Faculty Bibliography

OBJECTIVES/OBJECTIVE:In this study we aimed to investigate foetal and maternal pregnancy outcomes from a large multicentre cohort of women diagnosed with MCTD and anti-U1RNP antibodies. METHODS:This multicentre retrospective cohort study describes the outcomes of 203 pregnancies in 94 consecutive women ever pregnant who fulfilled the established criteria for MCTD with confirmed U1RNP positivity. RESULTS:The foetal outcomes in 203 pregnancies were as follows: 146 (71.9%) live births, 38 (18.7%) miscarriages (first trimester pregnancy loss of <12 weeks gestation), 18 (8.9%) stillbirths (pregnancy loss after 20 weeks gestation) and 11 (5.4%) cases with intrauterine growth restriction. Maternal pregnancy outcomes were as follows: 8 (3.9%) developed pre-eclampsia, 2 (0.9%) developed eclampsia, 31 (15.3%) developed gestational hypertension and 3 (1.5%) developed gestational diabetes. Women with MCTD and aPL and pulmonary or muscular involvement had worse foetal outcomes compared with those without. Moreover, we report a case of complete congenital heart block (0.45%) and a case of cutaneous neonatal lupus, both born to a mother with positive isolated anti-U1RNP and negative anti-Ro/SSA antibodies. CONCLUSION/CONCLUSIONS:In our multicentre cohort, women with MCTD had a live birth rate of 72%. While the true frequency of heart block associated with anti-U1RNP remains to be determined, this study might raise the consideration of echocardiographic surveillance in this setting. Pregnancy counselling should be considered in women with MCTD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mothers giving birth to children with manifestations of neonatal lupus (NL) represent a unique population at risk for the development of clinically evident pathologic autoimmunity since many are asymptomatic and only become aware of anti-SSA/Ro positivity (anti-Ro+) based on heart block in their fetus. Accordingly, we hypothesized that the microbiome in saliva is associated with the development of autoreactivity and in some cases the progression in health status from benign to overt clinical disease including Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The study comprised a clinical spectrum of anti-Ro+ mothers, all of whom gave birth to a child with NL: 9 were asymptomatic or had an undifferentiated autoimmune disease (Asym/UAS) and 16 fulfilled criteria for SS and/or SLE. Microbial diversity was reduced across all levels from kingdom to species for the anti-Ro+ mothers vs healthy controls; however, there were no significant differences between Asym/UAS and SS/SLE mothers. Relative abundance of Proteobacteria and more specifically class Betaproteobacteria decreased with clinical severity (healthy controls < Asym/UAS < SS/SLE). These ordered differences were maintained through the taxonomic hierarchy to three genera (Lautropia, Comamonas, and Neisseria) and species within these genera (L. mirabilis, N. flavescens and N. oralis). Biometric analysis comparing von Willebrand Factor domains present in human Ro60 with L. mirabilis proteins support the hypothesis of molecular mimicry. These data position the microbiome in the development of anti-Ro reactivity and subsequent clinical spectrum of disease.

