Faculty Bibliography

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

Passively acquired anti-Ro associated congenital heart block (CHB) likely results from pathologic crosstalk between inflammatory and fibrosing pathways eventuating in scarring. The target, Ro60, complexed with uridine rich ssRNA induces TLR7-dependent macrophage production of supernatants capable of trans-differentiating human fetal cardiac fibroblasts. This study addressed the molecular components responsible for the TLR-dependent profibrosing effects. In seeking the link between an inflammatory response of macrophages and the profibrotic effect on fibroblasts, a microarray analysis comparing the mRNA expression profile of macrophages stimulated with hY3 (ssRNA associated with Ro60) or immune complexes consisting of Ro60-hY3-IgG fraction containing anti-Ro60 antibodies (IC) in the presence or absence of IRS661 (antagonist of TLR7) was performed. Gene expression of the vasoconstrictor endothelin-1 (ET-1), recently shown to promote dermal fibrosis, was significantly upregulated by ~ 4 fold and confirmed by RT-PCR. Incubation of macrophages with either hY3 or IC increased secretion of ET-1 from 2.64 pg/mL (baseline) to 9.67 pg/mL (p <.0001) and 7.52 pg/mL (p =.0003) respectively. Pre-treatment with IRS661 decreased hY3-induced secretion to 3.83 pg/mL and IC to 3.98 pg/mL. The direct effect of ET-1 (100 nM) on these fibroblasts (shown to express both ETa and ETb receptors) resulted in a profibrosing phenotype as demonstrated by a) TGFbeta secretion (13 pg/mL vs. 772 pg/mL, p =.03), b) increased collagen release (18 ng/mL baseline vs. 772 ng/mL, p =.05, and c) expression of a-smooth muscle actin (alpha-smac). ET-1-induced TGFbeta secretion was significantly decreased (p =.03) by each of the following: BQ-123, an ETa antagonist (119 pg/mL), BQ-788, an ETb antagonist (145 pg/mL), and anti-ET-1 antibody (211 pg/mL), but not isotype control antibody (691 pg/mL). Similarly, ET-1-induced collagen secretion was significantly decreased (p =.05) by BQ-123 (147 ng/mL collagen), BQ-788 (247 ng/mL), anti-ET-1 antibody (253 ng/mL), and anti-TGFbeta antibody (239 ng/ml), but not isotype control (815 ng/mL). Predictably, pretreatment of fibroblasts with either BQ-123, BQ-788 or incubation of supernatants with anti-ET-1 antibody, but not isotype control, significantly inhibited the profibrosing readouts (increased TGFbeta, collagen and alpha-smac) induced by supernatants from macrophages stimulated with either hY3 or IC. Additionally, fibroblasts transfected with either ETa or ETb siRNA, but not scrambled siRNA, also significantly inhibited the profibrosing readouts induced stimulated macrophages. Immuno-histochemistry of a heart from a fetus dying with CHB revealed ET-1/2/3 antibody staining but not isotype IgG control in the septal region in areas showing calcification, fibrosis, and mononuclear cell infiltrates (not seen in an age-matched fetal heart). In conclusion, these data suggest that macrophage secretion of ET-1 may be one mechanism linking TLR inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB

Purpose: Organ injury induced by antibodies characteristic of Sjogren Syndrome and Systemic Lupus Erythematosus, while varied in the adult and fetus, may share in common a link between apoptosis and ultimate fibrosis. In congenital heart block (CHB), binding of maternal anti-Ro/La antibodies to apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Immunological staining of CHB hearts reveals AV node TGFb staining and TGFb activation promotes fibroblast trans-differentiation, a scarring phenotype. Since the uPA/uPAR system plays a role in TGFb activation, this study evaluated whether anti-Ro/La binding to apoptotic cardiocytes via plasmin activation stimulates TGFb and promotes a profibrosing phenotype. Methods and Results: Initial analysis showed increased TGFb in supernatants from co-cultures of healthy cards and apoptotic cards incubated with IgG fractions from mothers whose sera contain anti-Ro/La antibodies and who had a child with