Faculty Bibliography

Background/Purpose: Towards understanding the molecular mechanisms that link maternal anti-Ro antibodies to the development of conduction system disease in a second trimester fetus, single cell (scRNA-seq) and bulk RNA-seq were applied to a fetal heart dying with complete congenital heart block (CHB) and a gestational age-matched healthy heart from an elective termination.
Method(s): The CHB heart was obtained from a 20-week fetus identified to have complete block at 19 weeks. The mother (35 y/o Asian with SS on no hydroxychloroquine) declined dexamethasone or IVIG and elected to terminate, thus no exposure to maternal medications confounded interpretation of findings. Both hearts were obtained under identical conditions. Freshly collected single-cell suspensions were generated using a Langendorff preparation with cannulation and perfusion of the aorta with collagenase and trypsin enzymes. Two approaches were taken to mine the transcriptome in the resulting cell suspensions: agnostic evaluation applying 10X Genomics platform-based scRNA-seq and low input RNA-seq of flow sorted cells upon leukocytes (DAPI negative, CD45+) and fibroblasts (DAPI negative, CD45-, podoplanin-positive).
Result(s): For scRNA-seq, we obtained 2,693 and 5,408 high-quality scRNA-seq profiles from the control and CHB hearts, respectively. We applied a graph-based clustering method and identified 13 and 14 major clusters of cells from the control and CHB hearts, respectively, as visualized by t-distributed stochastic neighbor embedding (t-SNE). Differential gene expression analysis guided by established lineage markers revealed four cardiomyocyte clusters (CM1-CM4), three fibroblast clusters (FB1-FB3), endothelial cells (EC), erythroblasts (EB), macrophages (MAC), dendritic cells (DC), Tcells (TC) and B cells (BC). Ranked by abundance, the control heart exhibited CM>FB>EC>MAC>DC>EB, BC, TC; the CHB heart exhibited CM>FB>EC, MAC>TC, BC, EB. The CHB heart also contained natural killer cells (NK) and mast cells (MC, lowest abundance). Given the high abundance of MACs among the immune cells (control:108;CHB:606) and the consistent identification of MACs on histologic analysis of CHB hearts, differential expression analysis demonstrated overexpression of interferon-induced genes (4-fold or greater, i.e. log2(CHB-control)>2) in CHB MACs. In CHB, most cell types expressed high levels of ISG1, IFITM1 and IFITM3, whereas in the control only IFITM3 showed widespread expression. For SIGLEC1, expression was restricted to MACs and was expressed by 18% of CHB MACs and only 6% of control MACs. While the transcriptome using low input RNA-seq of anti-CD45 flow-sorted CHB leukocytes did not allow granular analysis of leukocyte subpopulations, expression of SIGLEC1 and interferon-related genes were increased in CHB versus control. Applying 10X Genomics, proliferating fibroblasts expressed MKI67 and TOP2A in CHB but not control fibroblasts.
Conclusion(s): This unprecedented opportunity to obtain CHB tissue absent any exposure to maternal medications support scRNA-seq's utility to survey landscape and heterogeneity not possible with low input RNA-seq of flow-sorted cells. IFNand SIGLEC1-positive macrophages may contribute to fibrosis

