RESUMO
AIMS: Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples. METHODS AND RESULTS: Three healthy foetal human hearts (19th to 22nd week of gestation) and one foetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single-cell suspensions followed by 10× Genomics- and Illumina-based single-cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse sub-populations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types of the CHB heart compared with the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contribute to extracellular matrix deposition and subsequent fibrosis. CONCLUSION: These data provide an information-rich resource to further our understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease.
Assuntos
Anticorpos Antinucleares/imunologia , Autoimunidade , Coração Fetal/imunologia , Coração Fetal/patologia , Bloqueio Cardíaco/congênito , Transcriptoma , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Idade Gestacional , Bloqueio Cardíaco/embriologia , Bloqueio Cardíaco/genética , Bloqueio Cardíaco/imunologia , Bloqueio Cardíaco/patologia , Humanos , RNA-Seq , Análise de Célula ÚnicaRESUMO
The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated noncoding Y ssRNA. The top 10 upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response [e.g., IFN-induced protein 44-like (IFI44L), of MX dynamin-like GTPase (MX)1, MX2, and radical S-adenosyl methionine domain containing 2 (Rsad2)]. Within the fibrotic pathway, transcript levels of endothelin-1 (EDN1), phosphodiesterase (PDE)4D, chemokine (C-X-C motif) ligand (CXCL)2, and CXCL3 were upregulated, while others, including adenomedullin, RAP guanine nucleotide exchange factor 3 (RAPGEF3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, and dual specificity phosphatase 1, were downregulated. Agnostic Database for Annotation, Visualization and Integrated Discovery analysis revealed a significant increase in inflammatory genes, including complement C3A receptor 1 (C3AR1), F2R-like thrombin/trypsin receptor 3, and neutrophil cytosolic factor 2. In addition, stimulated fibroblasts expressed high levels of phospho-MADS box transcription enhancer factor 2 [a substrate of MAPK5 (ERK5)], which was inhibited by BIX-02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top 10 most highly expressed transcripts in CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes.NEW & NOTEWORTHY Congenital heart block is a rare disease of the fetal heart associated with maternal anti-Ro autoantibodies which can result in death and for survivors, lifelong pacing. This study provides in vivo and in vitro transcriptome-support that injury may be mediated by an effect of Type I Interferon on fetal fibroblasts.
Assuntos
Anticorpos Antinucleares/metabolismo , Coração Fetal/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Bloqueio Cardíaco/congênito , Mediadores da Inflamação/metabolismo , Interferon Tipo I/metabolismo , Transcriptoma , Adulto , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Feminino , Coração Fetal/imunologia , Coração Fetal/patologia , Fibroblastos/patologia , Fibrose , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Bloqueio Cardíaco/genética , Bloqueio Cardíaco/imunologia , Bloqueio Cardíaco/metabolismo , Bloqueio Cardíaco/patologia , Humanos , Mediadores da Inflamação/imunologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Miocárdio , Comunicação Parácrina , Gravidez , TransfecçãoRESUMO
The etiology of idiopathic dilated cardiomyopathy (iDCM) remains unknown. Immune therapies have improved outcome in fetuses with DCM born to mothers with autoimmune disease (aDCM). The purpose of this retrospective study was to compare the myocardial B and T cell profiles in fetuses and neonates with idiopathic DCM (iDCM) versus autoimmune-mediated DCM (aDCM) and to describe the normal cell maturation within the human fetal myocardium. Of 60 fetal autopsy cases identified from institutional databases, 10 had aDCM (18-38 weeks), 12 iDCM (19-37 weeks) and 38 had normal hearts (11-40 weeks). Paraffin-embedded myocardium sections were stained for all lymphocyte (CD45), B cells (CD20, CD79a), T cells (CD3, CD4, CD7, CD8) and monocyte (CD68) surface markers. Two independent, blinded cell counts were performed. Normal hearts expressed all B and T cell markers in a bimodal fashion, with peaks at 22 and 37 weeks of gestation. The aDCM cohort was most distinct from normal hearts, with less overall T cell markers [EST -9.1 (2.6) cells/mm(2), p = 0.001], CD4 [EST -2.0 (0.6), p = 0.001], CD3 [EST -3.9 (1.0), p < 0.001], CD7 [EST -3.0 (1.1), p = 0.01] overall B cell markers [EST -4.9 (1.8), p = 0.01] and CD79a counts [EST -2.3 (0.9), p = 0.01]. The iDCM group had less overall B cell markers [EST -4.0 (1.8), p = 0.03] and CD79a [EST -1.7 (0.9), p = 0.05], but no difference in T cell markers. Autoimmune-mediated DCM fetuses have less B and T cell markers, whereas iDCM fetuses have less B cell markers compared with normal fetal hearts. The fetal immune system may play a role in the normal development of the heart and evolution of dilated cardiomyopathy.
