autoantibodies against L1 p40, and this protein can be detected in circulating granulocytes in both pediatric and adult SLE patients. P40 expression and autoantibody levels correlate with disease activity. Markers of neutrophil activation and death also correlate with these autoantibodies and with disease activity, suggesting that neutrophils express L1 and are a source of p40.
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Lupus Eritematoso Sistémico , Adulto , Autoanticuerpos , Niño , Humanos , Inmunoglobulina A , Inmunoglobulina G , NeutrófilosRESUMEN
More than 200 human disorders include various manifestations of autoimmunity. The molecular events that lead to these diseases are still incompletely understood and their causes remain largely unknown. Numerous potential triggers of autoimmunity have been proposed over the years, but very few of them have been conclusively confirmed or firmly refuted. Viruses have topped the lists of suspects for decades, and it seems that many viruses, including those of the Herpesviridae family, indeed can influence disease initiation and/or promote exacerbations by a number of mechanisms that include prolonged anti-viral immunity, immune subverting factors, and mechanisms, and perhaps "molecular mimicry". However, no specific virus has yet been established as being truly causative. Here, we discuss a different, but perhaps mechanistically related possibility, namely that retrotransposons or retroviruses that infected us in the past and left a lasting copy of themselves in our genome still can provoke an escalating immune response that leads to autoimmune disease. Many of these loci still encode for retroviral proteins that have retained some, or all, of their original functions. Importantly, these endogenous proviruses cannot be eliminated by the immune system the way it can eliminate exogenous viruses. Hence, if not properly controlled, they may drive a frustrated and escalating chronic, or episodic, immune response to the point of a frank autoimmune disorder. Here, we discuss the evidence and the proposed mechanisms, and assess the therapeutic options that emerge from the current understanding of this field.
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Autoinmunidad/genética , Susceptibilidad a Enfermedades , Retrovirus Endógenos , Retroelementos , Enfermedades Reumáticas/etiología , Elementos Alu , Animales , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/metabolismo , Genoma Humano , Humanos , Inmunidad , Elementos de Nucleótido Esparcido Largo , Provirus/genética , Enfermedades Reumáticas/diagnóstico , Enfermedades Reumáticas/metabolismoRESUMEN
OBJECTIVE: Long interspersed nuclear element 1 (LINE-1) encodes 2 proteins, the RNA binding protein p40 and endonuclease and reverse transcriptase (open-reading frame 2p [ORF2p]), which are both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with LINE-1 RNA and additional RNA binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether SLE patients also produce autoantibodies against LINE-1 p40. METHODS: Highly purified p40 protein was used to quantitate IgG autoantibodies in serum from 172 SLE patients and from disease controls and age-matched healthy subjects by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. RESULTS: Antibodies reactive with p40 were detected in the majority of patients and many healthy controls. Their levels were higher in patients with SLE, but not those with systemic sclerosis, compared to healthy subjects (P = 0.01). Anti-p40 reactivity was higher in patients during a flare than in patients with disease in remission (P = 0.03); correlated with the SLE Disease Activity Index score (P = 0.0002), type I interferon score (P = 0.006), decrease in complement C3 level (P = 0.0001), the presence of anti-DNA antibodies (P < 0.0001) and anti-C1q antibodies (P = 0.004), and current or past history of nephritis (P = 0.02 and P = 0.003, respectively); and correlated inversely with age (r = -0.49, P < 0.0001). SLE patient sera also reacted with p40-associated proteins. CONCLUSION: Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step toward SLE pathogenesis.
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Autoanticuerpos/inmunología , Proteínas de Unión al ADN/inmunología , Lupus Eritematoso Sistémico/inmunología , Factores de Edad , Anticuerpos Antinucleares/inmunología , Estudios de Casos y Controles , Complemento C1q/inmunología , Complemento C3/inmunología , Humanos , Elementos de Nucleótido Esparcido Largo/inmunología , Nefritis Lúpica/inmunología , Esclerodermia Sistémica/inmunología , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVES: Our aim is to use information from cytogenetic anomalies to identify candidate genes for autism. METHODS: We have identified a male patient with mental retardation and autism who has a balanced translocation involving chromosomes 6 and 7, described as t(6;7)(p11-p12;q22). This translocation was inherited from an apparently normal father. RESULTS: Using fluorescence in situ hybridization, we have localized the breakpoints on both the chromosomes; and using bioinformatic genomic analysis, we have identified a number of potential candidate genes at these loci. These include the neural pentraxin 2 gene, NPTX2, and a novel gene encoding a transmembrane protein, TMEM130, which contains a polycystic kidney domain on 7q22. On 6p12 the breakpoint directly interrupts isoform 2 of the human homologue of the mouse dystonin gene. We also performed a 250 K single nucleotide polymorphism microarray analysis and comparative genomic hybridization using a bacterial artificial chromosome microarray to look for minor genomic deletions or duplications in the proband's DNA. The single nucleotide polymorphism microarray analysis identified a number of copy number variants, remote from the translocation breakpoints, containing potential candidate genes. CONCLUSION: It is conceivable that one or more of the copy number variant regions or either of the two breakpoint locations and the dystonin gene, in particular, may be a new locus for a form of mental retardation, which may also include autistic features.
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Trastorno Autístico/genética , Rotura Cromosómica , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 7/genética , Discapacidad Intelectual/genética , Translocación Genética , Adolescente , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Cromosomas Artificiales Bacterianos , Análisis Citogenético , Análisis Mutacional de ADN , Femenino , Dosificación de Gen , Regulación de la Expresión Génica , Genoma Humano/genética , Humanos , Lactante , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Muscle-eye-brain disease (MEB), is caused by mutations in the POMGnT1 gene. We describe a white family with two siblings affected with congenital hypotonia early-onset glaucoma, and psychomotor delays. Brain magnetic resonance images (MRIs) showed hydrocephalus, bilateral frontal polymicrogyria, abnormal cerebellum, and characteristic flattened dystrophic pons. We identified novel POMGnT1 gene alterations in this family. Both affected siblings were found to be compound hetrozygotes and carried two missense changes inherited from their mother and one missense change (p.R442C) inherited from their father. Our findings further define the phenotypic spectrum of MEB and its occurrence in the US population.