Background/Purpose: Systemiclupus erythematosus (SLE) disproportionally affects women of childbearing age. Low disease activity for 6 months prior to conception leads to the best outcomes; however, there is little prospective data describing the relative frequency and predictors of flares during and after pregnancy under such conditions.
Method(s): Analyses used data from the PROMISSE study, a multicenter, prospective observational study (2003-2014) of 384 pregnant women meeting >=4 ACR SLE criteria. Subjects were enrolled <12 wks gestation and samples collected throughout pregnancy and post-partum. Exclusion criteria were multi-fetal pregnancy, active disease (prednisone >20 mg/ d), or renal disease (proteinuria >1 gm/24h, and creatinine > 1.2 mg/dL). Mild/moderate and severe flares were defined using the SELENA-SLEDAI Flare Index. Flares during pregnancy were assessed in all 384 patients, and post-partum flares in those with study visits 2-6 months post-partum. Logistic regression models were fit to the data to identify independent predictors of experiencing any type of flare during pregnancy and post-partum.
Result(s): Rates of Flare: 105 flares were recorded during pregnancy (3.8% 1st Trimester, 53.3% 2nd, 42.9% 3rd). Counting one flare per person, 100 of 384 patients (26%) flared at any point during pregnancy; 20.8% of patients had mild/ moderate flares and 6.25% had severe. 57 of 234 patients (24.4%) with a study visit 2-6 months post-partum flared; 22.7% had mild/moderate flares and 1.7% severe. Post-partum flares were mild, and 19 of 57 (33.3%) were treated; 13 with an increase in prednisone, 6 with NSAID or hydroxychloroquine, and 1 with mycophenolate mofetil. The rate of any type of flare was 0.39/person-year at any point during pregnancy and 0.84/person-year post-partum. The proportion of subjects who had any flares during and after pregnancy was similar, but the post-partum flares occurred over a shorter duration of follow-up. Correlates of Flare: Among baseline variables considered (Table) only age, ethnicity/race, low complement, and PGA were independently predictive of having any flare during pregnancy. Clinical features associated with adverse pregnancy outcome (platelet count, antihypertensive use, and LAC) were not predictive of flare. The mean time from the last visit during pregnancy to the post-partum visit was 20 weeks and ranged from 9 to 34 weeks. Neither baseline clinical variables nor clinical variables of the last visit during pregnancy were associated with occurrence of any post-partum flare.
Conclusion(s): Flares during pregnancy are correlated with clinical and serological activity during the first trimester. Flares during and after pregnancy are typically mild, infrequently require treatment, and occur at similar rates. (Table Presented)

OBJECTIVE:Cutaneous neonatal lupus (cNL) occurs in possibly 5%-16% of anti-Ro±anti-La antibody-exposed infants. Data suggest in utero exposure to hydroxychloroquine (HCQ) may prevent cardiac NL. The aim was to assess whether in utero exposure to HCQ decreases the risk of cNL and/or delays onset. METHODS:A multicentre case-control study was performed with 122 cNL cases and 434 controls born to women with a rheumatological disease who had documentation of maternal anti-Ro±anti-La antibodies at pregnancy and confirmation of medication use and the child's outcome. A secondary analysis was performed on 262 cNL cases, irrespective of maternal diagnosis, to determine if HCQ delayed time to cNL onset. RESULTS:Twenty (16%) cNL cases were exposed to HCQ compared with 146 (34%) controls (OR 0.4 (95% CI 0.2 to 0.6); p<0.01). Exposure to HCQ was associated with a reduced risk of cNL; exposure to anti-La antibody and female gender were associated with an increased risk of cNL. Exposure to HCQ remained significantly associated with a reduced cNL risk in the analyses limited to mothers with systemic lupus erythematosus and those who developed rash ≤1 month. When analysing all 262 cNL cases, HCQ-exposed infants were older (6.0 (95% CI 5.7 to 6.3) weeks) at cNL onset versus HCQ-non-exposed infants (4.4 (95% CI 3.9 to 5.0) weeks), but the difference was not statistically significant (p=0.21). CONCLUSION/CONCLUSIONS:Exposure to HCQ was associated with a reduced risk of cNL. Among cNL cases, those exposed to HCQ tend to have later onset of rash. Both findings suggest a protective effect of HCQ on cNL.

