Faculty Bibliography

Autoimmune associated congenital heart block (CHB) may result from pathogenic cross-talk between inflammatory and profibrosing pathways. Incubation of macrophages with immune complexes (IC) composed of Ro60, a target of the pathologic maternal autoantibodies necessary for CHB, hY3 ssRNA, and affinity-purified anti-Ro60 antibody induces the Toll-like receptor 7 (TLR7)-dependent generation of supernatants that provoke a fibrosing phenotype in human fetal cardiac fibroblasts. We show herein that these cells are a major source of TGFbeta and that endothelin-1 (ET-1) is one of the key components responsible for the profibrosing effects generated by stimulated macrophages. Supernatants from macrophages incubated with IC induced the fibroblast secretion of TGFbeta, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGFbeta was decreased by inhibitors of the ET-1 receptors ETa or ETb or by an anti-ET-1 antibody but not by an isotype control. Exogenous ET-1 induced a profibrosing phenotype, whereas fibroblasts transfected with either ETa or ETb siRNA were unresponsive to the profibrosing effects of the IC-generated macrophage supernatants. Immunohistochemistry of the hearts from two fetuses dying with CHB revealed the presence of ET-1-producing mononuclear cells in the septal region in areas of calcification and fibrosis. In conclusion, these data support a novel role of ET-1 in linking TLR7 inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB

Systemic lupus erythematosus (SLE) is characterized by multiple autoantibodies and complement activation. Recent studies have suggested that anti-nuclear antibody (ANA) positivity may disappear over time in some SLE patients. Anti-double-stranded DNA (dsDNA) antibody titers and complement levels may vary with time and immunosuppressive treatment, while the behavior of anti-extractable nuclear antigen (ENA) over time is less well understood. This study sought to determine the correlation between historical autoantibody tests and current testing in patients with SLE. Three hundred and two SLE patients from the ACR Reclassification of SLE (AROSE) database with both historical and current laboratory data were selected for analysis. The historical laboratory data were compared with the current autoantibody tests done at the reference laboratory and tested for agreement using percent agreement and Kappa statistic. Serologic tests included ANA, anti-dsDNA, anti-Smith, anti-ribonucleoprotein (RNP), anti-Ro, anti-La, rheumatoid factor (RF), C3 and C4. Among those historically negative for immunologic markers, a current assessment of the markers by the reference laboratory generally yielded a low percentage of additional positives (3-13%). However, 6/11 (55%) of those historically negative for ANA were positive by the reference laboratory, and the reference laboratory test also identified 20% more patients with anti-RNP and 18% more with RF. Among those historically positive for immunologic markers, the reference laboratory results were generally positive on the same laboratory test (range 57% to 97%). However, among those with a history of low C3 or C4, the current reference laboratory results indicated low C3 or C4 a low percentage of the time (18% and 39%, respectively). ANA positivity remained positive over time, in contrast to previous studies. Anti-Ro, La, RNP, Smith and anti-dsDNA antibodies had substantial agreement over time, while complement had less agreement. This variation could partially be explained by variability of the historical assays, which were done by local laboratories over varying periods of time. Variation in the results for complement, however, is more likely to be explained by response to treatment. These findings deserve consideration in the context of diagnosis and enrolment in clinical trials

Background- Cardiac manifestations of neonatal lupus include conduction disease and, rarely, an isolated cardiomyopathy. This study was initiated to determine the mortality and morbidity of cardiac neonatal lupus and associated risk factors in a multi-racial/ethnic US-based registry to provide insights into the pathogenesis of antibody-mediated injury and data for counseling. Methods and Results- Three hundred twenty-five offspring exposed to maternal anti-SSA/Ro antibodies with cardiac neonatal lupus met entry criteria. Maternal, fetal echocardiographic, and neonatal risk factors were assessed for association with mortality. Fifty-seven (17.5%) died, 30% in utero. The probability of in utero death was 6%. The cumulative probability of survival at 10 years for a child born alive was 86%. Fetal echocardiographic risk factors associated with increased mortality in a multivariable analysis of all cases included hydrops and endocardial fibroelastosis. Significant predictors of in utero death were hydrops and earlier diagnosis, and of postnatal death were hydrops, endocardial fibroelastosis, and lower ventricular rate. Isolated heart block was associated with a 7.8% case fatality rate, whereas the concomitant presence of dilated cardiomyopathy or endocardial fibroelastosis quadrupled the case fatality rate. There was a significantly higher case fatality rate in minorities compared with whites, who were at a lower risk of hydrops and endocardial fibroelastosis. Pacing was required in 70%; cardiac transplantation was required in 4 children. Conclusion- Nearly one fifth of fetuses who develop cardiac neonatal lupus die of complications predicted by echocardiographic abnormalities consistent with antibody-associated disease beyond the atrioventricular node. The disparity in outcomes observed between minorities and whites warrants further investigation