Assuntos
Linfócitos B/citologia , Cardiomiopatia Dilatada/imunologia , Coração Fetal/imunologia , Miocárdio/imunologia , Linfócitos T/citologia , Autopsia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Bases de Dados Factuais , Feminino , Coração Fetal/patologia , Humanos , Recém-Nascido , Modelos Lineares , Masculino , Miocárdio/patologia , Ontário , Estudos RetrospectivosRESUMO
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 , CoelhosRESUMO
In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-ß activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-ß, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-ß compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-ß Ab or TGF-ß inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-ß dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-ß activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-ß activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.
Assuntos
Apoptose/imunologia , Sítios de Ligação de Anticorpos , Coração Fetal/imunologia , Miócitos Cardíacos/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ribonucleoproteínas/imunologia , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Autoanticorpos/metabolismo , Transdiferenciação Celular/imunologia , Células Cultivadas , Cicatriz/imunologia , Cicatriz/patologia , Técnicas de Cocultura , Feminino , Coração Fetal/metabolismo , Coração Fetal/patologia , Fibrose , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
Neonatal lupus has become an important model of passively acquired autoimmunity since the seminal observation in the late 1970s that sera from nearly all mothers of children with isolated congenital heart block (CHB) contain specific autoantibodies.
Assuntos
Bloqueio Cardíaco/congênito , Apoptose/imunologia , Dexametasona/uso terapêutico , Progressão da Doença , Coração Fetal/imunologia , Glucocorticoides/uso terapêutico , Bloqueio Cardíaco/imunologia , Humanos , Imunoglobulinas Intravenosas/imunologia , Recém-Nascido , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Miocárdio/imunologia , Sistema de Registros , Ribonucleoproteínas/imunologiaRESUMO
Apart from complete and incomplete congenital heart block (CHB), new cardiac manifestations related to anti-SSA/Ro antibodies have been reported in children born to mothers bearing these antibodies. These manifestations include transient fetal first-degree heart block, prolongation of corrected QT (QTc) interval, sinus bradycardia, late-onset cardiomyopathy, endocardial fibroelastosis and cardiac malformations. Anti-SSA/Ro antibodies are not considered pathogenic to the adult heart, but a prolongation of the QTc interval has recently been reported in adult patients and is still a matter of debate. Treatment of CHB is not well established and needs to be assessed carefully. The risks and benefits of prenatal fluorinated steroids are discussed.
