Faculty Bibliography

Objective: The risk of fetal atrioventricular block (AVB) in anti-Ro exposed pregnancies approximates 2% with no prior affected pregnancies. Antibody titer may be contributory to AVB, but there are no data on the negative predictive value (NPV) of antibody titer identifying fetuses unlikely to develop AVB, thus averting costly serial echocardiographs currently recommended for all anti-Ro pregnancies. Using a commercial core laboratory specifying anti-Ro52 and anti-Ro60 antibodies, we sought to develop a threshold level identifying fetuses unlikely to develop AVB, thus averting costly echo surveillance currently recommend for all anti Ro pregnancies.
Study Design: We performed a retrospective multicenter (Children's Hospital Colorado fetal database and NYU Research Registry for Neonatal Lupus) review of women screened for anti-Ro antibodies by several local commercial laboratories because of rheumatic disease or a previous child with AVB. Serum from anti-Ro positive women was sent to a 2nd commercial core laboratory at Associated Regional and University Pathologists laboratories (Salt Lake City, UT) to determine anti-Ro52 and anti-Ro60 antibody (TheraDiag, France) levels. We calculated the NPV individually of anti-Ro52 and anti-Ro60 and the two combined (anti-Ro) using a logistic regression model and a parallel testing strategy.
Result(s): Of 332 women, 142 had a child with AVB and 190 never had a child with AVB. An anti-Ro60 threshold of <101 AU/mL achieved 90% NPV for AVB with 79 titers falling below the threshold (Figure). The logistic regression model with both anti-Ro52 and anti-Ro60 using a threshold of a predicted probability <22% achieved a 91% NPV with a larger sample size below the cutoff (n=87) than any other method (Table).
Conclusion(s): While we could not determine a cutoff for no AVB, the likelihood of AVB developing if the maternal anti-Ro60 titer <101 AU/mL is 10%. Our logistic regression model gives a likelihood of 9% to develop fetal AVB below the threshold. These data may help to guide surveillance of future anti-Ro positive pregnancies. [Formula presented] [Formula presented]
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Background/Purpose: Current management of lupus nephritis (LN) is guided by histopathological features on kidney biopsy and measurement of proteinuria. Urine proteomics is a non-invasive source of novel biomarkers which may better reflect the complex dynamic immunobiology of LN in real time. Two composite measures include CKD273, which can predict the risk of progression of chronic kidney disease in the general population, and LN120, which was designed to diagnose LN. Both are multidimensional urine proteomic classifiers consisting of 273 or 120 peptides, respectively, with major components including collagen fragments, abundant blood-derived proteins, and proteins involved in inflammation. We investigated the ability of these classifiers to predict traditional biopsy features and disease response in LN.
Method(s): A total of 31 adults with biopsy-proven LN were included in this study. All participants met the SLICC and 2019 EULAR/ACR Classification Criteria for SLE based on a spot urine protein-to-creatinine ratio of >0.5 and class III, IV, and/or V LN on renal biopsy. Urine samples were collected at week 0 (at the time of renal biopsy) and week 12 and then subjected to peptidome analysis using a capillary electrophoresis-mass spectrometry (CE-MS) platform. This peptidome data was used to calculate CKD273 and LN120 classifiers at each time point. LN response status was determined at week 52 based on proteinuria, creatinine, and prednisone dosage (no more than 10 mg daily). Spearman's rank correlation and t-tests were used to compare proteomic classifiers with renal biopsy characteristics and response.
Result(s): At week 0, both CKD273 and LN120, but not proteinuria, exhibited a moderate to strong correlation with histological activity index on renal biopsy (Figure 1; rho = 0.65 with p = 0.00024 for CKD273; rho = 0.47 with p = 0.013 for LN120). CKD273 also correlated with chronicity index (rho = 0.54, p = 0.0037). Neither classifier significantly correlated with lupus nephritis ISN class. With respect to response, CKD273 and LN120 were not significantly different between groups at week 0. However, a reduction in LN120 was observed in 100% of complete responders, 60% of partial responders, and 50% of non-responders at week 12 (Figure 2). The magnitude of this change in LN120 in complete responders versus non-responders did not reach statistical significance (p = 0.13), though this is potentially because of the small number of responders with CE-MS data available at both time points (n = 4). CKD273 did not significantly change with time in any response group (Figure 3).
Conclusion(s): This work provides proof of concept that urine proteomic classifiers can noninvasively predict histological activity and chronicity in LN. Complete responders, but not partial responders or non-responders, exhibited an impressive numerical decrease in LN120 by week 12, suggesting that proteomic scores may track with and predict a durable treatment response. Larger studies are needed to validate these findings

