Faculty Bibliography

Background: The recurrence rate of anti-SSA/Ro associated congenital heart block (CHB) is 17%. Reversal of 3^rd degree block has never been achieved. Prophylactic IVIG was considered based on two presumed mechanisms of efficacy, a) saturation of FcRn to accelerate maternal IgG catabolism and decrease placental transport b) elevation of macrophage FcRIIB expression to attenuate inflammatory fetal responses. Purpose: To evaluate IVIG efficacy and safety as a preventive therapy for CHB. Methods: A multicenter open-label study based on Simon's 2-stage optimal design was initiated. Enrollment criteria included: maternal anti-SSA/Ro antibody, a previous child with CHB/rash, nd or 3^rd degree CHB in three fetuses. Results: Twenty mothers were enrolled. Sixteen children had normal PR intervals throughout the study and no manifestations of neonatal lupus. One child developed a transient rash consistent with neonatal lupus and had normal PR intervals during pregnancy and normal EKG at birth. However, the pre-determined stopping rule was reached. CHB was detected in three fetuses, at 19, 20 and 25 weeks; none followed an abnormal PR interval. One of these mothers had two previous children with CHB. Antibody titers assessed before every IVIG infusion, and at 28 wks, 34 wks and delivery were compared with values obtained at baseline. No significant changes in maternal antibody titers to SSA/Ro, SSB/La, or Ro52 were detected over the course of therapy or at delivery (P>0.05 for all comparisons). There were no changes in maternal blood pressure, severe headaches, rashes, fever or any other adverse effects related to the infusions. Neonatal weight, height, and head circumference were derived from gestational age -specific growth curves to correct for prematurity when necessary. Four (21%) of the newborns, two with CHB and two healthy, were small for gestational age (<10th centile) and 3 healthy babies (16%) were born prematurely (<37 weeks of gestation). Conclusion: IVIG at doses consistent with replacement does not prevent the recurrence of CHB or reduce maternal antibody titers. Having established safety with this protocol and feasibility of patient enrollment, subsequent studies should address the efficacy of IVIG at higher doses to exploit an anti-inflammatory effect

Purpose: The original SELENA Flare Index (SFI) performed well in the SELENA trials to separate severe from mild/moderate flares. However, in current clinical trials it is important to also separate mild from moderate flares. Method: As part of the Lupus Foundation of America's International Flare Definition and Validation initiative, the SELENA Flare Index (SFI) was revised through a series of consensus conference calls. There was an initial review and discussion of the strengths and weaknesses of the SFI in SELENA and industry trials. Each call reviewed organ specific definitions of mild, moderate and severe flare. Definitions were discussed and re-reviewed until consensus was reached. Results:The revised SFI is organ-system based, and is not linked to the SLEDAI. For each organ system, suggested clinical manifestations are given for each category, but the intended treatment decision (if it is higher) overrides the clinical description in each category. Specifically, a mild flare is assigned if there is either no treatment, or initiation of hydroxychloroquine, prednisone <= 7.5 mg/day or a non-immunosuppressive therapy. Definition of a Moderate flare is consistent with use of prednisone > 7.5 mg/day but less than 0.5 mg/kg/day, or immunosuppressive therapy (other than cyclophosphamide), and Severe flare prednisone (or equivalent) >= 0.5 mg/kg/day, cyclophosphamide, biologic treatment, or hospitalization. Three mild flares are considered a moderate flare. Conclusion: The new SFI is organ system-based. The choice of recommended treatment overrides the clinical definition when the treatment choice is higher

