Faculty Bibliography

One of the strongest associations with autoantibodies directed to components of the SSA/Ro-SSB/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring, an alarming prospect facing 2% of primigravid mothers with these reactivities. This risk is 10-fold higher in women who have had a previously affected child with CHB. Anti-Ro/La antibodies are necessary but insufficient to cause disease. In vitro and in vivo experiments suggest that the pathogenesis involves exaggerated apoptosis, macrophage/myfibroblast crosstalk, TGFbeta expression and extensive fibrosis in the conducting system and in some cases surrounding myocardium. A disturbing observation is the rapidity of disease progression, with advanced heart block and life-threatening cardiomyopathy observed <2 weeks from normal sinus rhythm. Once 3rd degree (complete) block is identified, reversal has never been achieved, despite dexamethasone. Current strategies include the evaluation of an early echocardiographic marker of injury, such as a prolonged PR interval and the use of IVIG as a preventative measure for pregnancies of mothers with previously affected children

OBJECTIVE: To study the membrane expression of endothelial protein C receptor (mEPCR) in the renal microvasculature in lupus nephritis (LN) as a potential marker of injury and/or prognostic indicator for response to therapy. METHODS: mEPCR expression was analysed by immunohistochemistry in normal kidney and in 59 biopsies from 49 patients with LN. Clinical parameters were assessed at baseline, 6 months and 1 year. RESULTS: mEPCR was expressed in the medulla, arterial endothelium and cortical peritubular capillaries (PTCs) in all biopsies with LN but not in the cortical PTCs of normal kidney. Positive mEPCR staining in >25% of the PTCs was observed in 16/59 biopsies and associated with poor response to therapy. Eleven (84.6%) of 13 patients with positive staining for mEPCR in >25% of the PTCs and follow-up at 6 months did not respond to therapy, compared with 8/28 (28.6%) with mEPCR staining in < or =25% PTCs, P = 0.0018. At 1 year, 10 (83.3%) of 12 patients with positive mEPCR staining in >25% of the PTCs did not respond to therapy (with two progressing to end-stage renal disease) compared with 8/24 (33.3%) with positive staining in < or =25% of the PTCs, P = 0.0116. Although tubulo-interstitial damage (TID) was always accompanied by mEPCR, this endothelial marker was extensively expressed in the absence of TID suggesting that poor response could not be attributed solely to increased TID. mEPCR expression was independent of International Society of Nephrology/Renal Pathology Society class, activity and chronicity indices. CONCLUSION: Increased mEPCR expression in PTCs may represent a novel marker of poor response to therapy for LN

One of the strongest clinical associations with autoantibodies against components of the SSA/Ro-SSB/La ribonucleoprotein complex is the development of congenital heart block in an offspring, an alarming prospect facing 2% of primigravid mothers with these reactivities. This risk is increased tenfold in women who have had a previous child with congenital heart block. Accumulated evidence suggests that anti-SSA/Ro and anti-SSB/La antibodies are necessary but insufficient for fetal disease. Basic and clinical research is heavily focused on identifying fetal and environmental factors that convert disease susceptibility to disease development. A disturbing observation that has emerged from current research efforts is the rapidity of disease progression, with advanced heart block and life-threatening cardiomyopathy being observed less than 2 weeks after detection of a normal sinus rhythm. Once third-degree block is unequivocally identified, reversal has never been achieved, despite dexamethasone treatment. Accordingly, strategies aimed at preventing disease before irrevocable scarring ensues assume a high priority. One approach has been the implementation of serial echocardiography to monitor for a prolonged PR interval. Intravenous immunoglobulin is being evaluated as a potential prophylactic approach in mothers who have previously had an affected child