CHB (apo-CHB-IgG) compared to co-cultures of healthy cards and apoptotic cards incubated with control IgG (apo-nl-IgG). Using a luciferase bioassay of active TGFb, supernatants from co-cultures of healthy cards and apo-CHB-IgG cards exhibited increased levels of active TGFb compared to those cocultured with apo-nl-IgG cards (511pg/ml CHB-IgG RLU vs 217 nl-IgG RLU; p=0.007; n=7). Abrogation of RLU via anti-TGFb antibody or TGFb inhibitor (SB431542) confirmed TGFb activation was solely due to TGFb. Significantly increased uPA levels (903 pg/ml vs 508 pg/ml; p =0.01; n=5) and uPA activity (0.8 units vs 0.04 units; p=0.005; n=3) were demonstrated in supernatants generated from coculture of healthy cards and apo CHB-IgG cards compared to healthy cards and apo nl-IgG, respectively. To determine whether uPA activity was responsible for TGFb activation, coculture experiments were conducted in which the apo-CHB-IgG cards were treated with anti-uPAR or anti-uPA antibodies or the plasmin inhibitor aprotinin prior to coculturing with healthy cards. In all instances treatments attenuated TGFb activation and uPA activity. To evaluate the profibrotic role of the observed TGFb activation, supernatants from either apo-CHB-IgG or apo-nl-IgG cocultures with healthy cards were applied to serum deprived cardiac fibroblasts. Only supernatants derived from cocultures of healthy cards and apo-CHB-IgG cardiocytes promoted transdifferentiation as evidenced by increased SMAc staining, an effect that was decreased when fibroblasts were treated with supernatants where cocultures were pretreated with uPAR antibodies. Supporting the hypothesis that increased uPA activity is causally related to the pathogenesis of CHB, cord blood samples from 12 of 18 children with CHB exhibited increased uPA activity and uPAR cleavage compared to 4 of 14 non CHB children exposed to maternal anti-Ro antibodies. Conclusions: These data suggest that binding of anti-Ro/La antibodies to apoptotic cardiocytes by virtue of increased uPAR-dependent uPA activity trigger TGFb activation thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar

Purpose: To analyze whether quantitative scores on a multidimensional health assessment questionnaire (MDHAQ) provide clues to the likelihood of inflammatory versus non-inflammatory symptoms and concomitant fibromyalgia, an important challenge in clinical care, according to a global scale for noninflammatory symptoms completed by a rheumatologist in 50 patients with SLE seen in usual care. Methods: A cross-sectional study was performed in 50 consecutive SLE patients of one rheumatologist seen in usual care. On arrival at the clinic, patients completed a multidimensional health assessment questionnaire (MDHAQ) which includes scales for physical function (FN), 0-10 visual analog scales for pain (PN), global estimate (PTGL) and fatigue (FT), and a review of systems symptom checklist (SX). SLE patients also completed a self-report Systemic Lupus Assessment Questionnaire (SLAQ). The rheumatologist, unaware of MDHAQ and SLAQ scores, recorded a physician global estimate (MDGL) and an estimate of non-inflammatory symptoms, each scored on a 0-3 scale in 0.1 increments, as well as four SLE indices: SLEDAI-2K (SLE Disease Activity Index), BILAG (British Isles Lupus Assessment Group index), SLAM (SLE Activity Measure) with and without laboratory tests, and ECLAM (European Consensus Lupus Activity Measurement). SLE patients with scores of <0.5 on the noninflammatory symptom scale were regarded as low and those with scores >=0.5 high noninflammatory symptoms; the two groups were compared using the Mann-Whitney statistic. Results: The study included 45 women and 5 men, mean age 38.7 years, mean disease duration 7.3 years. Of the 50 patients, 16 had high and 34 low scores for non-inflammatory symptoms. Those with high scores for non-inflammatory symptoms had significantly higher scores for FN, PN, FT, PTGL, SX, SLAQ, and SLAM without laboratory tests, as well as significantly lower CRP. No significant differences were seen patients