One mechanism to molecularly explain the strong association of maternal anti-Ro60 Abs with cardiac disease in neonatal lupus (NL) is that these Abs initiate injury by binding to apoptotic cardiomyocytes in the fetal heart. Previous studies have demonstrated that beta(2)-glycoprotein I (beta(2)GPI) interacts with Ro60 on the surface of apoptotic Jurkat cells and prevents binding of anti-Ro60 IgG. Accordingly, the current study was initiated to test two complementary hypotheses, as follows: 1) competition between beta(2)GPI and maternal anti-Ro60 Abs for binding apoptotic induced surface-translocated Ro60 occurs on human fetal cardiomyocytes; and 2) circulating levels of beta(2)GPI influence injury in anti-Ro60-exposed fetuses. Initial flow cytometry experiments conducted on apoptotic human fetal cardiomyocytes demonstrated dose-dependent binding of beta(2)GPI. In competitive inhibition experiments, beta(2)GPI prevented opsonization of apoptotic cardiomyocytes by maternal anti-Ro60 IgG. ELISA was used to quantify beta(2)GPI in umbilical cord blood from 97 neonates exposed to anti-Ro60 Abs, 53 with cardiac NL and 44 with no cardiac disease. beta(2)GPI levels were significantly lower in neonates with cardiac NL. Plasmin-mediated cleavage of beta(2)GPI prevented binding to Ro60 and promoted the formation of pathogenic anti-Ro60 IgG-apoptotic cardiomyocyte complexes. In aggregate these data suggest that intact beta(2)GPI in the fetal circulation may be a novel cardioprotective factor in anti-Ro60-exposed pregnancies

Anti-Ro/SSA antibodies are associated with neonatal lupus (congenital heart block (CHB), neonatal transient skin rash, hematological and hepatic abnormalities), but do not negatively affects other gestational outcomes, and the general outcome of these pregnancies is now good, when followed by experienced multidisciplinary teams. The prevalence of CHB, defined as an atrioventricular block diagnosed in utero, at birth, or within the neonatal period (0-27 days after birth), in the offspring of an anti-Ro/SSA-positive women is 1-2%, of neonatal lupus rash around 10-20%, while laboratory abnormalities in asymptomatic babies can be detected in up to 27% of cases. The risk of recurrence of CHB is ten times higher. Most of the mothers are asymptomatic at delivery and are identified only by the birth of an affected child. Half of these asymptomatic women develop symptoms of a rheumatic disease, most commonly arthralgias and xerophtalmia, but few develop lupus nephritis. A standard therapy for CHB is still matter of investigation, although fluorinated corticosteroids have been reported to be effective for associated cardiomyopathy. Serial echocardiograms and obstetric sonograms, performed at least every 1-2 weeks starting from the 16th week of gestational age, are recommended in anti-Ro/SSA-positive pregnant women to detect early fetal abnormalities that might be a target of preventive therapy

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-beta activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-beta, 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-beta 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-beta Ab or TGF-beta inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-beta 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-beta 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-beta activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar

