Faculty Bibliography

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

No new drugs have been approved for the treatment of systemic lupus erythematosus (SLE) by the Food and Drug Administration for the last 30 years. One barrier has been the lack of validated biomarkers and surrogate endpoints. Validation of SLE biomarkers in the past have been methodologically flawed. We put forth a conceptual framework and five critical criterion for validating putative biomarkers and bio-surrogates in this heterogeneous multi-system disease with protean manifestations. Using the example of a putative biomarker for end-stage lupus nephritis, we performed computer simulations for planning a biomarker bio-repository to support the validation process. 'Random time window' sampling where a biomarker is obtained in an interval randomly selected from the total follow-up time for that subject creates survival bias. This can be avoided by the 'fixed calendar window' design, in which biomarkers are measured within the same, pre-specified period for all cohort members who remain at risk during that period. In lupus nephritis where the incidence rate of end-stage renal disease is relatively low, to accumulate 300 instances of end-stage renal disease, at risk patients would have to be followed for about 5,000 person-years, implying 500 subjects followed, on average, for about 10 years. Increasing the number of biomarker determinations per subject from one to five reduces the required number of subjects by 10-15%, while further increases in the number of observations per subject yielded much smaller gains. The large numbers of subjects required for a bio-repository, makes it essential to maximize the efficiency of study designs and analyses and provides the strongest rationale for collaboration and the use of standardized measures to ensure comparability

OBJECTIVE: To evaluate responses to mycophenolate mofetil (MMF) and intravenous cyclophosphamide (CYC) in lupus nephritis in a multiethnic population. METHODS: This was a retrospective study of all patients with systemic lupus erythematosus (SLE) that underwent kidney biopsy at New York University Medical Center. Patients with followup of at least 6 months were included. Clinical response was defined as complete (return to +/- 10% of normal) or partial (improvement of 50% in abnormal renal measurements). RESULTS: Ninty-nine patients were included in the study: 86% females, 86% non-Caucasian, age 34.2 +/- 1.1 years, 62% with proliferative nephritis (PN; ISN/RPS-III and IV), and 32% with membranous nephritis (MN; ISN/RPS-V). Of the 70 patients with PN, 37 were treated with CYC and 33 with MMF. The baseline characteristics of the 2 treatment groups were different in the incidence of ISN/RPS-IV, values of serum creatinine and serum albumin, and type of insurance (p < 0.05). The response rate was greater in the MMF than in the CYC group (70% vs 41%). Responses to MMF were different in Asians (11/11), Caucasians (4/5), African Americans (3/5), and Hispanics (5/11). Responses to CYC had a similar distribution (Asians 6/10, Caucasians 4/5, African Americans 4/9, Hispanics 1/11). In the MN group (N = 23) responses were similar to the PN group (73% MMF and 38% CYC). After adjusting for race, serum creatinine, serum albumin, type of insurance, and class of nephritis, in a logistic regression model, response to MMF was superior to CYC: OR 6.2 (95% CI 1.9-20.2). Hispanics had worse outcome than Caucasians (OR 0.17). Longterm followup suggested no difference in maintenance with MMF or CYC. CONCLUSION: After controlling for the fact that less severe nephritis is preferentially treated with MMF, we found overall that response to MMF was superior to CYC. In this US population, ethnicity was observed to have an influence on response