estimated as high and low scoring patients for SLEDAI, BILAG, SLAM, ECLAM, C3, C4, antiDsDNA, or ESR. Fewer than 50% of low patients had FN, PN, PTGL, or FT >=2, while 100% of high patients had FT >2, and 94% PTGL >2. All patients with high non-inflammatory symptoms (16/16) reported more than 5 SX, compared to 15/34 (44%) low patients, and 12/16 (75%) high patients reported >10 SX, compared to 6/34 (18%) low patients. (Table Presented) Conclusion: High scores for dysfunction, pain, fatigue, global estimates, and number of symptoms are common in SLE patients with high versus low levels of non-inflammatory symptoms. SLE indices do not distinguish between patients with high versus low levels of non-inflammatory symptoms. A simple global scale to estimate non-inflammatory symptoms may be informative in therapeutic decisions, particularly if consistent with patient questionnaire patterns

Purpose: To analyze agreement levels between patient (PT) and physician (MD) assessments in 50 patients with SLE seen in usual care, including a) global PT and MD estimates of status; b) patient self-report scores on the SLAQ (Systemic Lupus Activity Questionnaire) and MDHAQ (Multidimensional Health Assessment Questionnaire) for physical function (FN), pain (PN), patient global estimate (PTGL), fatigue (FT), RAPID3 (FN, PN, and PTGL), and review of systems checklist (SX); c) physician-scored indices SLEDAI-2K (SLE Disease Activity Index), BILAG (British Isles Lupus Assessment Group index), SLAM (SLE Activity Measure) and ECLAM (European Consensus Lupus Activity Measurement). Methods: A cross-sectional study was performed in 50 consecutive SLE patients of one rheumatologist. Patients completed the SLAQ and MDHAQ, including PTGL. The rheumatologist scored a physician global estimate (MDGL) (scored 0-3 in 0.1 increments) without knowledge of PTGL, and completed the SLEDAI 2K, BILAG, SLAM, and ECLAM. Agreement levels of various measures were analyzed using Spearman rank order correlations. Results: The study included 45 women and 5 men, mean age 38.7 years, mean disease duration 7.3 years, 36% Caucasian, 18% Black, 26% Hispanic, 18% Asian. The mean MDGL (1.10+/-0.62), PTGL (3.11 +/-2.81) and SLE indices (SLEDAI 5.02+/-3.75; BILAG 4.60+/-4.31; SLAM 3.86+/-2.92; ECLAM 1.97+/-1.37) indicated mild/moderate lupus activity. The correlation between MDGL and PTGL of rho=0.14 was not statistically significant. Correlations between MDGL and SLE indices were significant, rho=0.60-0.72 (p<0.001). Correlations between PTGL and patient measures also were significant, rho=0.58-0.87 (p<0.001). However, PTGL was correlated at lower levels with SLE indices - significantly with BILAG and SLAM (0.35-0.40; p<0.01), and not significantly with SLEDAI or ECLAM. MDGL was not correlated significantly with any patient measure or index. (Table Presented) Conclusion: MDGL and PTGL are not correlated significantly, an observation made previously. MDGL was correlated significantly with all physician-derived indices, and PTGL was correlated significantly with all patient-derived measures and indices. By contrast, MDGL was correlated at much lower, nonsignificant levels with patient-derived measures and indices, and PTGL was correlated at lower levels with physician-derived indices. Further analysis of these discordances may clarify the clinical relevance of various measures in clinical trials, and may lead to improved care and compliance in patients with SLE