Background/Purpose : Pregnancy counseling of all anti-Ro positive women includes advice regarding the development of congenital heart block (CHB), albeit the risk is only 2% for primigravida women or those with previously unaffected offspring. Despite this low risk, the prevailing surveillance recommendation is weekly echocardiography. While evidence from basic research laboratories support that high titers of antibodies confer clinically meaningful risk, unfortunately the majority of commercial laboratories use the BioPlex assay, which provides positive and negative values with limited information on actual levels because the sera or plasma are not diluted past a specified cutoffgiven cost (e.g. values of anti-Ro inclusive of Ro52 or Ro60 by laboratories such as Quest or LabCorp provide positive as 1-8 or > 8 units with no further information). The present study was initiated to assess whether the Bio-Plex assay used by many commercial laboratories provides adequate stratification of risk for counseling regarding management. Methods : The study group comprised healthy non-pregnant donors (N = 9), healthy pregnant donors (N = 62), women testing positive for anti-Ro by commercial BioPlex but without CHB children (N = 60 SLE and 2 SS), and women with CHB children (N = 83). Anti-Ro60 reactivity was assessed using native antigen and anti-Ro52 using recombinant protein. Sera were applied to coated microtiter plates at serial dilutions ranging from 1:1000 -1:50,000 for 1h at RT and run in duplicate. Tested samples were multiplied by the dilution factor which gives an OD in the range of 0.3-0.8. Results were considered positive at 123 ELISA units (EU) for Ro60 and 215 EU for Ro52 as this represented the mean +3 SD of the values obtained for healthy control sera. Results : Of the 83 CHB mothers tested, 74 had titers of Ro60 and Ro52 > 1000 EU, in 1 anti-Ro60 was > 1000 EU and anti-52 Ro between 215 -1000, in 3 anti-Ro52 was > 1000 EU and anti-Ro60 between 300 -1000, and 1 mother had anti-Ro60 > 1000 EU and was negative for anti-Ro52. Albeit all positive, the sera from 4 CHB mothers obtained 15 years after the birth of the affected child were < 1000 EU for both anti-Ro60 and Ro52. With these results setting thresholds ( > 1000 EU in either Ro60 or Ro52 for CHB risk), we assessed patients testing positive for anti-Ro based on the BioPlex assay. Of 42 patients with values of > 8 on BioPlex testing, 14 had titers > 1000 EU for both anti-Ro60 and Ro52, 7 had anti-Ro60 > 1000 EU, and 8 had anti-Ro52 > 1000 EU. Thus, 13 of 42 (25%) with commercial Ro > 8 did not meet the threshold EU for CHB risk. Of 20 patients considered positive for anti-Ro by BioPlex with values between 1-8, none had levels of either anti-Ro60 or Ro52 at 1000 EU. No patient or healthy control testing negative by the BioPlex assay was positive for CHB risk in our ELISA. Conclusion : These data suggest that commercial testing using the BioPlex assay may fall short of stratifying risk for CHB. Women with positive values < 8 are not likely at risk, obviating the cost and burden of weekly fetal echo surveillance. Moreover, even those considered high titer on commercial testing may be at low risk supporting the need for more quantitative commercial testing than is currently available. (Figure Presented)

Background/Purpose : Lupus nephritis (LN) complicates up to 60% of patients with systemic lupus erythematosus (SLE) and carries a high morbidity and mortality. The definitive diagnosis is based on kidney biopsy. This is invasive and not always readily available, thus delaying treatment. Sometimes multiple biopsies are required over the course of the disease. Importantly, while renal pathology is accurate at describing the morphology of renal disease, the underlying biology and molecular pathways are not thoroughly assessed. Urine proteomics is a non-invasive strategy that may provide insights regarding ongoing renal disease. Methods : One thousand proteins were quantified (RayBiotech Kiloplex assay) on a total of 112 longitudinal urine samples from 32 SLE patients with active LN and 7 healthy controls (HC) enrolled in the Accelerating Medicines Partnership (AMP). All patients underwent treatment as directed by their own physicians. Differentially excreted proteins at baseline (SLE vs HC, proliferative vs membranous LN, responders vs non responders) were identified using a linearmodel with moderated t statistic. Response to treatment was defined based on proteinuria at 1 year as complete (< 0.5g/24h) or partial (50% reduction but >0.5/24h). In the longitudinal analysis, a mixed model was employed to identify markers associated with proteinuria. Pathway