Objectives/UNASSIGNED:The study aimed to determine if serum albumin at 12 months predicts long-term renal outcome at 48 months. Data from the NYU SAMPLE (Specimen and Matched Phenotype Linked Evaluation) Lupus Registry were used to compare the performance of albumin, anti-double-stranded DNA, C3/C4, proteinuria and haematuria. Methods/UNASSIGNED:82 patients with SLE with data at time of renal biopsy, at 12 months and at a second visit, and up to 48 months were included. The significance of each biomarker as a predictor of an adverse renal outcome (ARO), defined as doubling of serum creatinine, as creatinine >4 mg/dL if initial >2.5 mg/dL or ESRD, was evaluated in univariate and exploratory multivariable Cox proportional hazards models. Hazard ratios (HRs) for ARO with 95% CIs were generated. The receiver operating characteristic (ROC) curves at 48 months were used to identify the optimal cut-off point for albumin and proteinuria to predict ARO. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for albumin and proteinuria. Results/UNASSIGNED:Serum albumin and proteinuria had statistically significant HRs for ARO (0.140 and 1.459, respectively). The model with both albumin and proteinuria indicated no additional independent contribution of proteinuria to albumin alone. The ROC curves identified cut-offs of 3.7 g/dL for albumin and 0.964 urine protein to creatinine ratio for proteinuria. Albumin had a sensitivity of 94%, specificity of 87%, PPV of 64% and NPV of 98%. Conclusions/UNASSIGNED:This study demonstrates serum albumin >3.7 g/dL is a predictor of a favourable long-term renal outcome. These results support the inclusion of albumin as an outcome in lupus nephritis trials and treat-to-target guidelines.

Objective/UNASSIGNED:To survey an international sample of providers to determine their current practices for the prevention, screening, and treatment of congenital heart block (CHB) due to maternal Ro/SSA antibodies. Methods/UNASSIGNED:A survey was designed by the organizing committee of the 9th International Conference of Reproduction, Pregnancy and Rheumatic Diseases. It was sent to attendants of the conference and authors of recent publications or abstracts at ACR 2012, 2013 or 2014 on rheumatic diseases and pregnancy. Results/UNASSIGNED:In anti-Ro/SSA positive women, 80% of 49 respondents recommended screening by serial fetal echocardiogram (ECHO), with most starting at week 16 (59%) and stopping at week 28 (25%), although the time to stop varied widely. For women without a prior infant with neonatal lupus, respondents recommend every other week (44%) or weekly (28%) fetal ECHOs. For women with a prior infant with neonatal lupus, 80% recommend weekly fetal ECHOs. To prevent CHB, HCQ was recommended by 67% of respondents and most would start pre-pregnancy (62%). Respondents were asked about medications to treat varying degrees of CHB in a 20-week pregnant, anti-Ro and La positive SLE patient. For first degree, respondents recommended starting dexamethasone (53%) or HCQ (43%). For second degree, respondents recommended starting dexamethasone (88%). For third degree, respondents recommended starting dexamethasone (55%) or IVIg (33%), although 27% would not start treatment. Conclusion/UNASSIGNED:Despite the absence of official guidelines, many physicians with a focus on pregnancy and rheumatic disease have developed similar patterns in the screening, prevention and treatment of CHB.

Background/UNASSIGNED:We examined the usefulness of erythrocyte-bound C4d (EC4d) to monitor disease activity in SLE. Methods/UNASSIGNED:Data and blood samples were collected from three different studies, each of which included longitudinal evaluations using the Physicians Global Assessment (PGA) of disease activity and the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) SLE Disease Activity Index (SLEDAI), which was assessed without anti-double-stranded DNA (dsDNA) and low complement C3/C4 (clinical SELENA-SLEDAI). EC4d levels were determined using flow cytometry; other laboratory measures included antibodies to dsDNA, C3 and C4 proteins. Relationships between clinical SELENA-SLEDAI, PGA and the laboratory measures were analysed using linear mixed effect models. Results/UNASSIGNED:The three studies combined enrolled 124 patients with SLE (mean age 42 years, 97% women, 31% Caucasians and 34% African-Americans) followed for an average of 5 consecutive visits (range 2-13 visits). EC4d levels and low C3/C4 status were significantly associated the clinical SELENA-SLEDAI or PGA in each of the three study groups (p<0.05). Multivariate analysis revealed that EC4d levels (estimate=0.94±0.28) and low complement C3/C4 (estimate=1.24±0.43) were both independently and significantly associated with the clinical SELENA-SLEDAI (p<0.01) and PGA. EC4d levels were also associated with the clinical SELENA-SLEDAI (estimate: 1.20±0.29) and PGA (estimate=0.19±0.04) among patients with chronically low or normal C3/C4 (p<0.01). Anti-dsDNA titres were generally associated with disease activity. Conclusion/UNASSIGNED:These data support the association of EC4d with disease activity regardless of complement C3/C4 status and its usefulness in monitoring SLE disease. Additional studies will be required to support these validation data.