Assuntos
Anticorpos Antinucleares/imunologia , Eletrocardiografia , Cardiopatias/etiologia , Imunidade Materno-Adquirida , Miocárdio/patologia , Adulto , Idade de Início , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Betametasona/uso terapêutico , Bradicardia/etiologia , Bradicardia/imunologia , Cardiomiopatias/epidemiologia , Cardiomiopatias/etiologia , Cardiomiopatias/imunologia , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Dexametasona/uso terapêutico , Fibroelastose Endocárdica/etiologia , Fibroelastose Endocárdica/imunologia , Feminino , Coração Fetal/imunologia , Coração Fetal/patologia , Coração Fetal/fisiopatologia , Bloqueio Cardíaco/congênito , Bloqueio Cardíaco/tratamento farmacológico , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/imunologia , Cardiopatias Congênitas/etiologia , Cardiopatias Congênitas/imunologia , Cardiopatias/congênito , Cardiopatias/imunologia , Cardiopatias/fisiopatologia , Humanos , Lactente , Recém-Nascido , Síndrome do QT Longo/congênito , Síndrome do QT Longo/imunologia , Lúpus Eritematoso Sistêmico/congênito , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Estudos Multicêntricos como Assunto , Miocárdio/imunologia , Gravidez , Complicações na Gravidez/tratamento farmacológico , Complicações na Gravidez/imunologia , Estudos ProspectivosRESUMO
Congenital heart block (CHB) associated with maternal anti-SSA/SSB antibodies: a report of four cases. CHB detected in utero is strongly associated with maternal antibodies to SSA (Ro) and SSB (La). Their pathogenic role in the development of CHB has been established in several studies. The mothers of affected infants frequently had autoimmune disease (systemic lupus erythematosus, Sjögren's syndrome) or were entirely asymptomatic. It is very difficult to identify pregnant asymptomatic mothers carrying anti-SSA/SSB antibodies. We report four cases of infants born to asymptomatic mothers with anti-SSA/SSB antibodies, three of them developed isolated congenital cardiac heart block and one with no evidence of CHB. All three CHB are detected during pregnancy between 16 and 24 weeks of gestation. All maternal sera contained antibodies to SSA alone or the both SSA and SSB. Three of four subsequent pregnancies were complicated by heart block. One child affected died in utero. While the two other newborns with CHB required pacemaker insertion during the first 3 months of life. Although the association of anti-SSA/SSB with CHB is widely accepted, the precise mechanism by which these antibodies cause cardiac conduction abnormalities remains to be defined. Antibodies to SSA/SSB have been proposed to be a serologic marker for neonatal lupus syndrome and CHB. Fetal and neonatal diseases are presumed to be due to the transplacental passage of these IgG autoantibodies from the mother into the fetal circulation. Since these antibodies may have a pathogenic role in CHB, screening of infants with isolated CHB or neonatal lupus and their mothers for the presence of anti-SSA and anti-SSB is strongly recommended.
Assuntos
Anticorpos Antinucleares/imunologia , Autoantígenos , Doenças Autoimunes/imunologia , Bloqueio Cardíaco/congênito , Imunidade Materno-Adquirida , Isoanticorpos/imunologia , Complicações na Gravidez/imunologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas/imunologia , Adulto , Bradicardia/embriologia , Bradicardia/imunologia , Estimulação Cardíaca Artificial , Feminino , Morte Fetal/etiologia , Doenças Fetais/imunologia , Coração Fetal/imunologia , Coração Fetal/patologia , Bloqueio Cardíaco/imunologia , Bloqueio Cardíaco/terapia , Humanos , Recém-Nascido , Masculino , Gravidez , Antígeno SS-BRESUMO
Few diseases exemplify the integration of research from bench to bedside as well as neonatal lupus, often referred to as a model of passively acquired autoimmunity. In essence, this disease encompasses two patients, both the mother and her child. The signature histologic lesion of autoimmune-associated congenital heart block is fibrosis of the conducting tissue, and in some cases the surrounding myocardium. It is astounding how rapid and, in most cases, irreversible is the fibrotic response to injury. The mechanism by which maternal anti-SSA/Ro-SSB/La antibodies initiate and perpetuate inflammation, and eventuate in scarring of the atrioventricular node, is not yet defined. In vitro and in vivo studies suggest that one pathologic cascade leading to scarring may be initiated via apoptosis, resulting in translocation of SSA/Ro-SSB/La antigens and subsequent surface binding by maternal autoantibodies. These opsonized cardiocytes are phagocytosed by macrophages, which secrete factors that transdifferentiate fibroblasts into myofibroblasts, a scarring phenotype. Dissecting the individual components in this fibrotic pathway should provide insights into the rarity of irreversible injury and should form the basis of rational approaches to prevention and treatment.