OBJECTIVE: To evaluate the frequency of anti-alpha-enolase antibodies in the sera of mothers whose children have congenital heart block (CHB), given provocative results in which alpha-enolase, a membrane protein, was recognized by monoclonal antibodies reactive with the peptide p200 of 52 kDa Ro/SSA in a neonatal rat heart library. METHODS: An ELISA using a recombinant alpha-enolase protein was developed. Sera from 100 anti-Ro52+ CHB mothers in the Research Registry for Neonatal Lupus, 50 patients with systemic lupus erythematosus (SLE; 7 anti-Ro52+), and 48 healthy controls were tested for anti-alpha-enolase reactivity. RESULTS: There were no significant differences in the median values obtained from CHB mothers, patients with SLE, or controls at each of the dilutions tested. Only 7 (7%) at 1:100 dilution and 2 (2%) at 1:1000 dilution of 100 CHB sera were 3 standard deviations above the mean value obtained for controls. Preincubation with recombinant Ro52 did not inhibit anti-alpha-enolase reactivity. CONCLUSION: The low frequency of anti-alpha-enolase antibodies in the sera of CHB mothers and the absence of apparent cross-reactivity with Ro52 suggest that antibodies to Ro52 are not likely to mediate CHB via binding to alpha-enolase

Purpose: A major barrier to understanding and treating lupus nephritis (LN) is the paucity of sensitive and validated biomarkers. Recent evidence has suggested that two markers found on the endothelium of LN biopsies merit focus. Adiponectin is expressed on the endothelium of all vessels in biopsies from patients with LN but decreased in areas of fibrosis and/or inflammation. Adiponectin knockout mice suggest that adiponectin may be a key regulator of proteinuria. Increased expression of membrane EPCR in LN biopsies predicts a poor response to therapy. Based on these vascular clues, this study leveraged the LN induction trial comparing intravenous cyclophosphamide (IVC) and mycophenolate mofetil (MMF) to evaluate the relationship between clinical response of LN and levels of adiponectin and sEPCR as a proxy for vascular protective molecules. Methods: The endothelial markers, adiponectin, sEPCR, e-selectin, and nitric oxide were measured in 109 plasma from 48 patients enrolled in the LN induction trial. Response was evaluated based on reaching a primary endpoint with a prespecified decrease in urine protein/creatinine ratio and stabilization or improvement in serum creatinine. The span of the induction period was 24 weeks. Sample collection included visits 4 (4wks), 7 (15 wks) and 9 (24 wks). Results: There was a consistent trend toward increased levels of plasma adiponectin in responders vs nonresponders (19.2 +/- 6.8 vs 16.4 +/- 9.1 at visit 4; 13.0 +/- 5.2 vs 11.7 +/- 6.5 at visit 7; 13.7 +/- 7.8 vs 10.9 +/- 4.9 at visit 9). In patients with subnephrotic proteinuria (<3g/day urine protein; 63% of the total), plasma adiponectin was similarly increased in responders vs nonresponders at all visits. Moreover, when combining data across all visits nonreponders had significantly lower adiponectin (p=0.0032). There was a tendency of sEPCR to decrease in responders vs nonresponders (243 +/- 164 vs 284 +/- 167 at visit 4; 338+/- 271 vs 341 +/- 197 at visit 7; 260 +/- 104 vs 368 +/- 216 at visit 9). In comparing MMF vs IVC, sEPCR, levels were significantly higher in the IVC group when data was combined over all visits (p=0.005). Consistent with evidence that therapy in the responder arm mobilizes vascular protective molecules, levels of nitric oxide changed in the predicted direction (62 +/- 47 vs 68 +/- 66 at visit 4; 39 +/- 46 vs 52 +/- 65 at visit 7; 27 +/- 33 vs 92 +/- 55 at visit 9; p=0.02). Combining data across all visits, nonresponders had significantly higher NO levels than responders (p=0.046). The differences in plasma NO were not accounted for by clearance as fractional excretion of NO did not differ between responders and nonresponders. Levels of sE selectin did not track with response. Conclusion: These results demonstrate longitudinal associations between increased adiponectin and decreased sEPCR levels as a combined biomarker of renal response. Accordingly, vascular protection tracks with favorable outcomes