OBJECTIVE:To characterize and compare the factors associated with tubulointerstitial inflammation (TII) and tubulointerstitial scarring, defined as interstitial fibrosis and/or tubular atrophy (IF/TA), in patients with lupus nephritis (LN). METHODS:We identified SLE patients with a renal biopsy consistent with LN between 2005 and 2017. Clinical data was collected from the medical records. Multivariable logistic regression models were fitted to assess factors associated with TII and with IF/TA (moderate-to-severe vs. none-or-mild). RESULTS:at the time of biopsy. In addition, the presence of moderate-to-severe TII and older age were also associated with moderate-to-severe IF/TA. None of the routinely available serologic markers, including anti-dsDNA, anti-Ro/La antibodies and low complement were associated with tubulointerstitial damage (TID). CONCLUSIONS:The use of hydroxychloroquine was strongly associated with less inflammation, while the finding of TII, proliferative LN, and low eGFR were the major determinants of the presence of tubulointerstitial scarring. Identifying modifiable factors is critical for the development of better preventive and therapeutic strategies directed towards ultimate improvement in survival in patients with lupus-related kidney disease.

Objectives/UNASSIGNED:To describe glucocorticoid (GC) use in the SLICC inception cohort and to explore factors associated with GC use. In particular we aimed to assess temporal trends in GC use and to what extent physician-related factors may influence use. Methods/UNASSIGNED:Patients were recruited within 15 months of diagnosis of SLE from 33 centres between 1999 and 2011 and continue to be reviewed annually. Descriptive statistics were used to detail oral and parenteral GC use. Cross sectional and longitudinal analyses were performed to explore factors associated with GC use at enrolment and over time. Results/UNASSIGNED:We studied 1700 patients with a mean (s.d.) follow-up duration of 7.26 (3.82) years. Over the entire study period, 1365 (81.3%) patients received oral GCs and 447 (26.3%) received parenteral GCs at some point. GC use was strongly associated with treatment centre, age, race/ethnicity, sex, disease duration and disease activity. There was no change in the proportion of patients on GCs or the average doses of GC used over time according to year of diagnosis. Conclusion/UNASSIGNED:GCs remain a cornerstone in SLE management and there have been no significant changes in their use over the past 10-15 years. While patient and disease factors contribute to the variation in GC use, between-centre differences suggest that physician-related factors also contribute. Evidence-based treatment algorithms are needed to inform a more standardized approach to GC use in SLE.

BACKGROUND:Immunoglobulin M (IgM) autoreactivity to malondialdehyde (MDA) protein modifications is part of the natural antibody repertoire in health and may have beneficial functions. In contrast, IgG anti-MDA are increased in chronic inflammation and autoimmunity and may instead have pathogenic properties. METHODS:Herein, we investigated serum IgG anti-MDA levels by enzyme-linked immunosorbent assay (ELISA) in 398 systemic lupus erythematosus (SLE) patients in the Swedish Karolinska SLE cohort and compared these to findings in 225 US SLE patients from New York University and Johns Hopkins University. RESULTS:In two independent cohorts, IgG anti-MDA levels correlated positively with disease activity by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; p < 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p < 0.0001) for high anti-MDA IgG levels with active disease (SLEDAI ≥ 6). Furthermore, IgG anti-MDA correlated directly with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), soluble tumor necrosis factor receptors (sTNFR-1, sTNFR-2), and vascular cell adhesion molecule 1 (VCAM-1) measurements, and inversely with complement factors (C1q, C2, C3, C4). Importantly, IgG anti-MDA levels were significantly elevated in SLE patients with active nephritis (p = 0.0005) and correlated with cystatin C estimated glomerular filtration rate and albuminuria. CONCLUSIONS:Elevated IgG anti-MDA in SLE patients was associated with high disease activity, with active lupus nephritis, and with biomarkers of systemic inflammation. This natural antibody reactivity may have potential prognostic utility, and may also actively contribute to pathogenesis.