Assuntos
Nó Atrioventricular/patologia , Autoantígenos , Doenças Autoimunes/congênito , Bloqueio Cardíaco/congênito , Imunidade Materno-Adquirida , Lúpus Eritematoso Sistêmico/congênito , RNA Citoplasmático Pequeno , Adulto , Anticorpos Antinucleares/imunologia , Nó Atrioventricular/embriologia , Nó Atrioventricular/imunologia , Doenças Autoimunes/complicações , Doenças Autoimunes/embriologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Feminino , Coração Fetal/imunologia , Coração Fetal/patologia , Fibroblastos/patologia , Fibrose , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/imunologia , Bloqueio Cardíaco/patologia , Humanos , Recém-Nascido , Inflamação , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/embriologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Gravidez , Ribonucleoproteínas/imunologia , Antígeno SS-BRESUMO
Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16-24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-alpha from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.
Assuntos
Anticorpos Antinucleares/metabolismo , Apoptose/imunologia , Sítios de Ligação de Anticorpos , Coração Fetal/imunologia , Macrófagos/metabolismo , Miocárdio/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Biotinilação , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Precipitação Química , Técnicas de Cocultura , Feminino , Coração Fetal/citologia , Coração Fetal/metabolismo , Coração Fetal/ultraestrutura , Humanos , Macrófagos/imunologia , Macrófagos/ultraestrutura , Microscopia Eletrônica de Varredura , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , GravidezRESUMO
Congenital heart block is a serious condition with significant mortality due in most cases to the transplacental transfer of autoantibodies from an otherwise asymptomatic mother. Although SSA/Ro and SSB/La autoantibodies have been implicated, attention has focused recently on autoantibodies to envelope proteins of endogenous retrovirus-3 (ERV-3). We have recently identified in 1% of the caucasian population a natural knock out of ERV-3 due to a premature stop mutation generating a severely truncated form of the protein [corrected]. If a pregnant female homozygous for the truncated form of the ERV-3 carries a foetus expressing the entire protein, the mother might be expected to acquire high titre immunity, while the foetus homozygous for the truncated form would not be expected to immunise its mother. In order to test whether this naturally occurring model could shed light on the pathogenesis of CHB, we determined the status of the ERV-3 stop polymorphism in 12 mothers of CHB infants [corrected]. The fact that none was homozygous for the stop mutation tends to rule out a role for the stop polymorphism of the mothers in the generation of the CHB disease, but does not exclude that other polymorphisms might be involved [corrected].
Assuntos
Anticorpos Antivirais/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Retrovirus Endógenos/imunologia , Coração Fetal/imunologia , Bloqueio Cardíaco/congênito , Imunidade Materno-Adquirida , Isoanticorpos/imunologia , Modelos Imunológicos , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Autoanticorpos/sangue , Autoantígenos/biossíntese , Autoantígenos/genética , Autoimunidade , Retrovirus Endógenos/genética , Epitopos/imunologia , Feminino , Doenças Fetais/imunologia , Genes env , Genótipo , Bloqueio Cardíaco/embriologia , Bloqueio Cardíaco/imunologia , Sistema de Condução Cardíaco/imunologia , Sistema de Condução Cardíaco/patologia , Humanos , Recém-Nascido , Isoanticorpos/sangue , Camundongos , Mutação Puntual , Polimorfismo Genético , Gravidez , Regiões Terminadoras Genéticas , Trofoblastos/imunologia , Trofoblastos/virologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genéticaRESUMO
Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.