Background/Purpose: Based on inhibition of viral replication and limited reports on clinical efficacy, hydroxychloroquine (HCQ) was initially considered as a prophylaxis and treatment of COVID-19. Despite this optimism, more extensive reports have significantly dampened the promise of efficacy, however cardiac toxicity has surfaced raising attention to this complication. Although HCQ is generally considered safe during pregnancy based on studies in patients with systemic lupus erythematous and other rheumatic conditions, this initiative leveraged a unique opportunity to evaluate neonatal electrocardiograms (ECGs) in the context of HCQ levels to address any potential cardiotoxicity.
Method(s): Neonatal ECGs and HCQ blood levels were available in a recently completed study evaluating the efficacy of HCQ 400mg daily to prevent the recurrence of congenital heart block associated with anti-SSA/Ro antibodies. The ECGs of affected newborns who met the primary outcome of advanced block were not included in this safety study so that the results only reflect those infants with no clinical cardiac disease. Using the Bazett formula to correct for heart rate, corrected QT (QTc) intervals were calculated and compared to age-matched normal values. For reference, the median (2nd percentile - 98th percentile) values for QTc were 413 (378-448) msec in males, and 420 (379-462) msec in females. QTc intervals were recorded in the absence of knowledge of the HCQ levels. Values exceeding 448 msec for males and 462 msec for females were considered abnormal. Levels of HCQ were assessed during each trimester of pregnancy and in the cord blood, providing unambiguous assurance of drug exposure.
Result(s): There were 45 ECGs available for interpretation within the first 4 months of life in unaffected infants. Overall, there was no correlation between cord blood levels of HCQ and the QTc (R = 0.02, P = 0.86) or the average value of HCQ levels obtained during each individual pregnancy and cord blood and the QTc (R = 0.04, P = 0.80), as shown in Figure 1A and Figure 1B. Likewise there was no correlation between the average of the maternal HCQ levels obtained at each trimester and delivery plus cord levels and the QTc on the ECGs of the 31 infants evaluated on day of life 1-4 (R = 0.08, P = 0.63) or those of the 14 children older than 4 days (R = 0.01, P = 0.95). Maternal values of HCQ were sustained throughout pregnancy and delivery (Figure 2). Mean QTc values were nearly identical between those in the highest and lowest quartiles of cord blood HCQ levels (P = 0.57) and between the highest and lowest quartiles of average HCQ levels during pregnancy (P = 0.54) (Figure 3A and 3B). Among these 45 infants, only 5 had prolongation of the QTc (11%; 95% CI: 4% - 24%), 2 marked and 3 marginal. No arrhythmias occurred in any neonate that was not known to have heart block.
Conclusion(s): In aggregate, these data provide reassurances that the maternal use of HCQ is not associated with a high incidence of QTc prolongation in the neonate