Purpose: Exploration of pathogenic mechanisms coupling anti-Ro/La antibodies to fetal cardiac injury has focused on the protein target of the maternal immune response. The relevance of Interferon Type I and Toll like receptor (TLR) signaling in SLE supports the potential importance of Ro associated ssRNAs in the cascade to congenital heart block (CHB). Objective: To address the hypothesis that ssRNA induces macrophage activation via TLR ligation following uptake of a complex of Ro60, hY3 or mutant pre 5S (m-pre5S) RNA and anti-Ro with release of proinflammatory and profibrotic factors which result in scarring of the conduction system and endocardial fibroelastosis. Method: In vitro conditions included evaluation of human macrophages for secretion of TNFa (inflammatory component) and transdifferentiation of human fetal cardiac fibroblasts and collagen production (fibrosing component) as well as staining of human fetal hearts. Results:The TLR component was evaluated by transfection of ssRNA. Treatment of IFNg-primed macrophages with hY3 or m-pre5S RNA (2.5mg each) significantly stimulated TNFa release (1,121+/-373pg/mL p=0.02, N=14, and 1,072+/-338pg/mL respectively vs resting macrophages 92+/-40 pg/mL, p=0.01, N=14), an effect not observed with transfected ssRNA41 (control RNA) or modified hY3 RNA (base substitution of hY3, A/U). Both theTLR7/8 antagonist IRS661(32ng/mL) and chloroquine (10mM) significantly decreased TNFa release induced by either hY3 or m-pre5S RNA (IRS661: 159+/-77pg/mL p=0.03, N=9 for hY3, and 71+/-29pg/mL p=0.03, N=9 for pre-5S; chloroquine: 267+/-89pg/mL p=0.03, N=9 for hY3, and 180+/-70pg/mL p=0.03, N=9 for pre-5S). Immune complexes generated by incubation of an IgG fraction from a CHB mother with native Ro60-hY3 (CHB IgG Ro60-hY3) significantly increased TNFa secretion compared to CHB IgG Ro60-ssRNA41 or normal IgG (healthy donor absent anti-Ro) incubated with Ro60-hY3 (687+/-248pg/mL vs 246+/-103pg/mL p=0.05, N=3 vs 18+/-11pg/mL p<0.001, N=3). Fibrosis was evaluated using the identical supernatants (sups). Transdifferentiation of fibroblasts (SMAc staining, N=5) was markedly increased by incubation with sups generated from macrophages + hY3 or m-pre5s RNA, CHB IgG Ro60-hY3, not control ssRNAs or normal IgG Ro60-hY3. IRS661 and chloroquine each abrogated induced SMAc. Collagen secretion was stimulated by sups of macs + hY3 (766+/-82ng/mL, p=0.003 vs macs) compared to ssRNA41 (150+/-30ng/mL, p=NS vs macs) or hY3-A/U (197+/-27ng/mL, p=NS vs macs), and macs + CHB IgG Ro60-hY3 (650ng/mL) compared to normal IgG Ro60-hY3 (200ng/mL). TLR7 expressing mononuclear cells were observed in a CHB heart, not normal heart, and localized near the AV groove at a site enriched in fibrosis. Conclusion: These data support a novel injury model in CHB whereby endogenous ligand, Ro60 associated ssRNA, forges a nexus between TLR ligation and fibrosis instigated by binding of anti-Ro to the target protein accessible via apoptosis

Purpose: Maternal anti-Ro antibodies are highly associated with heart block and/or cardiomyopathy (cardiac neonatal lupus (NL) in an offspring. Monozygotic twin studies and the 10 fold increased recurrence rate of cardiac NL in a subsequent pregnancy implicate a strong genetic influence on risk of disease. Here, we report the first genome-wide association study of cardiac NL. Methods: Caucasian children (n=117) with cardiac NL were identified from an extensive collection of DNA in the U.S. Research Registry for Neonatal Lupus (RRNL). Two criteria were required: 1) cardiac NL defined as heart block (1st, 2nd,3rd, degree) documented by electrocardiogram (if 1st degree), echocardiogram, history of pacemaker, or statement in the medical record; and/or presence of cardiac injury which included autopsy evidence of a mononuclear infiltrate in the endocardium, myocardium and pericardium and/or endocardial fibroelastosis on echocardiogram always associated with cardiac dysfunction 2) maternal antibodies to 52kD SSA/Ro, 60kD SSA/Ro, or 48kD SSB/La. In 96%, 2nd or 3rd degree block was present. Cardiac NL subjects were genotyped using the Illumina 370K SNP platform and merged with 3351 Illumina out-of-study controls from SLEGEN (Harley 2008). Standard quality control and admixture adjusted tests of association were computed. Results: The HLA region (6p21) showed strong evidence of association. Two HLA SNPs in high linkage disequilibrium were rs3135353 (p_dom= 3.99E-08; OR = 2.87; 2.8 kb from HLA-DRB5) and rs3129963 (p_dom= 8.96E-07; OR = 2.57; 75 kb from HLA-DRA). Outside the HLA region, rs1810636 near 20p13 showed significant association to cardiac NL (p_rec= 1.53E-12, OR = 4.07). In proximity to this region are the DNA sequences of five noncoding RNAs that associate with the spliceosome. An additional suggestive association at 5q11.2 includes rs 2432143 (p_dom=5.87E-05, OR=2.28) within the integrin, alpha 1, a receptor involved in cell-cell adhesion, which may play a role in inflammation and fibrosis. Importantly, none of the fifteen prominent non-HLA polymorphisms reported in SLE association studies were associated with cardiac NL at less than 1E-5. Conclusion: These analyses are the first genome-wide association study for cardiac NL and identify several strong statistical associations. These associations, including the HLA region, corroborate the genetic influences on cardiac NL and may provide a basis for exuberant fibrosis of the conduction system