Assuntos
Apoptose , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Coração Fetal/imunologia , Proteínas Fetais/imunologia , Bloqueio Cardíaco/congênito , Imunidade Materno-Adquirida , Miocárdio/imunologia , Complicações na Gravidez/imunologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas/imunologia , Reações Antígeno-Anticorpo , Antígenos de Superfície/imunologia , Aorta/embriologia , Autoanticorpos/sangue , Transporte Biológico , Núcleo Celular/imunologia , Separação Celular/métodos , Células Cultivadas , Colagenases/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Coração Fetal/citologia , Coração Fetal/efeitos dos fármacos , Bloqueio Cardíaco/etiologia , Humanos , Proteínas Musculares/imunologia , Miocárdio/citologia , Naftoquinonas/farmacologia , Gravidez , Isoformas de Proteínas/imunologia , Estaurosporina/farmacologia , Antígeno SS-BAssuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Coração Fetal/imunologia , Bloqueio Cardíaco/congênito , Cardiopatias Congênitas/embriologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas/imunologia , Feminino , Coração Fetal/patologia , Bloqueio Cardíaco/imunologia , Sistema de Condução Cardíaco/anormalidades , Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/imunologia , Cardiopatias Congênitas/imunologia , Humanos , Gravidez , Antígeno SS-BRESUMO
Neonatal lupus erythematosus (NLE) is a rare disorder characterized by congenital heart block and/or cutaneous disease and less commonly is associated with hepatic and hematological abnormalities. The majority of affected infants acquire Ro/SS-A (Ro) autoantibodies transplacentally from the maternal circulation and it is generally thought that these antibodies are pathogenic. Mothers at increased risk of having a child affected by NLE should be followed closely during pregnancy to detect the development of fetal heart block, as this is associated with significant morbidity and mortality. Mothers of affected infants are at increased risk of developing a rheumatic disease if they have not already done so.
Assuntos
Doenças Autoimunes/congênito , Lúpus Eritematoso Sistêmico/congênito , Lúpus Eritematoso Sistêmico/imunologia , Anticorpos Antinucleares/imunologia , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/imunologia , Coração Fetal/imunologia , Bloqueio Cardíaco/diagnóstico , Bloqueio Cardíaco/imunologia , Humanos , Recém-Nascido , Placenta/imunologia , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/imunologiaRESUMO
Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sjögren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8.7 to 14.9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9.4 to 14.9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development.
Assuntos
Anticorpos Antinucleares/imunologia , Bloqueio Cardíaco/congênito , Bloqueio Cardíaco/imunologia , Laminina/imunologia , Adulto , Idoso , Afinidade de Anticorpos , Western Blotting , Células Cultivadas , Reações Cruzadas/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Coração Fetal/imunologia , Imunofluorescência , Humanos , Imunidade Materno-Adquirida , Pessoa de Meia-Idade , Miocárdio/imunologiaRESUMO
A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these G1N2-expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this "primary" ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, G1N2-expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express G1N2, while upon the completion of septation, no G1N2-expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of G1N suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the "primary" interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from G1N2-expressing myocytes in the interventricular septum, while the "compact" atrioventricular node develops at the junction of the band of G1N2-positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the ("penetrating") atrioventricular bundle. A "dead-end tract" represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the G1N2-ring in the human embryonic heart, indicating a phylogenetically conserved origin of the conduction system in vertebrates.
Assuntos
Antígenos/metabolismo , Coração Fetal/imunologia , Músculos/imunologia , Nó Atrioventricular/embriologia , Nó Atrioventricular/imunologia , Coração Fetal/embriologia , Idade Gestacional , Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/imunologia , Humanos , Imuno-Histoquímica , Músculos/embriologiaRESUMO
Two different effects of maternal autoantibodies presented in a third-trimester pregnancy. The first was complete fetal heart block, demonstrated ultrasonographically, which correlated with the presence of anti-Ro and anti-La antibodies in the maternal serum. The second effect was decidual vasculopathy and thrombosis, a morphologic finding in the placenta that caused massive placental infarction and intrauterine death. The placental pathology correlated with the presence of anticardiolipin antibodies in the maternal serum at the time of stillbirth.