OBJECTIVE: Individualized therapy based on genetic background and monitoring of metabolites can optimize drug safety and efficacy. Such an approach is available for azathioprine (AZA), the thiopurine antimetabolite. AZA exerts therapeutic effects when metabolized to the active thiopurine nucleotide, 6-thioguanine (6-TGN). In inflammatory bowel disease (IBD), 6-TGN levels in the target range of 235-400 pmol/8x10(8) red blood cells (RBC) are associated with a high likelihood of response. Our objective was to evaluate whether drug escalation based on metabolite levels improves efficacy and maintains safety in patients with systemic lupus erythematosus (SLE). METHODS: We conducted a 6-month open-label dose-escalation clinical study of patients with active SLE treated with azathioprine dosed by body weight and metabolite levels. The primary endpoint was >or=50% improvement in any one parameter of disease activity, or 50% decrease in glucocorticoid dose. RESULTS: Of 50 patients enrolled in the study, 21 achieved clinical responses, 13 of whom had 6-TGN<235 pmol/8 x10(8) RBC. Ten patients had no clinical response at 6 months, yet achieved either therapeutic IBD 6-TGN levels (>235, n=4) or received maximum AZA dose>or=3.5 mg/kg (n=6). In 19 patients the drug was discontinued prematurely due to side effects or SLE activity. For those patients in whom either liver function test or white blood cell count abnormalities were encountered, metabolites guided attribution to drug or disease activity. CONCLUSION: Clinical responses in SLE can occur at levels of 6-TGN lower than the target range established for IBD. During followup, measurements of AZA metabolites may provide a rational approach to safety

This review focuses on events subsequent to planning a pregnancy and addresses three components of concern for women with systemic lupus erythematosus: maternal, placental, and fetal. Flare rates are generally low for patients who are clinically stable at conception. For patients who have never had renal disease, there is no frm evidence that they will develop active renal disease simply due to being pregnant. For patients who begin pregnancy with an abnormal creatinine (> 2 mg/dl is ill advised), risks include hypertension, preeclampsia, high rate of fetal loss, and possible further deterioration of renal function. Discontinuation of angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and mycophenalate is mandatory. Elevated levels of sVEGF-1 may be a harbinger of preeclampsia. For patients with anti-phospholipid antibodies detected in the frst trimester of pregnancy, the lupus anticoagulant per se may be the strongest predictor of pregnancy complications. For women with anti-SSA/Ro antibodies the risk of having a child with congenital heart block is 2% which rises to a recurrence rate of 18%. Information on current approaches to prevention and treatment of heart complications of neo-natal lupus is provided

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: 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 the intensity and frequency of 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: The BILAG index was scored every 4 wks for 52 wks after the first study drug treatment. Only pts who achieved response (BILAG C, D, or E scores for all domains before wk 52) were included in this analysis. A severe flare was defined as >= 1 BILAG A or > 3 BILAG B domain scores in a pt who previously achieved response; an A flare was defined as >= 1 new BILAG A domain scores; a moderate flare was defined as 2 BILAG B domain scores following response. Kaplan-Meier estimates were used to assess time to flare. Annualized flare rates were calculated using the number of flares a patient experienced during the study; the p-value was based on Poisson regression model. Only flares that occurred after the protocol-mandated prednisone taper at 12 wks were incorporated into the analysis of prednisone usage during flares. Results: A response was achieved in 58 (66.0%) of 88 PLA-treated and 127 (75.1%) of 169 RTX-treated pts during the study, and these patients qualified for subset analysis. Whereas the times to first moderate or severe flare were not different in the two treatment groups, there was a trend towards a prolonged time to first A flare in the RTX group (HR=0.612; p=0.052). Annualized severe and moderate flare rates were similar in both groups, but the mean annualized A flare rate in the RTX group was significantly lower than in the PLA group (0.86 vs 1.41; p=0.038). Prednisone was increased in 20 of 101 (20.7%) of the A flares and was not significantly different between the RTX and PLA groups (24% vs 14%; p=0.204). Pts with the highest initial prednisone dosing (> 60 mg/day) had lower treatment rates (14.0%). In one month, 44/81 (54.3%) of the untreated A flare improved to B or better. Conclusion: There was no difference between PLA and RTX in preventing moderate or severe flares after a response was achieved. However, RTX treatment was associated with a lower annualized A flare rate, and a trend towards prolonging the time to A flare. Prednisone rescue following A flares was low compared to observational cohorts, possibly reflecting a reluctance to reinitiate prednisone, or reduced BILAG A severity, in this trial

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