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1.
Ann Rheum Dis ; 81(8): 1151-1161, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35470161

RESUMO

OBJECTIVE: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. METHODS: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. RESULTS: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. CONCLUSIONS: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.


Assuntos
Bloqueio Atrioventricular , Auxilinas , Animais , Anticorpos Antinucleares , Bloqueio Atrioventricular/genética , Autoanticorpos , Coração Fetal , Estudo de Associação Genômica Ampla , Bloqueio Cardíaco/congênito , Lúpus Eritematoso Sistêmico/congênito , Camundongos
2.
Ann Rheum Dis ; 71(3): 448-54, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22084395

RESUMO

BACKGROUND: Congenital heart block (CHB) may develop in fetuses of women with anti-Ro/La autoantibodies following placental transfer of maternal autoantibodies and disruption of the fetal atrioventricular (AV) conduction system. Animal models of CHB currently rely on immunisation or transfer of anti-Ro/La antibodies purified from mothers of children with CHB, which does not allow precise identification of the disease-inducing antibody specificity. OBJECTIVE: To determine the ability of different anti-Ro52 monoclonal antibodies to induce cardiac electrophysiological abnormalities in vivo and affect the calcium homoeostasis of cardiomyocytes in vitro. METHODS: Monoclonal antibodies recognising different domains of Ro52 were generated and injected into pregnant rats, and ECG was recorded on newborn pups. Cultures of rat neonatal cardiomyocytes were established to assess the effect of the different anti-Ro52 monoclonal antibodies on calcium homoeostasis. RESULTS: First-degree AV block and bradycardia developed after maternal transfer of antibodies specific for amino acids 200-239 of Ro52 (p200), while pups exposed to antibodies targeting N- or C-terminal epitopes of Ro52 did not show any electrocardiogram abnormalities. Addition of an anti-p200 antibody to cultured cardiomyocytes induced calcium dyshomoeostasis in a time- and dose-dependent manner, while addition of other Ro52 antibodies had no effect. CONCLUSION: These data for the first time show unambiguously that antibodies specific for amino acids 200-239 of Ro52 can induce cardiac conduction defects in the absence of other autoantibodies, and may therefore be the main initiators of cardiac pathology in the pool of anti-Ro52 antibodies in mothers of children with CHB.


Assuntos
Anticorpos Monoclonais/imunologia , Bloqueio Atrioventricular/congênito , Ribonucleoproteínas/imunologia , Animais , Animais Recém-Nascidos , Especificidade de Anticorpos/imunologia , Bloqueio Atrioventricular/imunologia , Autoanticorpos/imunologia , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Eletrocardiografia , Epitopos/imunologia , Feminino , Homeostase/imunologia , Troca Materno-Fetal/imunologia , Miócitos Cardíacos/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/imunologia , Ratos
3.
J Immunol ; 185(6): 3574-82, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20696861

RESUMO

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1(av1) (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1(l)). Different anti-Ro52 Ab fine specificities were generated in RT1(av1) versus RT1(l) animals. Maternal and fetal influence was determined in an F(2) cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1(l) than RT1(av1) pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1(l) pups more frequently developed heart block than RT1(av1) pups after passive transfer of RT1(av1) anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52-positive pregnancies.


Assuntos
Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/imunologia , Autoanticorpos/biossíntese , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Troca Materno-Fetal/imunologia , Ribonucleoproteínas/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/genética , Bloqueio Atrioventricular/congênito , Linhagem Celular , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade/imunologia , Troca Materno-Fetal/genética , Dados de Sequência Molecular , Gravidez , Ratos , Ratos Endogâmicos Lew , Ribonucleoproteínas/administração & dosagem
4.
PLoS One ; 9(10): e109128, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25343256

RESUMO

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Retículo Endoplasmático/genética , Proteínas de Membrana/biossíntese , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Citoplasma , Desmossomos/metabolismo , Junções Comunicantes/genética , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Miocárdio/patologia , Fosforilação
5.
PLoS One ; 8(9): e72668, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039792

RESUMO

BACKGROUND: Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.