enrichment analysis was performed using the genes coding for the differentially excreted analytes using Ingenuity Pathway Analysis (IPA) and other publicly available pathway libraries. Results : There were 186 proteins increased in SLE patients (Fig. 1). The most enriched pathway was TNFa (p< 0.001). We found 74 differentially excreted proteins comparing proliferative and pure membranous LN. CD4, MCP-1, MIP-1a, RANTES, IL-16, and IL-7, markers involved in CD4 T cell and monocyte biology, were enriched in proliferative disease. A few targets were exclusively identified in either class (i.e. CD4 in proliferative nephritis). We used a longitudinal model to identify specific urine proteins associated with worse proteinuria as a marker of severity. Proteinu-ria was associated with 105 markers (FDR < 0.05), the strongest association being CD163 (p = 10-9), a phagocyte marker. IPA implicated several pathways involving fibrosis, acute phase response, LPS/IL1, RXR, ICOS signaling and macrophage/fibroblasts (Fig. 2). Next, we identified 27 differentially excreted proteins in non-responders. IPA revealed that tretinoin, GM-CSF, TNF, and IL1 were among the top upstream regulators (Fig. 3). Conclusion : There is an inflammatory signature in the urine of patients with LN implicating monocyte and TNFa pathways. These signatures are associated with proliferative disease, worse proteinuria, and non-response to treatment. Of note, TNFa is involved in LN and has therapeutic potential. In phase 1 of AMP, monocytes were the main urine cell type identified by singe cell RNA sequencing in patients with LN. These results suggest that urine proteomics might identify and infer active pathological mechanisms in LN, paving the way for a more personalized approach to treatment. Further work in Phase 2 of AMP is being pursued to validate and extend these findings

Background/Purpose : Autoantibody production precedes SLE or SS by years, including anti-Ro. Anti-Ro + mothers of children with congenital heart block (CHB) are a unique population at risk for pathologic autoimmunity, as many are asymptomatic (Asym/UAS) and become aware of autoantibodies due to fetal disease and yet have a 10-year progression rate to SS/SLE of 20%-30%. We hypothesized that variation in the oral microbiome correlates with transition to SLE or SS and pathogenicity involves sequence homology between Ro60 and bacterial von Willebrand factor type A domain protein (vWFA).
Method(s): The oral microbiome of 25 anti-Ro + mothers of CHB children (Asym/UAS, N=9; SS/SLE, N=16) and 7 healthy controls (HC) were processed using 16S ribosomal RNA sequencing. Analysis of variance methods compared the centered log ratio transformed relative abundances for 1) HC vs. anti-Ro + mothers, and 2) assuming an ordering of severity from HC < Asym/UAS < SS/SLE. To adjust for multiple comparisons, a taxonomic stepdown method coupled with false discovery rate (FDR) was used. The Basic Local Alignment Search Tool evaluated homology of Ro60 at aa 371-381 and peptides of vWFA. Results : Sequencing 16S rRNA identified microorganisms from 2 kingdoms, 16 phyla, 25 classes, 41 orders, 70 families, 164 genera, and 166 species. The Shannon Index (H) revealed that for each taxonomic level except species, there were significant reductions in diversity in the anti-Ro + mothers relative to HC (P <= 0.05). There were global differences in the microbiota of these mothers relative to HC (perMANOVA P=0.00049). The phylum Actinobacteria was more abundant in the anti-Ro + mothers vs HC (P FDR =0.0231). Within Actinobacteria , the class Coriobacteriia and subsequent lower taxonomic levels down to Atopobium parvulum , all exhibited increases in relative abundance in the anti-Ro + mothers compared to HC. There was a significant reduction in the relative abundance as clinical severity increased within one of the most frequent phyla, Proteobacteria (P FDR =0.030; mean+/-SD; HC 0.24+/-0.07; Asym/UAS 0.19+/-0.12; SS/SLE 0.11+/-0.08). The difference in the relative abundances between Asym/UAS and SS/SLE within Proteobacteria was significant (P=0.042). Within Proteobacteria , the common class Betaproteobacteria also showed reduced relative abundance with increasing clinical severity (P FDR =0.0037; HC 0.11+/-0.04; Asym/UAS 0.072+/-0.07; SS/ SLE 0.031+/-0.04). These ordered differences were maintained down the taxonomic hierarchy to the genus ( Lautropia , P FDR =0.0072) and species within this genus ( L. mirabilis , P FDR =0.012). Next, sequences of vWFA secreted by these taxa were evaluated. For a comparison of Ro60 T cell epitope, FLLAVDVSASMNQ, the vWFA, VLVVFDNSSSMTA vWFA of A. parvulum was not a fit due to the aromatic and polar aa at positions 5 and 9, respectively. In contrast, the vWFA of L. mirabilis , LLLLLDVSGSMAG, was identical at 7 of the first 11 aa. Conclusion : These data provide evidence that the microbiome differs along a clinical spectrum of autoimmunity. In part, the data refiect a path involving depletion of L. mirabilis , which is secondary to a pathologic role of anti-Ro along with an expansion of A. parvulum , an opportunistic taxon

