Natural killer cells and type II interferon in Ro/SSA and La/SSB autoantibody-exposed newborns at risk of congenital heart block.

OBJECTIVE: Congenital heart block (CHB) with immune cell infiltration develops in the fetus after exposure to maternal Ro/La autoantibodies. CHB-related serology has been extensively studied, but reports on immune-cell profiles of anti-Ro/La-exposed neonates are lacking. In the current study, we characterised circulating immune-cell populations in anti-Ro/La+mothers and newborns, and explored potential downstream effects of skewed neonatal cell populations. METHODS: In total, blood from mothers (n=43) and neonates (n=66) was sampled at birth from anti-Ro/La+ (n=36) and control (n=30) pregnancies with or without rheumatic disease and CHB. Flow cytometry, microarrays and ELISA were used for characterising cells and plasma. RESULTS: Similar to non-pregnant systemic lupus erythematosus and Sjögren-patients, anti-Ro/La+mothers had altered B-cell subset frequencies, relative T-cell lymphopenia and lower natural killer (NK)-cell frequencies. Surprisingly, their anti-Ro/La exposed neonates presented higher frequencies of CD56dimCD16hi NK cells (p<0.01), but no other cell frequency differences compared with controls. Type I and II interferon (IFN) gene-signatures were revealed in neonates of anti-Ro/La+ pregnancy, and exposure of fetal cardiomyocytes to type I IFN induced upregulation of several NK-cell chemoattractants and activating ligands. Intracellular flow cytometry revealed IFNγ production by NK cells, CD8+ and CD4+ T cells in anti-Ro/La exposed neonates. IFNγ was also detectable in their plasma. CONCLUSION: Our study demonstrates an increased frequency of NK cells in anti-Ro/La exposed neonates, footprints of type I and II IFN and an upregulation of ligands activating NK cells in fetal cardiac cells after type I IFN exposure. These novel observations demonstrate innate immune activation in neonates of anti-Ro/La+pregnancy, which could contribute to the risk of CHB.

Hydroxychloroquine to Prevent Recurrent Congenital Heart Block in Fetuses of Anti-SSA/Ro-Positive Mothers.

BACKGROUND: Experimental and clinical evidence support the role of macrophage Toll-like receptor signaling in maternal anti-SSA/Ro-mediated congenital heart block (CHB). OBJECTIVES: Hydroxychloroquine (HCQ), an orally administered Toll-like receptor antagonist widely used in lupus including during pregnancy, was evaluated for efficacy in reducing the historical 18% recurrence rate of CHB. METHODS: This multicenter, open-label, single-arm, 2-stage clinical trial was designed using Simon's optimal approach. Anti-SSA/Ro-positive mothers with a previous pregnancy complicated by CHB were recruited (n = 19 Stage 1; n = 35 Stage 2). Patients received 400 mg daily of HCQ prior to completion of gestational week 10, which was maintained through pregnancy. The primary outcome was 2° or 3° CHB any time during pregnancy, and secondary outcomes included isolated endocardial fibroelastosis, 1° CHB at birth and skin rash. RESULTS: By intention-to-treat (ITT) analysis, 4 of 54 evaluable pregnancies resulted in a primary outcome (7.4%; 90% confidence interval: 3.4% to 15.9%). Because 9 mothers took potentially confounding medications (fluorinated glucocorticoids and/or intravenous immunoglobulin) after enrollment but prior to a primary outcome, to evaluate HCQ alone, 9 additional mothers were recruited and followed the identical protocol. In the per-protocol analysis restricted to pregnancies exposed to HCQ alone, 4 of 54 (7.4%) fetuses developed a primary outcome as in the ITT. Secondary outcomes included mild endocardial fibroelastosis (n = 1) and cutaneous neonatal lupus (n = 4). CONCLUSIONS: These prospective data support that HCQ significantly reduces the recurrence of CHB below the historical rate by >50%, suggesting that this drug should be prescribed for secondary prevention of fetal cardiac disease in anti-SSA/Ro-exposed pregnancies. (Preventive Approach to Congenital Heart Block With Hydroxychloroquine [PATCH]; NCT01379573).

Type I IFN system activation in newborns exposed to Ro/SSA and La/SSB autoantibodies in utero.