Assuntos
Autoanticorpos/imunologia , Canais de Cálcio Tipo T/imunologia , Epitopos/imunologia , Bloqueio Cardíaco/congênito , Sequência de Aminoácidos , Animais , Nó Atrioventricular/efeitos dos fármacos , Nó Atrioventricular/metabolismo , Autoanticorpos/sangue , Autoantígenos/imunologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/genética , Mapeamento de Epitopos , Espaço Extracelular , Feminino , Coração Fetal/efeitos dos fármacos , Coração Fetal/imunologia , Coração Fetal/metabolismo , Expressão Gênica , Bloqueio Cardíaco/genética , Bloqueio Cardíaco/imunologia , Humanos , Masculino , Troca Materno-Fetal/imunologia , Camundongos , Dados de Sequência Molecular , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Peptídeos/imunologia , Gravidez , Coelhos
6.
J Invest Dermatol ; 129(8): 2000-10, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19194477

RESUMO

Ro52 is an E3 ubiquitin ligase with a recently identified regulatory role in inflammation. The protein is targeted by autoantibodies in rheumatic diseases, and Ro52 autoantibodies are specifically associated with cutaneous lupus erythematosus (CLE) and photosensitivity. The aim of this study was to investigate cutaneous Ro52 expression in CLE patients and to examine whether UVR might modulate Ro52. Ro52 expression was assessed by immunohistochemistry in biopsies derived from CLE lesions (n=25), nonlesional (n=7), and healthy control skin using four anti-Ro52 mAbs generated by us. Ro52 expression was also analyzed in psoriatic, lichenoid, and eczematous lesions. It was increased in the epidermis of spontaneous CLE lesions as compared with unaffected skin of patients and healthy controls. High epidermal Ro52 expression was also observed in other inflammatory dermatoses investigated. Ro52 was upregulated in experimentally photoprovoked CLE lesions as observed by immunohistochemistry in sequential biopsies, which was confirmed in vitro both at the mRNA and protein levels by exposing cultured patient-derived primary keratinocytes to UVR. In conclusion, Ro52 expression is upregulated in keratinocytes in inflammatory skin conditions and in response to UVR. High Ro52 expression might lead to the breaking of tolerance and the generation of Ro52 autoantibodies in genetically susceptible subjects. Further, the upregulation of Ro52 in keratinocytes after sun exposure might also be a triggering factor for skin lesions in patients with Ro52 antibodies.


Assuntos
Dermatite/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Ribonucleoproteínas/análise , Pele/imunologia , Pele/efeitos da radiação , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Queratinócitos/imunologia , Masculino , Pessoa de Meia-Idade , Ribonucleoproteínas/genética , Raios Ultravioleta
7.
J Clin Immunol ; 28(3): 220-31, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18071879

RESUMO

Interferon-alpha (IFN-alpha) has been implicated in the pathogenesis of Sjögren's syndrome and systemic lupus erythematosus. Ro52, which was recently identified as an E3 ligase with anti-proliferative and pro-apoptotic properties, is a major autoantigen targeted in both these conditions. Microarray analyses have indicated up-regulation of Ro52 by IFN-alpha, and the objective of the present study was to address the potential link between IFN-alpha and Ro52. To investigate the influence of IFN-alpha on Ro52 protein levels and cellular localization, we generated a panel of monoclonal antibodies to different domains of Ro52. These novel monoclonal antibodies were characterized by immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay using cell lysates, recombinant Ro52 protein, and synthetic peptides. Ro52 was up-regulated in HeLa cells and human B cells at the messenger RNA and protein levels in response to IFN-alpha stimulation as detected by reverse transcriptase polymerase chain reaction and Western blot. After up-regulation, Ro52 translocated from the cytoplasm to the nucleus. The nuclear translocation of Ro52 was observed after staining with generated monoclonal antibodies specific for both the RING, coiled-coil, and B30.2 domains of Ro52 and the nuclear translocation of Ro52 preceded IFN-alpha-induced apoptotic cell death detected by caspase-3 and TUNEL staining in the treated cultures. In conclusion, our data show that IFN-alpha first induces up-regulation of Ro52 protein and then prompts translocation of the up-regulated Ro52 protein in to the nucleus. The translocation precedes apoptosis of the IFN-alpha exposed cells, suggesting a role for Ro52 in mediating the anti-proliferative or pro-apoptotic effects of the autoimmune-related cytokine IFN-alpha.


Assuntos
Anticorpos Monoclonais/metabolismo , Autoantígenos/metabolismo , Interferon-alfa/fisiologia , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo , Regulação para Cima/imunologia , Transporte Ativo do Núcleo Celular/imunologia , Animais , Especificidade de Anticorpos , Apoptose/imunologia , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Proliferação de Células , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos , Ribonucleoproteínas/fisiologia , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
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