Assuntos
Autoanticorpos/imunologia , Bloqueio Cardíaco/etiologia , Infarto/etiologia , Troca Materno-Fetal/imunologia , Placenta/irrigação sanguínea , Adulto , Anticorpos Antinucleares/análise , Anticorpos Antinucleares/imunologia , Cardiolipinas/imunologia , Feminino , Morte Fetal/etiologia , Coração Fetal/imunologia , Coração Fetal/fisiopatologia , Bloqueio Cardíaco/sangue , Bloqueio Cardíaco/imunologia , Humanos , Imunidade Materno-Adquirida/imunologia , Infarto/sangue , Infarto/imunologia , Gravidez , Complicações Cardiovasculares na Gravidez/sangue , Complicações Cardiovasculares na Gravidez/etiologia , Complicações Cardiovasculares na Gravidez/imunologia , Trombose/etiologiaRESUMO
Congenital complete heart block (CCHB) is a rare but potentially fatal disease of infants born to mothers with autoimmune disease where maternal autoantibodies to Ro (SS-A) are thought to cross the placenta and damage fetal cardiac tissue. We have adopted a novel approach to demonstrate the localization and specificity of maternal autoantibodies deposited in fetal heart. We raised an anti-idiotype against maternal anti-La antibodies, which reacted strongly with the surface immunoglobulin on the myocardial fibres from a CCHB heart but not a control fetal heart of the same age. Maternal immunoglobulin eluted from the CCHB heart reacted with La (SS-B) by ELISA. Using monoclonal and affinity-purified antibodies to La and affinity-purified anti-Ro antibodies, both antigens were identified on the surface of the fibres of the affected heart. Surface co-expression of immunoglobulin, complement and Class II antigen, consistent with a local immune response, was also found. This is the first definitive demonstration of Ro and La antigens and specific maternal anti-La antibody and idiotype on the surface of myocardial fibres in CCHB. It suggests that induction of Ro and La antigens on the surface of myocardial fibres during fetal development may be critical in the localization of the specific autoantibodies and subsequent evolution of congenital complete heart block.
Assuntos
Anticorpos Antinucleares , Autoantígenos , Bloqueio Cardíaco/congênito , RNA Citoplasmático Pequeno , Doenças Autoimunes/complicações , Doenças Autoimunes/imunologia , Feminino , Coração Fetal/imunologia , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/imunologia , Humanos , Idiótipos de Imunoglobulinas , Recém-Nascido , Masculino , Miocárdio/imunologia , Gravidez , Complicações na Gravidez/imunologia , Ribonucleoproteínas/imunologia , Antígeno SS-BRESUMO
The presence of anti-Ro (SS-A) antibody in maternal serum has been associated with congenital heart block. Using human sera monospecific for anti-Ro (SS-A) antibodies in an indirect immunofluorescence assay, Ro (SS-A) antigen was found to be present in the nuclei of myocardial cells and cells of the cardiac conduction system. The possible relationship between the location of Ro (SS-A) antigen, anti-Ro (SS-A) antibody, and heart block is discussed.
Assuntos
Autoantígenos/análise , Coração Fetal/patologia , Bloqueio Cardíaco/imunologia , Sistema de Condução Cardíaco/patologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas , Autoantígenos/imunologia , Colinesterases/metabolismo , Coração Fetal/imunologia , Imunofluorescência , Idade Gestacional , Sistema de Condução Cardíaco/imunologia , Humanos , Soros Imunes/análiseRESUMO
After sensitization by rabbit anti-rat-heart serum (AHS), Wistar rats were injected with 9 ml/kg of AHS on day 9 of gestation. The incidence of malformations was 31.5% and that of cardiovascular malformations was 21.8%. The main malformations were microphthalmia, ventricular septal defect, pulmonary stenosis, and general edema. No antinuclear antibody was detected in the maternal sera by the fluorescent antibody technique. No complete heart block was found in the fetuses by electrocardiography. The fluorescent antibody technique demonstrated localization of rat IgG and rat C3 on the maternal myocardium, but a light-microscopic study revealed no myocarditis or endocarditis in either the maternal or the fetal heart. The types of malformation were similar to those observed after a single administration of antikidney serum, which presumably involves yolk sac dysfunction. These results show that both the existence of the immune complexes of antibodies against AHS and the administration of AHS on day 9 are necessary to cause yolk sac dysfunction or other teratogenic changes.