Objectives: SLE patients (pts) with moderate-to-severe disease activity (>=1 BILAG A or >=2 BILAG B domain scores) despite background immunosuppressives and corticosteroids, were randomized to placebo (PLA) or rituximab (RTX). Although differences in primary and secondary outcome measures were not observed, an exploratory analysis was performed to evaluate the imObjectives: SLE patients (pts) with moderate-to-severe disease activity (>=1 BILAG A or >=2 BILAG B domain scores) despite background immunosuppressives and corticosteroids, were randomized to placebo (PLA) or rituximab (RTX). Although differences in primary and secondary outcome measures were not observed, an exploratory analysis was performed to evaluate the impact of RTX on disease flares. Our objectives were to assess in those pts who achieved response whether RTX affected: 1) time to moderate or severe flares, 2) annualized flare rates, and 3) prednisone usage during flares. Methods: Pts who achieved response (BILAG C, D, E scores for all domains before wk52) on monthly BILAG assessments were included in this analysis. Flares, defined as increased disease activity following achievement of response, were stratified as follows: Severe flare: >=1 BILAG A or >3 BILAG B domain scores; A flare: >=1 new BILAG A domain scores; Moderate flare: 2 BILAG B domain scores. Kaplan-Meier estimates were used to assess time-to-flare. Annualized flare rates were calculated using number of flares per patient with a Poisson regression model. Only flares that occurred after the protocol-mandated prednisone taper at 12 wks were included in the analysis of prednisone use during flares. Results: Responses were achieved in 58/88 (66.0%) PLA-treated and 127/169 (75.1%) RTX-treated pts during the study. No difference was found between rituximab and placebo in preventing or delaying moderate to severe flares. However, when BILAG A flares alone were examined, rituximab reduced the risk of an A flare after achieving a response by 52 weeks (hazard ratio=0.612; p=0.0524) and lowered the annualized A flare rate (0.86 +/- 1.47 (SD) vs 1.41 +/- 2.14; p=.038). Eighty-four of 169 (49.7%) rituximab-treated patients achieved low disease activity without subsequent A flares versus 31/88 (35.2%) patients in the placebo group (p=0.027). Prednisone rescue for A flares was similar in rituximab- (24%) and placebo-treated (14%) patients (p=.204). Conclusions: No conclusions about rituximab efficacy can be drawn from this post hoc analysis. This exploratory analysis suggests that assessment of BILAG A flares may distinguish potential treatment effects with more sensitivity than BILAG B flares, which capture modest changes in disease activity. If confirmed in other studies, this observation may help in the design of more robust clinical trial protocols

Objectives: Isolated congenital heart block (CHB) is highly associated with maternal anti-Ro/SSA antibodies. CHB carries a substantial mortality, approaching 30%, and morbidity, with over 60% of surviving affected children requiring lifelong pacemakers. To date, complete atrioventricular (AV) block is irreversible. This is a rare disease and factors beyond requisite maternal autoantibody are unknown. Data from twin studies and the 10-fold increased recurrence rate of cardiac neonatal lupus (NL) in subsequent pregnancies indicate a strong genetic contribution to risk. We posit that fetal genes influence the response to maternal autoantibodies. Methods: Children of European ancestry (n=116) with cardiac NL were identified from the U.S. Research Registry for Neonatal Lupus. Cases were genotyped using the Illumina 370K SNP platform and merged with 3351 controls from the International Consortium on Systemic Lupus Erythematosus Genetics (SLEGEN). Standard quality control and admixture-adjusted tests of association were computed. Results: Outside the HLA region, a strong association was detected at 21q22, upstream from the transcriptional regulator ERG (rs743446, p=5.45E-06, OR = 2.40). Within the HLA, associated regions include PSORS1C1 (rs3130544, p = 1.94E-07, OR = 2.77) and a missense mutation (proline to serine) at TCF19 (rs7750641, p = 1.58E-07, OR = 2.79), at Class I; several variants in the MICB-NFKBIL1- LTA-TNF-LTB-AIF1 region at Class III (rs2230365, p= 1.00E-03, OR=0.46; rs2857595, p= 1.96E-09, OR = 2.37; rs3128982, p= 6.40E-06, OR=1.86; and rs3099844, p= 4.52E-10, OR= 3.34; and the C6orf10 locus at class II (rs3115553, p=2.69E-05, OR=1.81; rs6457536, pa=1.74E-05, OR=1.84; and rs7775397 (p = 1.35E-09, OR=3.30). These are consistent with our previous results (Clancy 2002). With the exception of the HLA region, no loci previously implicated in autoimmune diseases achieved genome-wide significance in the CHB children. Conclusion: These results suggest that genetic variation near ERG, PSORS1C1, LTA/TNF/LTB and C6orf10 in the fetus may promote an abnormal tissue response initiated by exposure to maternal autoantibodies. Identification of risk loci is an incremental step towards discovery of a fetal genetic component that contributes to the anti-SSA/Ro associated development of life-long cardiac damage