OBJECTIVE: In utero exposure of the fetus to Ro/La autoantibodies may lead to congenital heart block (CHB). In the mother, these autoantibodies are associated with activation of the type I interferon (IFN)-system. As maternal autoantibodies are transferred to the fetus during pregnancy, we investigated whether the type I IFN-system is activated also in newborns of anti-Ro/La positive mothers, and whether fetal IFN activation is affected by maternal immunomodulatory treatment. METHODS: Blood drawn at birth from anti-Ro/La positive mothers, their newborns and healthy control pairs was separated into plasma and peripheral blood mononuclear cells (PBMC). PBMC were analysed directly or cultured. mRNA expression was analysed by microarrays, cell surface markers by flow cytometry, and IFNα levels by immunoassays. RESULTS: We observed increased expression of IFN-regulated genes and elevated plasma IFNα levels not only in anti-Ro/La positive women, but also in their newborns. CD14+ monocytes of both anti-Ro/La positive mothers and their neonates showed increased expression of Sialic acid-binding Ig-like lectin-1, indicating cellular activation. Notably, the IFN score of neonates born to mothers receiving immunomodulatory treatment was similar to that of controls, despite persistent IFN activation in the mothers. In both maternal and neonatal PBMC, IFNα production was induced when cells were cultured with anti-Ro/La positive plasma. CONCLUSIONS: Ro/La autoantibody-exposed neonates at risk of CHB have signs of an activated immune system with an IFN signature. This study further demonstrates that neonatal cells can produce IFNα when exposed to autoantibody-containing plasma, and that maternal immunomodulatory treatment may diminish the expression of IFN-regulated genes in the fetus.

Cell atlas of the foetal human heart and implications for autoimmune-mediated congenital heart block.

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.

Comparative effects of sodium channel blockers in short term rat whole embryo culture.

This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the l-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the l-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB>bupivacaine>AZA>lidocaine>nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart.

Maternal antibody-associated fetal second-degree heart block and atrial flutter: case report and review.

Exposure to maternal anti-Ro (SS-A) and anti-La (SS-B) antibodies is a well-described risk factor for the development of fetal atrioventricular (AV) block. The role of maternal fluorinated steroids in the treatment and prevention of antibody-mediated fetal AV block is controversial. Fetal atrial flutter has rarely been described in association with maternal antibodies. This report describes a case of fetal exposure to maternal anti-Ro antibodies with associated second-degree AV block and atrial flutter. Interestingly, the reported patient had 2:1 AV conduction during both normal atrial rates (consistent with AV node conduction disease) and episodes of flutter (consistent with physiologic AV node functionality). The fetus was treated with transplacental digoxin and dexamethasone, which resolved both rhythm disturbances. The case report is followed by a brief discussion of AV block and atrial flutter associated with maternal antibody exposure.

Antidepressants cause bradycardia and heart block in GD 13 rat embryos in vitro.

This study investigated the effects of a range of antidepressant drugs on the heart of gestation day 13 rat embryos in vitro. The general hypothesis was that the drugs would adversely affect the function of the embryonic heart since they all have some cardiac ion channel blocking activity in addition to their main pharmacological effect on neurotransmitters. The results showed that all the tested drugs caused bradycardia in a generally concentration-dependent manner. At higher concentrations most of the drugs caused some degree of heart block consistent with sodium channel blockade and some drugs also showed negative inotropy associated with blockade of the L-type calcium channel. One drug, trazodone, caused arrhythmia consistent with blockade of the hERG (human ether-a-go-go related gene) potassium channel. In general the effects on the embryonic rat heart were only seen at "free drug" concentrations much greater than those likely to occur in pregnant women taking antidepressant medication. The least margin of safety was seen with the tricyclic antidepressants and the serotonin antagonist and reuptake inhibitor trazodone.

Hypoplastic left heart syndrome with restrictive atrial septum and advanced heart block documented with a novel fetal electrocardiographic monitor.

Hypoplastic left ventricle with congenital heart block has been reported previously in a fetus with concurrent left atrial isomerism and levo-transposition of the great arteries. We present the unusual case of an infant diagnosed in utero with hypoplastic left heart syndrome, a restrictive atrial septum and advanced heart block but with D-looping of the ventricles and no atrial isomerism. In addition, fetal heart rhythm was documented with the assistance of a new fetal electrocardiographic monitor.

Genetic origins of pediatric heart disease.

Pediatric heart disease comprises many forms of cardiovascular disease in the young including cardiovascular malformations (CVM), cardiomyopathies, vasculopathies, e.g., Marfan syndrome, and cardiac arrhythmias. CVM are an important component of pediatric heart disease and constitute a major portion of clinically significant birth defects. In the past decade, the complementary nature of genetic, developmental, and biochemical approaches have contributed to extraordinary advances in understanding the origins of pediatric heart disease. Results of the studies of the cardiac transcription factor, NKX2.5, illustrate these accomplishments and at the same time provide a forecast of the nature of future genetic studies to better understand the origins of pediatric heart disease.

[Intrauterine treatment of incomplete fetal heart block in a mother with Sjögren syndrome]. / Intrauterine Therapie eines inkompletten AV-Blocks bei einer Mutter mit Sjögren-Syndrom.