Purpose: Coagulation is one of the first pathways to be elicited by vascular injury, and its activation is followed by proinflammatory phenomena, in part due to loss of the anti-inflammatory activity of both the Protein C pathway and membrane Endothelial Protein C receptor (mEPCR). It has been recently demonstrated that mEPCR is highly expressed in the cortical peritubular capillaries of kidneys from patients (pts) with active lupus nephritis compared to normal human kidney. Profound upregulation of mEPCR was observed even in areas absent tubulointerstitial damage. This study addressed the hypothesis that changes in the microvasculature extend beyond the clinically targeted organ and that dysregulation is a fundamental characteristic of SLE. Methods: The study included SLE pts in whom renal disease was considered active as assessed by proteinuria and urinary sediment. Renal biopsies were performed in all pts. Thirty skin biopsies from non-lesional nonsunexposed skin (buttocks) were obtained in 26 pts (23 females, 3 males) and five healthy controls (4 females, 1 male). The paraffin skin sections were individually stained with specific antibodies against mEPCR and adiponectin (protective markers), ICAM-1 (proinflammatory) and CD31 (pan endothelial marker). Immunohistochemistry (IHC) was scored by counting peroxidase-brown labeled blood vessels (10-20 microns in diameter) without knowledge of the clinical information associated with the biopsy. The number of blood vessels with an intensity of at least 1+ were quantitatively scored with ranges 1-12. To account for the number of blood vessels per slide, the CD31 count had to be 12 to be included in the analysis. Results: The 28 renal biopsies comprised the following ISN/RPS classifications: 4 Class III, 7 Class IV, 8 Class V, I Class VI, 3 Class III/V, 3 Class IV/V. Nineteen percent of the pts had a GFR <60 (mean GFR, 82 ml/min). Abnormal laboratory values for complement and anti-dsDNA antibodies were reported in 72% and 75% of pts, respectively. Nephrotic range proteinuria was present in 37%. For IHC skin assessments of the controls, the mean score for mEPCR was 1 (highest 2), ICAM-1 was 4 (highest 7) and adiponectin was 1 (highest 2). In 17/25 (68%) of the SLE non-lesional non-sun exposed skin sections, mEPCR was expressed above the highest control. In 16/30 (53%) ICAM-1 staining exceeded 7. In contrast, only 6/25 (19%) expressed adiponectin above 2. For each specific stain there were no apparent differences between biopsy class, degree of proteinuria, presence of anti dsDNA or low complement levels. However, pts with mEPCR staining above 2 had higher GFR measurements than those with staining < 2 (88 ml/min +/- 31 versus 53 +/- 32, p= 0.0168). In contrast, GFR was unrelated to ICAM-1 and adiponectin expression. Conclusion: These data are consistent with the notion that there is widespread activation of the microvasculature. The capacity of endothelial cells to utilize anticoagulation pathways is not restricted to the kidney and expression of mEPCR in the microcirculation likely represents an attempt to limit microvascular inflammation in kidney and skin

Objective: In patients with systemic lupus erythematosus (SLE), IRF5 genotype is associated with anti-Ro autoantibodies. It is not clear whether this association with autoantibody profile is specific to SLE disease status. To examine this question, we assessed IRF5 genotypes in a large cohort of individuals who all had high titer anti-Ro autoantibodies and carried a variety of diagnoses including healthy/asymptomatic, Sjogren's syndrome (SS), and SLE. Methods: We studied 85 European-ancestry individuals recruited to the Research Registry for Neonatal Lupus who all had high titer anti-Ro autoantibodies and a child with neonatal lupus. The diagnoses of these subjects were as follows: 15 healthy/asymptomatic, 10 SLE, 19 SLE/SS, 20 SS, and 21 undifferentiated autoimmune disease (UAS). IRF5 SNPs were genotyped using Taqman primer-probe sets to define previously reported European-derived SLE risk and protective haplotypes. Results: The IRF5 SLE-risk haplotype defined by the rs10488631 C allele was enriched in subjects of all diagnoses except SS when compared to large published data sets of European-American controls [OR=2.30 (1.50-3.32), p=8.4x10^-5]. Each of the asymptomatic, SLE, SLE/SS, and UAS mothers showed a very similar increase in rs10488631 allele frequency when examined individually (ORs range from 2.00 to 3.15, no significant differences between the asymptomatic, SLE, SLE/SS and UAS groups). The rs10488631 C allele frequency in the subjects diagnosed with SS was essentially the same as