Background/Purpose : Based on encouraging bench to bedside results including experimental evidence supporting Toll-like receptor signaling in the pathogenesis of CHB, a case control study demonstrating CHB risk reduction in hydroxychoroquine (HCQ) exposed fetuses of anti-Ro positive SLE women, and a historical cohort study supporting a reduction in recurrence rate, an open label single arm Phase 2 clinical trial was initiated to evaluate whether HCQ reduces the CHB recurrence rate (pi) below the historical recurrence rate of 18%. Methods : A two-stage trial design (N=19 first stage; N=54 second stage) using Simon's optimal approach was employed to allow for early stopping due to absence of treatment efficacy. The null hypothesis, H 0 :pi <= 18%, would be rejected and HCQ considered efficacious at the end of the trial if <= 5 of 54 mothers with anti-Ro and a previous CHB child had a subsequent child with 2 nd or 3 rd degree block (primary outcome). The protocol required HCQ initiation or maintenance at 400mg by 10 wks gestation. Mothers underwent serial echocardiograms, with bloods drawn each trimester and delivery for cord blood to measure antibody and HCQ levels. Results : Sixty five mothers (all with previous CHB child and anti-Ro52 or Ro60 > 1,000 EU; 47.9% with anti-La; 71.4% White; 47.6% SLE and/or SS; 42.9% started HCQ solely for CHB prevention; 41% prior CHB child died, 3.2% had > 1 CHB child) signed consent. Ten were considered screen failures (2 miscarriages < 12 wks, 7 wherein dating of conception placed HCQ initiation at > 10 wks, 1 given dexamethasone (dex) 1mg at 10 wks) and 1 was lost to follow up before delivery leaving 54 pregnancies evaluable with serial fetal echos and birth or one yr EKG or echo results known. In Stage I, 2/19 fetuses had CHB, and the study proceeded to Stage II. By intention to treat analysis, 4/54 pregnancies resulted in CHB (7.4%; p = 0.02 for H 0 ), all at 19-20 wks. Three presented with 2 nd degree block, one reverted to NSR at birth following dex and two progressed to 3 rd degree despite dex and IVIG (one electively terminated). One presenting with 1 st degree was treated with dex prophylactically (eliminating this case from evaluating HCQ exposure alone), progressed to 2 nd but reverted to NSR at birth. At 2 yrs, the 2 in NSR had intermittent 2 nd degree on Holter monitor. In 8 mothers potentially confounding medications, IVIG and/or dex, were prescribed after enrollment for lupus flare, cardiac concerns apart from advanced block (APCs, echo brightness, 1 st degree block), and/or physician decision to consider additional prophylaxis. To evaluate HCQ alone, 9 additional mothers were enrolled, one whose fetus developed 3 rd degree block at 19 wks. Including only pregnancies exposed to HCQ alone prior to confirmed 2 nd or 3 rd degree block, 4/54 developed CHB (7.4%; p = 0.02). In total 5/63 pregnancies (7.9%) resulted in advanced block. HCQ levels in the second trimester confirmed a 98% adherence rate. Anti-Ro levels remained > 1,000 EU (considered vulnerable for CHB) throughout pregnancy. No CHB developed in any of the 7 mothers screened out because of low dose or delayed start of HCQ. Conclusion : These prospective data from a single-arm clinical trial support that HCQ significantly reduces the recurrence of CHB below the historical rate

Background/Purpose : The impact of renal injury in lupus nephritis (LN) is widespread with consequences to resident cells in other tissue beds, even non-lesional, non-sun exposed skin. Faithful reflection of a relevant renal tissue pathway in a more readily accessible compartment would allow for less invasive diagnostic alternatives. While ongoing studies are exploiting single cell RNA sequencing to link phenotype to biotype and identify cell specific pathways in the kidney, this study was initiated to address the hypothesis that these pathways may be reflected in uninvolved skin which is more likely to be serially biopsied. Methods : Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of nonlesional, non-sun-exposed skin from the buttocks of 5 healthy controls, 4 SLE patients without LN and 18 SLE patients with proteinuria (with skin biopsies obtained within 