BACKGROUND: Isolated fetal heart block is considered as an immunological disorder in the majority of cases. Mothers of affected fetuses often suffer from connective tissue disease (Sjögren syndrome or Lupus erythematodes). All of them test positive for anti-SS-A (anti Ro) and/or anti-SS-B (anti La) antibodies. Once established, third-degree congenital heart block is permanent and often requires a pacemaker. CASE: We report on a pregnancy in a mother with Sjögren syndrome which was complicated by the development of incomplete fetal heart block, diagnosed by pulsed wave Doppler echocardiography. We started oral dexamethasone treatment to reduce immune-mediated fetal cardiac damage and to prevent complications like hydrops fetalis. CONCLUSION: Detection of isolated fetal heart block is possible with pulsed Doppler sonography, but there are no clear recommendations for treatment.

Atrial and ventricular rate response and patterns of heart rate acceleration during maternal-fetal terbutaline treatment of fetal complete heart block.

Terbutaline is used to treat fetal bradycardia in the setting of complete heart block (CHB); however, little is known of its effects on atrial and ventricular beat rates or patterns of heart rate (HR) acceleration. Fetal atrial and ventricular beat rates were compared before and after transplacental terbutaline treatment (10 to 30 mg/day) by fetal echocardiography in 17 fetuses with CHB caused by immune-mediated damage to a normal conduction system (isoimmune, n = 8) or a congenitally malformed conduction system associated with left atrial isomerism (LAI, n = 9). While receiving terbutaline, 9 of the 17 fetuses underwent fetal magnetocardiography (fMCG) to assess maternal HR and rhythm, patterns of fetal HR acceleration, and correlation between fetal atrial and ventricular accelerations (i.e., AV correlation). Maternal HR and fetal atrial and ventricular beat rates increased with terbutaline. However, terbutaline's effects were greater on the atrial pacemaker(s) in fetuses with isoimmune CHB and greater on the ventricular pacemaker(s) in those with LAI-associated CHB. Patterns of fetal HR acceleration also differed between isoimmune and LAI CHB. Finally, despite increasing HR, terbutaline did not restore the normal coordinated response between atrial and ventricular accelerations in isoimmune or LAI CHB. In conclusion, the pathophysiologic heterogeneity of CHB is reflected in the differing effect of terbutaline on the atrial and ventricular pacemaker(s) and varying patterns of HR acceleration. However, regardless of the cause of CHB, terbutaline augments HR but not AV correlation, suggesting that its effects are determined by the conduction system defect rather than the autonomic control of the developing heart.

Juvenile-onset hypergammaglobulinemic purpura and fetal congenital heart block.

Waldenström's hypergammaglobulinemic purpura (HGP) is a rare chronic disorder characterized by recurrent purpura on the legs, a polyclonal increase in serum gamma-globulin, an elevated erythrocyte sedimentation rate and a positive rheumatoid factor. A 30-year-old primigravid woman with 14 years of HGP was found to have fetal bradycardia at 25 weeks' gestation. Laboratory investigations demonstrated positive anti-Ro/SSA and anti-La/SSB antibodies in the maternal serum. Cesarean delivery was performed at 39 weeks, and a 2750-g female infant was born with complete atrioventricular block. Fortunately, the neonatal period has been uneventful without need for pace-making. Maternal HGP exacerbated just after delivery, but resolved within 1 week without treatment. Physicians should be aware of the possible presence of neonatal lupus-related anti-Ro/SSA and anti-La/SSB autoantibodies in patients with HGP. Screening for these autoantibodies is important and could be used as a marker to identify and manage high-risk pregnancies.

Prenatal 2D ultrasonic diagnosis of fetal complete atrioventricular block and bilateral hydroceles with occult maternal SS-A.

OBJECTIVE: We present a case of fetal complete atrioventricular block, diagnosed by the M mode of two-dimensional ultrasound, that was complicated with polyhydramnios and bilateral hydrocele. CASE REPORT: The fetus was delivered at 38 weeks' gestation by caesarean section. Postpartum autoimmune survey disclosed positive anti-Ro antibodies (SS-A) for both the mother and the newborn. Severe neonatal bradycardia with complete atrioventricular block was identified. CONCLUSION: Antenatal evaluation for a maternal history of autoimmune disease (especially systemic lupus erythematosus) and prevention of fetal bradycardia by reducing immune-mediated injuries on the cardiac conduction system are important.

[Congenital heart block and autoantibodies]. / Kongenit hjerteblok og autoantistoffer.

Maternal IgG autoantibodies against Ro52 (part of the anti-SSA specificity) are correlated with congenital heart block (CHB). Most cases revert spontaneously and can be monitored by Doppler echocardiography, but fetuses that progress into second-degree block require treatment with, e.g., corticosteroids. A third-degree block is irreversible and necessitates lifelong pacemaking. It may prove beneficial to screen all expectant mothers for anti-Ro52 autoantibodies. Decisions about this will require further studies of the diagnostic performance of the Ro52 autoantibody test.