Purpose: The mechanisms underlying premature atherosclerosis in SLE are not understood. The endothelium merits focus since it provides the physiologic boundary which limits extravasation and diapedesis of inflammatory cells. Methods: One hundred and nineteen patients with SLE, predominantly non-Caucasian, and 71 healthy controls matched for age, sex and race, underwent carotid ultrasonography and donated blood for evaluation of circulating endothelial cells (CEC), soluble endothelial protein C receptor (sEPCR) and gene polymorphism at A6936G, soluble E-selectin, and adiponectin. Results: Carotid plaque was more prevalent among patients than controls (43% vs 17%, p=0.0002). Mean CCA IMT was greater in patients compared to controls (0.59mm+/-0.19 vs 0.54mm+/-0.11, p=0.03). Levels of CEC (19 vs 3 CECs/mL, p<0.0001) and sE-selectin (64 vs 36 ng/ml, p<0.0001) were significantly elevated in patients compared to controls. Unexpectedly, adiponectin was also significantly higher in patients compared to controls (16 ug/mL versus 11 ug/mL, p=0.0001) but no differences were seen in the levels of sEPCR or the distribution of genotype. Independent predictors of plaque status using logistic regression models included: age (p<0.0001; OR=2.1 per 10 year increase; 95% CI: 1.5-3.0), SLE status (p=0.015; OR=3.4 for SLE vs control; 95% CI: 1.3-9.1), sE-selectin (p=0.016; OR=1.2 per 10 unit increase; 95% CI: 1.0-1.4) and adiponectin (p=0.050; OR=1.5 per 10 unit increase; 95% CI: 1.0-2.4). Comparing SLE patients with and without plaque, there were no differences in cardiac CRP, complement, anti-dsDNA ab, CEC, sEPCR levels and EPCR SNP. However, sE-selectin and adiponectin levels were significantly higher in SLE with plaque compared to those without (sE-selectin 78 vs 52 ng/ml; p=0.006; adiponectin 18 vs 14 ug/ml; p=0.033). The estimated odds ratios for plaque in the final logistic regression model were: OR_selectin= 1.3 per 10 ng/ml increase (95% CI: 1.1-1.5) and OR_adiponectin=1.8 per 10 ug/ml increase (95% CI: 1.1-3.0). SELENA-SLEDAI scores were similar between groups, and the proportion of patients with SLEDAI<= 4 did not segregate with the absence of plaque. Neither past nor current medications significantly associated with plaque. In the stable subjects (SLEDAI <=4), age (p=0.007), sE-selectin (p=0.02) and adiponectin (p=0.02) remained associated with plaque. The prevalence of plaque was greatest in the stable patients with high sE-selectin plus high adiponectin (55%; p =0.0009) confirming the multivariable analyses. Sixty-two patients donated blood at a second visit. High sE-selectin and adiponectin were sustained in plaque patients compared to non-plaque patients (p=0.0009 and p=0.0011 respectively). Conclusion: These results confirm that SLE patients, irrespective of race, are at increased risk for premature atherosclerosis and support the hypothesis that endothelial perturbation is contributory even in the absence of clinically measurable disease activity