controls (OR=1.05). Interestingly, the rs3807306 C allele was significantly increased in the SS patients as compared to subjects from the other 4 diagnostic categories [OR=2.34 (1.14-4.79), p=0.026]. Conclusions: There was a significant increase in the frequency of the IRF5 SLE-risk haplotype in subjects with anti-Ro antibodies with varying diagnoses which was of comparable magnitude to the anti-Ro/IRF5 association observed in SLE (OR<2). Interestingly, the SS cohort was distinct from the other neonatal lupus mothers with respect to IRF5 genotype, and a different SNP was associated with SS status. These data suggest that the genetic association of IRF5 with autoimmunity is strongly influenced by serologic profile, and that the anti-Ro association with IRF5 genotype extends beyond the SLE disease state to some degree

Purpose: The classic cardiac manifestations of neonatal lupus (cardiac-NL) include a spectrum of conduction dysfunction (1^st, 2^nd, or 3^rd degree heart block (CHB)) and more rarely cardiomyopathy which can be absent any conduction disease. This study was undertaken to update the mortality data of cardiac-NL in a large US based cohort and identify associated risk factors to further understand the pathogenesis of anti-SSA/Ro mediated injury and provide evidence based data for counseling women with these antibodies. Methods: Three hundred and one children with cardiac-NL (295 with CHB and 6 with isolated cardiomyopathy) enrolled in the Research Registry for Neonatal Lupus (RRNL) had sufficient medical records for review. The RRNL database was analyzed for the following potential mortality maternal risk factors: age at pregnancy, race/ethnicity, anti-SSB/La antibody status, health status and fetal risk factors: time of diagnosis, exposure to maternal non-fluorinated and fluorinated steroids during pregnancy, method of delivery, and gender. In addition, morbidity was assessed by the frequency of pacemaker placement and cardiac transplant. Results: Follow up of the children ranged from in utero death to adulthood. Of the 301 children with cardiac-NL, 53 (17.6%) died. Thirty percent died in utero or at birth, 41% died prior to six months of postnatal life and the remaining 29% died after 6 months. Mortality was higher among children born to non-Caucasian mothers compared to Caucasian mothers (33% vs 15% p=.0003). Maternal age was equivalent between the groups (29.1 dead vs 29.7 live). The maternal presence of anti-SSB/La antibodies was 74% for those whose children died and 63% for those whose children survived, which was not significant. A maternal diagnosis of Sjorgen's Syndrome and/or Systemic Lupus Erythematosus was not significantly associated with cardiac-NL death (53% in death vs 44%) suggesting that prior knowledge of maternal antibody status did not influence mortality. With regard to fetal factors, 86% of those who died were diagnosed with cardiac-NL during pregnancy compared to 85% who survived. For those diagnosed during pregnancy there was a trend towards early gestational diagnosis for those children that died compared to those that survived (21 vs 23.5 weeks p=.09). There was also a trend toward higher exposure to maternal fluorinated steroids after the diagnosis in children that died (53% vs 40% p=.09), however there was no difference in maternal use of non-fluorinated steroids in those that died vs those that survived (19% vs 16%). Most fetuses were delivered by C-section and this was not significantly associated with death (70% dead vs 75% live). Female gender was also not associated with outcome (49% who died were female vs 52% live). Eighty-five percent of children received a pacemaker, 43% within 9 days of birth, 20% between 9 days and one year. Five children (2%) received a heart transplant. Conclusion: Based on data from this large cohort, 17.6% of children born with cardiac-NL die from complications of the disease. Eighty-five percent required pacing and two percent required cardiac transplantation. Mortality was more prevalent in children born to non Caucasian mothers