Background/Purpose: Treatment of lupus nephritis relies on renal histopathological features. However, renal biopsies do not capture patient-specific active biological pathways. Urine proteomic biomarkers could revolutionize the diagnosis and management of lupus nephritis by predicting active intrarenal biological pathways and can be noninvasively monitored over time.
Method(s): One thousand proteins were quantified (RayBiotech) in a total of 112 longitudinal urine samples from 30 SLE patients with active lupus nephritis and 7 healthy controls (HC). The proteins and molecular pathways detected in the urine proteome at the time of biopsy were then analyzed with respect to lupus nephritis class, response to treatment after 1 year, histopathological features (activity and chronicity indeces), and trajectory over time (baseline and week 12, 26, and 52). The intrarenal expression of candidate biomarkers was evaluated using single cell transcriptomics of renal biopsies from patients with active lupus nephritis.
Result(s): There were 237 proteins (FDR < 10%) enriched in the urine of patients with lupus nephritis reflecting several molecular pathways involving chemotaxis, extracellular matrix remodeling, and activation of neutrophils and platelets. Hierarchical clustering using urine proteomics segregated SLE patients into 2 groups, with 80% of complete responders clustering together. This finding could not be similarly reproduced using standard features including baseline proteinuria, creatinine, histologic activity or chronicity scores, or class, indicating unique informative features of urine proteomics (Fig. 1). Patients with proliferative lupus nephritis (class III or IV) had stronger activation of chemotaxis pathways. IL-16 was the urinary protein most significantly increased in proliferative disease compared to membranous (FC 6, p=0.002) (Fig. 2A). Assessment of urine proteins that correlated with histologic activity kidney highlighted IL-16 as the single most strongly correlated protein with histologic activity (r=0.69, p=9.5.10-5; Fig. 2B). IL-16 concentration was independent of the amount of proteinuria and progressively diminished over time in patients who were responding to immunosuppression (Fig. 2C). Single cell RNA sequencing revealed significant intrarenal expression of IL16 by all infiltrating immune cells and highlighted IL16 as the second most expressed cytokine in lupus nephritis kidneys out of a compendium of 236 cytokines (Fig. 3A-B).
Conclusion(s): Urine proteomics can noninvasively identify active and biologically relevant pathways in lupus nephritis. Integrated urine proteomics and renal single cell transcriptomics revealed that IL-16, a CD4 ligand with chemotactic and proinflammatory functions, was one of the most expressed cytokine in lupus nephritis. As a urine proteomic biomarker, IL-16 may predict renal histological activity and could be monitored over time to assess response to immunosuppression. Urinary IL-16 is independent of proteinuria thus potentially providing actionable clinical information that is not captured by currently used biomarkers. Further studies are ongoing to validate these findings

Background - Based on inhibition of viral replication and limited reports on clinical efficacy, hydroxychloroquine (HCQ) is being considered as prophylaxis and treatment of COVID-19. Although HCQ is generally considered safe during pregnancy based on studies in patients with systemic lupus erythematous and other rheumatic conditions, there may still be reluctance to institute this antimalarial during pregnancy for the sole purpose of antiviral therapy. Methods - To provide data regarding any potential fetal/neonatal cardiotoxicity, we leveraged a unique opportunity in which neonatal electrocardiograms (ECGs) and HCQ blood levels were available in a recently completed study evaluating the efficacy of HCQ 400mg daily to prevent the recurrence of congenital heart block associated with anti-SSA/Ro antibodies. Results - Forty-five ECGs were available for QTc measurement, and levels of HCQ were assessed during each trimester of pregnancy and in the cord blood, providing unambiguous assurance of drug exposure. Overall, there was no correlation between cord blood levels of HCQ and the neonatal QTc (R = 0.02, P = 0.86) or the mean of HCQ values obtained throughout each individual pregnancy and the QTc (R = 0.04, P = 0.80). In total 5 (11%; 95% CI: 4% - 24%) neonates had prolongation of the QTc > 2SD above historical healthy controls (2 markedly and 3 marginally) but ECGs were otherwise normal. Conclusions - In aggregate, these data provide reassurances that the maternal use of HCQ is associated with a low incidence of infant QTc prolongation. However, if included in clinical COVID-19 studies, early postnatal ECGs should be considered.