Purpose: To investigate the effect of IVIg on the idiotype (id)-antiidiotype (anti-id) network in pregnant women with anti-Ro/SSA and anti-La/SSB antibodies who had a previous history of a child with CHB (high risk pregnancies). Method: The interaction of IVIg with the synthetic peptides was investigated by direct ELISA assays as well as inhibition experiments. Ten mothers who previously gave birth to a child with CHB were treated during a subsequent pregnancy with IVIG using a protocol in the Preventive IVIG Therapy for Congenital Heart Block (PITCH) Study. All individuals exhibited ELISA reactivity against epitope 349-364 of La/SSB in their initial samples. Sequential sera were drawn from all mothers during pregnancy (before each IVIg infusion at 12, 15, 18, 21, 25 and 28, 34, delivery), and evaluated for antibodies against the epitope 349-364 (pep) of La/SSB (idiotypic), as well as its complementary epitope (cpep) and anti-349-364 Fab2 fragments (both anti-idiotypic). Anti-349-364 reactivity was also evaluated after removal of anti-Id antibodies. Results: IVIg contains low affinity antibodies, most probably polyreactive antibodies, binding to both pep and cpep. The reactivity of anti-cpep (anti-id) antibodies appeared to be higher, compared to anti-pep antibodies. A partial inhibition of idiotype-antiidiotype binding (30%), was observed after IVIg treatment of purified antibodies. Following dosing of IVIg the maternal antibody titres to the major epitope of La/SSB decreased by 20% to 50% in four patients, remained stable in five and increased in one by 30%. In contrast, there was a substantial increase of anti-Id reactivity in all patients, ranging from 20% to 250%. After removal of anti-Id antibodies the Id reactivity was increased (up to 300%) in all patients. None of the children developed any conduction abnormality. Conclusion: This study supports the novel finding that IVIg administration decreases autoantibody titres and enhances the anti-idiotypic antibody response in pregnant women with anti-Ro/SSA and anti-La/SSB antibodies

Purpose: Two percent of neonates born to mothers with SSA/Ro autoantibodies have congenital heart block (CHB). It has been hypothesized that macrophage engagement of Toll-like receptors (TLR) via binding to the ssRNA moiety of the target autoantigen exposed on the surface of the fetal cardiocytes is involved in the pathogenesis. Using a RNA-based TLR agonist and an antagonist of TLR7 and TLR9, activation of TLR was studied by TNFalpha release. Method: Macrophages were derived from CD14+ monocytes of healthy donors cultured in suspension with growth medium RPMI1640/10% FBS and 10ng/ml GM-CSF for at least 7 days. Cells were plated at 4x10⁵ per well into 12-well plates and adhered macrophages were treated with TLR7 or TLR8 agonists in the absence or presence of antagonist for 24 hours. The levels of TNFalpha in culture supernatants were measured by ELISA. Cytotoxicity of compounds for macrophages was assessed in MTS assay. Expression of TLR7 and TLR8 mRNA was determined in RT-PCR. Results: Macrophages from different donors showed variability in the levels of TLR7 and TLR8 mRNA expression and corresponding variability in response to TLR agonists.TLR7 agonist R837 (imiquimod, Invivo Gen) at 5mg/ml induced statistically significant TNFalpha release from 31.6 to 457.7+/-85.9pg/ml (p=0.0033, N=7). RNA-based TLR8 agonist at 20mg/ml induced statistically significant TNFa release from 31.6 to 621+/- 124.6pg/ml (p=0.0029, N=7). The novel TLR antagonist at 80mg/ml (nontoxic for macrophages) reduced the induced TNFalpha release by TLR8 agonist (20mug/ml) from 621+/-124.6 to 11.4+/-5.9pg/ml (N=3). Endosomal acidification neutralizing agent, chloroquine at a nontoxic concentration of 5mg/ml inhibited the induced TNFa secretion by R837 (5mg/ml) from 457.7+/-85.9 to 184.6+/-31.07 (p=0.0086, N=6) and by TLR8 agonist (20mug/ml) from 621+/-124.6 to 4.4 pg/ml (N=2). No toxicity was seen in macrophages exposed for 24 hours to TLR antagonist at concentrations up to 100 mug/ml (MTS assay). In contrast, after a 24-hour exposure to 21mg/ml chloroquine only 75% of the macrophages were viable in MTS assay. The LC₅₀ value for chloroquine was 40mug/ml. Conclusion: Variability of TLR7 and TLR8 expression in isolated human macrophages from different donors may be relevant to the inflammatory cascade to CHB and discordant disease in fetuses exposed to anti-Ro antibodies. Given the profound TLR inhibition, antimalarial compounds, already in use during human pregnancy, and TLR antagonist studied herein may be relevant candidates for study in the prevention of disease

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