First-trimester fetal heart block and increased nuchal translucency: an indication for early fetal echocardiography.

Prevalence of congenital heart disease increases with nuchal translucency (NT) thickness. First-trimester fetal bradycardia may result from heart block associated with complex congenital heart disease. We report two cases detected in the first trimester of pregnancy, in which both fetuses showed an increased nuchal translucency and bradycardia. Fetal karyotype was normal in both fetuses. First-trimester fetal echocardiography was performed and, in both cases, complex congenital heart disease was diagnosed. We discuss the added role of fetal heart rate in first-trimester ultrasound screening, in fetuses with increased nuchal translucency and normal karyotype. We stress, as well, the importance of echocardiography performed in the first trimester as a potential tool for early diagnosis in selected cases.

Neonatal cholestasis associated with fetal arrhythmia.

We evaluated the consequence of different types of fetal arrhythmia in the development of neonatal cholestasis. The charts of 38 children born at St. Justine Hospital between 1993 and 2001 with sustained and hemodynamically significant fetal arrhythmias were studied: 19 with supraventricular tachycardia, 14 with atrial flutter, and 5 with atrio-ventricular (AV) block. Six of these 38 children presented with cholestasis. The average duration of arrhythmia was 15.7 days in the noncholestatic group, compared with 40.3 days in the cholestatic group ( P <.05). The three infants with supraventricular tachycardia who developed cholestasis survived and resolved their cholestasis, whereas 2 of 3 infants with AV block died. No infant with atrial flutter developed cholestasis. We conclude that newborns who developed tachyarrhythmia during their fetal life can show transient neonatal cholestasis. In patients with AV block, severe and irreversible liver failure could be observed. In addition, extensive collapse of the stroma and the absence of hepatocytes (foie vide) also were observed in a patient with anti-Ro antibodies.

Late neonatal lupus erythematosus onset in a child born of a mother with primary Sjögren's syndrome.

BACKGROUND: The neonatal lupus syndrome can be present as congenital heart block (CHB) or as neonatal lupus erythematosus (NLE), both seldom passively acquired autoimmune diseases. CHB starts around week 20 of pregnancy and is a lifelong event, whereas NLE is self limiting and usually starts at the 6th week of the child's age-the maximum recorded up to week 20. CASE REPORT: An asymptomatic mother with primary Sjogren's syndrome and anti-SSA/Ro52, anti-SSA/Ro60, and anti-SSB/La autoantibodies is described who, at gestational week 23 during her first pregnancy, was diagnosed as having a male fetus with CHB due to third degree atrioventricular block. The boy from the second pregnancy developed skin eruptions which clinically and by biopsy were compatible with NLE at week 20+1 post partum. CONCLUSIONS: Our case of NLE, starting at week 20+1 of age, seems to be the latest reported clinical case of NLE. Development of CHB and NLE in two consecutive boy pregnancies is unusual.

Transplacental fetal treatment improves the outcome of prenatally diagnosed complete atrioventricular block without structural heart disease.

BACKGROUND: Untreated isolated fetal complete atrioventricular block (CAVB) has a significant mortality rate. A standardized treatment approach, including maternal dexamethasone at CAVB diagnosis and beta-stimulation for fetal heart rates <55 bpm, has been used at our institutions since 1997. The study presents the impact of this approach. METHODS AND RESULTS: Thirty-seven consecutive cases of fetal CAVB since 1990 were studied. Mean age at diagnosis was 25.6+/-5.2 gestational weeks. In 33 patients (92%), CAVB was associated with maternal anti-Ro/La autoantibodies. Patients were separated into those diagnosed between 1990 and 1996 (group 1; n=16) and those diagnosed between 1997 and 2003 (group 2; n=21). The 2 study groups were comparable in the clinical presentation at CAVB diagnosis but did differ in prenatal management (treated patients: group 1, 4/16; group 2, 18/21; P<0.0001). Overall, 22 fetuses were treated, 21 with dexamethasone and 9 with beta-stimulation for a mean of 7.5+/-4.5 weeks. Live-birth and 1-year survival rates of group 1 were 80% and 47%, and these improved to 95% for group 2 patients (P<0.01). The 21 patients treated with dexamethasone had a 1-year survival rate of 90%, compared with 46% without glucocorticoid therapy (P<0.02). Immune-mediated conditions (myocarditis, hepatitis, cardiomyopathy) resulting in postnatal death or heart transplantation were significantly more common in untreated anti-Ro/La antibody-associated pregnancies compared with patients treated with steroids (0/18 versus 4/9 live births; P=0.007). CONCLUSIONS: A standardized treatment approach, including transplacental fetal administration of dexamethasone and beta-stimulation at heart rates <55 bpm, reduced the morbidity and improved the outcome of isolated fetal CAVB.