Background/Purpose: Neonatal Lupus (NL) is a model of passively acquired autoimmunity conferred by exposure to maternal anti-Ro antibodies with major manifestations being congenital heart block (CHB) and/or cutaneous disease. This study was initiated to address the development of de novo autoimmunity in these children and identify associated clinical and genetic risk factors.
Method(s): In a retrospective cohort study of enrollees in the Research Registry for Neonatal Lupus (RRNL), 511 children exposed to anti-Ro in utero responded to a follow up questionnaire focused on symptoms of autoimmunity. Self-reported diseases were confirmed via medical record review. Bivariate analyses were performed with potential risk factors for the development of autoimmune disease (AD) and included the NL status per se, a disease severity score based on mortality risk factors, and maternal AD (inclusive of lupus, Sjogren's syndrome, psoriasis, rheumatoid arthritis, or thyroid disease). A subset of 99 CHB, 9 cutaneous, and 55 unaffected anti-Ro exposed RRNL individuals were genotyped at Class II HLA DRB1 and DQB1 four-digit alleles, which were assigned by imputation (HIBAG) or sequencing. Generalized estimating equations (logit link, exchangeable correlation) were used to test for associations between HLA alleles and the development of AD.
Result(s): Of the respondents, 182 offspring had CHB, 95 had cutaneous only NL and 234 were siblings without NL. Females comprised 53% and 80% were Caucasian. The mean age was 14.2+/-9.7; 4% age 0-2 years, 48% 2-13 years, and 47% > 13 years. An AD developed in 38 offspring (20 CHB, 7 cutaneous NL, 11 non-NL siblings; Table 1). The most prevalent AD was thyroid disease. The development of an AD was significantly associated with presence of CHB vs. cutaneous only or non-NL siblings (11% vs. 5%, p=0.033). The maternal health status did not influence the development of an AD in the child (7% mothers with AD vs. 6% asymptomatic mothers, p=0.67). Mean NL severity score was higher in offspring with AD (3.8+/-4.8 vs. 2.2+/-4.0, p= 0.031). Other markers of fetal CHB disease severity were associated with subsequent AD development, including in-utero exposure to fluorinated steroids (15% vs. 6%, p=0.088) and beta agonists such as terbutaline (23% vs. 9%, p=0.043). In the study of 163 RRNL cases with HLA data (20 with AD, 143 without), HLA DRB1*03:01 (OR 3.4, CI 1.46-7.90, p=0.0045), DQA1*05:01 (OR 3.39, CI 1.16-9.92, p=0.0262), and DQB1*02:01 (OR 4.28, CI 1.73-10.62, p=0.0017) were associated with increased risk of AD (of note, these loci are in high linkage disequilibrium). In contrast, these alleles were not significantly associated with development of CHB (99 CHB vs. 64 without).
Conclusion(s): The development of an autoimmune disease was more common in anti-Ro exposed children with CHB, greater NL severity, and MHC Class II haplotypes. These factors may relate to an inherent susceptibility to inflammation and fibrosis, occuring in utero and later in life

Background/Purpose: Patients with systemic lupus erythematosus (SLE) are at increased risk of premature atherosclerosis and thrombosis. Hydroxychloroquine (HCQ) is widely used in the treatment of SLE and has been considered of benefit for overall vascular health albeit studies to address this benefit at the cellular level have been limited. Accordingly, this study was initiated to investigate the relationship between HCQ use and dose with platelet activity, the platelet transcriptome, and vascular functional readouts.
Method(s): Patients fulfilling ACR or SLICC criteria for SLE were consecutively recruited for platelet evaluation with the only exclusion being on nonsteroidal anti-inflammatory medications, aspirin or anticoagulants. At enrollment, blood was collected for hematology analysis using the Sysmex XN-1000 analyzer, platelet aggregation via the Helena AggRAMTM system, and platelet RNA isolation and storage. Microvascular function was assessed via sublingual sidestream darkfield imaging. Brachial artery reactivity testing was used to evaluate large vessel function. Stored platelet RNA was isolated and analyzed by RNA sequencing (Illumina HiSeq4000 Sequencing).
Result(s): Among 132 SLE subjects, 108 were on HCQ. Mean age was 39.9 +/- 13.0 and 97% were female. Lupus disease activity at the time of blood draw assessed by the SELENA-SLEDAI activity index was 3.44 (range 0-20). Demographics and SLE disease activity did not differ between those on versus off HCQ (Table 1). Platelet count and size were not different between groups (Figure 1A). Platelet aggregation in response to submaximal ADP at multiple concentrations was lower in participants on HCQ (Figure 1B). Consistently, there was an inverse relationship between HCQ dosing and platelet aggregation in response to ADP (2uM: R=-0.213, P=0.037; 1uM: R=-0.310, P=0.0025; 0.4uM: R=-0.376, P=0.00018; Figure 1C). Since no subjects were on aspirin (or any other antiplatelet therapy at enrollment), aggregation in response to arachidonic acid (AA) was robust and similar between groups. However, after incubating platelets with aspirin (3mM) in vitro, platelet aggregation in response to AA was lower in the HCQ group compared to non-HCQ group (P=0.035, Figure 1B). To investigate the potential mechanisms of HCQ induced lower platelet aggregation, we evaluated platelet RNA sequencing in 49 subjects (8 no HCQ, 41 on HCQ). Positive regulation of pathways related to platelet activation (and in particular, P-selectin expression) was inversely related to HCQ, especially with higher doses (Figure 1E). In terms of vascular function, subjects on HCQ had improved microvascular function as noted by an increased proportion of sublingual capillaries filled with RBCs (P=0.011) and smaller perfused boundary region (PBR, P=0.010). HCQ dosing correlated with PBR (R=-0.599, P=0.002, Figure 1H) and RBC Filling (R=-0.592, P=0.002, Figure 1I). BART also trended positively with HCQ dose (R=0.385, P=0.094; Figure 1J).
Conclusion(s): These findings suggest that HCQ may provide benefit for vascular health in SLE as supported by ex vivo experiments demonstrating decreased platelet aggregation and downregulation of platelet functional pathways as well as improved vascular readouts