Purpose: Perhaps with the exclusion of the elusive RBC cast, there is no single laboratory test that can unambiguously predict the presence of active renal disease in patients with SLE. Evaluation of urinary cytology is a valuable tool in identifying early transplant rejection with urinary lymphocytes (>20/10 high power field) preceding the rise in creatinine. This study addressed the value of urinary cytology as a marker of active renal disease in a consecutive sampling of SLE patients. Method: Seventy spot urine specimens were obtained from the first 50 patients fulfilling 4 ACR criteria seen in an outpatient setting. These were subjected to cytological evaluation in addition to protein/creatinine ratios. Results: Of the 50 patients, 19 have lupus nephritis defined by a kidney biopsy, 11 of which were active at the time of study as defined by a 24 hour protein excretion of >1 gram. As expected, the average protein/creatinine ratio on the spot urinalysis (UA) of patients with active nephritis compared to patients without active nephritis was 2.71 compared to.41. Clinical data on these patients further substantiated the presence of active renal disease; mean serum albumin (3.3 vs. 4.1), creatinine (1.36 vs 1.11), complement C3 (84 vs. 107), and C4 (16 vs. 21), and anti-dsDNA (228 vs. 52). Examination of the urinary cyto-diagnostic sediments (evaluated per 10 high power fields) revealed that in patients with active nephritis, there were significantly more acantocytes (dysmorphic RBCs), target, and isomorphic RBCs per high powered field compared to inactive patients 8/0, 27/1, 127/7 respectively. However, acantocytes, known to have the highest predictive value for glomerular injury, were only present in 2 patients with active nephritis, whereas all the others had none. The urinary neutrophils were higher in patients with active disease (235 vs. 63) but there was a high incidence of vaginal contamination (substantiated by the presence squamous epithelial cells in 9 of the 17 samples from active nephritis patients, and 33 of 53 inactive), After eliminating the contaminated samples, the PMNs were 131 vs 58 p=NS. The most striking difference between groups was in the number of lymphocytes, 81 vs 3 which remained significant even after removing the contaminated samples (92 vs 5, p=0.008). Of all the patients without active nephritis only 1 had urinary lymphocytes above 20 (21). Since contamination with vaginal fluid can have an impact on the presence of isomorphic RBCs we also examined the average number of RBCs after eliminating the contaminated samples, 62 vs. 12, p=NS. Conclusion: The presence of increased lymphocytes, not total WBC, on cytological urine exam suggests the diagnosis of active nephritis which may provide insight to activity vs chronicity, a critical clinical dilemma. The value of total RBCs and WBCs is diminished by contamination with vaginal fluid implying caution regarding scoring of renal domains

Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to correlate with development of congenital heart block. The aim of the present study was to evaluate the clinical relevance and predictive value of p200-antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and American women were included in the study. Sera originated from 202 mothers with an infant affected by second- or third-degree atrioventricular block (AVB), 177 mothers with rheumatic disease having infants with normal heart rate and female blood donors (n = 136). A novel serological assay for Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3.8% respectively was developed. Mothers of children affected by AVB II-III had significantly higher p200-antibody levels than mothers with rheumatic disease having children with normal heart rate (P < 0.001). In the Swedish cohort, a distinction between foetuses with normal conduction, AVB I, AVB II and III was possible. A significant difference in anti-p200 levels between AVB I and AVB II-III groups compared with foetuses with normal conduction (P < 0.05 and P < 0.01) was observed. Using p200-antibodies as a second step analysis in Ro52-positive pregnancies increased the positive predictive value for foetal cardiac involvement (AVB I, II or III) from 0.39 (0.27-0.51) to 0.53 (0.37-0.68). In conclusion, Ro52 p200-antibodies may occur in women with unaffected children, but levels are significantly higher in mothers of children with congenital heart block and are suggested as a relevant marker in evaluating the risk for foetal AV block