24 hrs of the kidney biopsy). Histology revealed Class III ( n=6 ), Class III/V or IV/V mixed ( n=11 ), Class V ( n=1 ), and nephrosclerosis ( n=1 ). Dissociation of cryostored skin biopsies with collagenase and trypsin enzymes was followed by scRNA-seq using the 10x Genomics platform using V2 and V3 reagents. Results : We obtained 8,019 and 17,655 high-quality scRNA-seq profiles from single cell suspensions of control and SLE non-lesional, non-sun-exposed skin, respectively. A graph-based clustering method was applied and identified major clusters of cells as visualized by t-distributed stochastic neighbor embedding (tSNE). Differential gene expression analysis guided by established markers revealed these cell clusters as keratinocyte (KC), one smooth muscle cell cluster (SMC), fibroblast (FB), melanocyte (MEL), vascular endothelial cells (VEC), lymphatic endothelial cells (LEC), macrophages-dendritic cells (MAC-DC), T cells (TC) and sweat gland cells (SGC) (Figure 1A). Ranking cells by abundance, the result of the SLE skin cells was KC >FB >VEC >LEC, SMC, MAC-DC, TC, MEL and SGC. Overall, samples processed using the recent V3 single cell reagent kit showed higher genes and transcript captures compared to V2. However, these samples also captured more mitochondrial transcripts (Figure 1B). An analysis of gene expression changes in KC, SMC, and VSC from the LN patients versus controls demonstrated overexpression of interferon stimulated genes. However, the degree of interferon response varied in these cell types with KCs (basal KC, p=0.00312 and hair follicle KC, p=0.000012) showing the highest response followed by VECs (p=0.0043) and SMCs (p=0.0068). In addition to the interferon response signature, VECs from the LN patients also showed upregulation of MHC-II genes such as HLA-DRB5 and HLA-DRB1, suggesting increased antigen presentation capacity (Figure 1C). Conclusion : scRNA-seq identifies major skin cell types and further clustering identifies rarer cell populations. KCs, SMCs, and VECs from the skin of LN patients reveal diverse IFN response states and additionally VECs also show higher antigen presentation potential. The V3 upgrade of 10x Genomics single cell reagents capture more genes and UMIs per cell, but also higher mitochondrial content compared to the V2 version

Background/Purpose : The EULAR/ACR 2019 Classification Criteria for SLE have been validated in an international cohort of 696 SLE patients and 574 non-SLE patients with a sensitivity of 96.1% and a specificity of 93.4%. We comparatively evaluated the performance characteristics of the SLE classification systems in subsets of the validation cohort with regard to gender, race/ethnicity, and disease duration. Methods : 21 SLE expert centers from 16 countries submitted up to 100 SLE cases and 100 SLE mimicking controls each, using a standardized form without knowledge of the new criteria system to form the validation cohort. Cases and control diagnosis (SLE or not SLE) were independently verified by 3 SLE experts. The EULAR/ACR 2019 classifi-cation criteria validation cohort consisted of female (n=1,098) and male (n=172) patients; Asian (n=118), Black (n=68), Hispanic (n=124) and White (n=941) patients; and patients with an SLE duration of less than 1 year (n=34), 1-3 years (n=196), 3-5 years (n=157), and 5 or more years (n=879). Sensitivity and specificity with 95% confidence intervals (CI) were estimated for the EULAR-ACR 2019 criteria, the SLICC 2012 criteria and the ACR 1997 criteria. Results : As shown in Table 1, most of the point estimates for sensitivity and specificity in subsets lay within the 95% confidence intervals of the sensitivity and specificity of the EULAR/ACR 2019 criteria validation. In particular, sensitivity and specificity for all race/ethnicity groups were within the confidence intervals or even higher. Formally, the sensitivity was slightly lower for male patients, corresponding to a higher specificity, but the male 95% confidence intervals (0.86-0.98 for sensitivity, 0.90-0.99 for specificity) overlapped with those of the full cohort. Sensitivity appeared independent of disease duration at least from year 1 on, with all 95% confidence intervals overlapping (for the first year after diagnosis 0.52-1.00 for sensitivity, 0.69-0.97 for specificity). Conclusion : The point estimates of sensitivity and specificity suggest that the EULAR/ACR 2019 SLE classification criteria perform well in diverse race/ethnicity groups, in men and in early disease. These results now need to be independently validated in larger groups of African American/Black, Asian, and Hispanic patients, male patients and in early disease