BACKGROUND:Experimental and clinical evidence support the role of macrophage Toll-like receptor signaling in maternal anti-SSA/Ro-mediated congenital heart block (CHB). OBJECTIVES/OBJECTIVE:Hydroxychloroquine (HCQ), an orally administered Toll-like receptor antagonist widely used in lupus including during pregnancy, was evaluated for efficacy in reducing the historical 18% recurrence rate of CHB. METHODS:This multicenter, open-label, single-arm, 2-stage clinical trial was designed using Simon's optimal approach. Anti-SSA/Ro-positive mothers with a previous pregnancy complicated by CHB were recruited (n = 19 Stage 1; n = 35 Stage 2). Patients received 400 mg daily of HCQ prior to completion of gestational week 10, which was maintained through pregnancy. The primary outcome was 2° or 3° CHB any time during pregnancy, and secondary outcomes included isolated endocardial fibroelastosis, 1° CHB at birth and skin rash. RESULTS:By intention-to-treat (ITT) analysis, 4 of 54 evaluable pregnancies resulted in a primary outcome (7.4%; 90% confidence interval: 3.4% to 15.9%). Because 9 mothers took potentially confounding medications (fluorinated glucocorticoids and/or intravenous immunoglobulin) after enrollment but prior to a primary outcome, to evaluate HCQ alone, 9 additional mothers were recruited and followed the identical protocol. In the per-protocol analysis restricted to pregnancies exposed to HCQ alone, 4 of 54 (7.4%) fetuses developed a primary outcome as in the ITT. Secondary outcomes included mild endocardial fibroelastosis (n = 1) and cutaneous neonatal lupus (n = 4). CONCLUSIONS:These prospective data support that HCQ significantly reduces the recurrence of CHB below the historical rate by >50%, suggesting that this drug should be prescribed for secondary prevention of fetal cardiac disease in anti-SSA/Ro-exposed pregnancies. (Preventive Approach to Congenital Heart Block With Hydroxychloroquine [PATCH]; NCT01379573).

AIMS/OBJECTIVE:Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for its normal functioning. We investigated fetal human heart single-cell transcriptomes from midgestational healthy and anti-Ro associated congenital heart block (CHB) samples, respectively. METHODS AND RESULTS/RESULTS:Three healthy fetal human hearts (19th-22nd week of gestation) and one fetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single cell suspensions followed by 10x Genomics- and Illumina-based single cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse subpopulations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types the CHB heart compared to the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contributing to extracellular matrix deposition and subsequent fibrosis. CONCLUSION/CONCLUSIONS:These data provide an information-rich resource to further understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease. TRANSLATIONAL PERSPECTIVE/UNASSIGNED:This study provides a single cell transcriptomic atlas of cells obtained from healthy second trimester fetal hearts to further understand human heart development and impart insight into autoimmune associated congenital heart block. In addition to myocytes and fibroblasts, previously uncharacterized subpopulations of endothelial cells were identified. Leveraging an unprecedented opportunity, healthy heart transcriptomes were compared to an age matched anti-SSA/Ro exposed fetal heart with third degree block in which no maternal medications were taken. Differential gene expression analysis revealed a remarkable interferon response in many cell types of the diseased heart. In addition, matrisome transcripts were enriched in the stromal cells likely contributing to the extracellular matrix deposition and thereby fibrosis, a signature lesion of heart block. Thus, targeting the interferon pathway merits therapeutic consideration.

Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has both a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by interferon-gamma. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, it was observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8 T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8 cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways may be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.