Faculty Bibliography

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 : Since one of the strongest associations with antibodies (abs) to SSA/Ro (Ro60) is the development of congenital heart block (CHB), this model provides an exceptional opportunity to define novel insights that link maternal abs with an inflammatory cellular response which eventuates in fibrotic replacement of the AV node. We recently compiled risk genes based on an agnostic transcriptomic survey of macrophages isolated from hearts of fetuses dying with CHB and healthy aged matched fetuses electively terminated, noting that IFN related genes (IRGs), including IFN induced Protein with Tetratricopeptide Repeats 1(IFIT1) and Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1), are highly upregulated in the CHB hearts. Accordingly, this study addressed the hypothesis that IRGs contribute to CHB pathogenesis. Methods : hY3 RNA, a noncoding ssRNA and TLR7/8 agonist, was used as a proxy of the Ro60 immune complex. Human derivatives included healthy peripheral blood macrophages and fibroblasts isolated from a healthy human fetal heart. Neutralizing IFNalpha and IFNbeta abs were used to assess the contribution of the respective cytokines to the model. Macrophage readouts included the expression of IFIT1 and SIGLEC1 transcripts (qPCR, units, fold change based on 2-DELTADELTACT, relative expression of transcript normalized to GAPDH) and myofibroblast phenotype (EdU imaging and SMAc by IF, respectively). Results : As expected, exposure of macrophages to IFNalpha resulted in a significant upregulation of IFIT1 and SIGLEC1 compared to untreated macrophages (70+/-25 vs 1, and 17+/-9, vs 1, respectively with both N=3, P< 0.05). Similarly, exposure to IFNbeta also resulted in the upregulation of these transcripts (254+/-237 vs 1, p=0.03, and 21+/-14 vs 1, respectively with both N=4, p< 0.03). The expression of these transcripts by IFNalpha-and IFNbeta-treated macrophages was completely attenuated by co-treatment using respective Type I IFN-specific neutralizing antibodies. In parallel, transfection of human macrophages with hY3 also resulted in upregulation of IFIT1 (112+/-30 vs 1, p=0.02, N=3) and SIGLEC (13+/-7 vs 1, N=3). To confirm TLR7/8 dependency of IRGs, the addition of TLR7/8 antagonist IRS661 to our in vitro model resulted in a significant decrease of IFIT1 expression to 14% (14+/-10, n=6) and SIGLEC1 to 54% (7+/-5, n=7, both P=0.03). Co-treatment with neutralizing antibody against IFNalpha reduced the expression of IFIT1 to 9% (10+/-9, n=3) and SIGLEC1 to 35% (5+/-3, n=3). Co-treatment with neutralizing antibody against IFNbeta also reduced the expression of IFIT1 to 24% (24+/-6, n=2) and SIGLEC1 to 59% (3+/-5, n=3). For a survey of direct effects of type I IFN, IFNalpha and IFNbeta were shown sharing the capacity to stimulate fibroblast proliferation (EdU, % positive) yielding a result of untreated (16%), IFNalpha (40%), and IFNbeta (48%). In addition, exposure of human fibroblasts to IFNalpha as well as IFNbeta induced expression of the myofibroblast marker, SMAc (IF) versus no expression by the untreated fibroblasts. Conclusion : These results suggest that type I IFN contributes to the inflammatory and profibrosing milieu associated with the development of CHB. Feed forward expression of IFN related genes in response to TLR signaling may provide new targets towards the prevention of disease

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 : 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

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

Background/Purpose : Osteoporosis and bone fractures are a frequent cause of morbidity in systemic lupus erythematosus (SLE), and are felt to be related both to disease activity and glucocorticoid (GC) exposure. Dual energy X-ray absorptiometry (DEXA) is the standard tool to assess bone density, but it does not measure bone quality or strength and is not a robust predictor of fractures in SLE. Clinical 3T MRI scans have been shown to assess information about bone not captured by DEXA. This study aims to evaluate factors associated with bone structure measured by DEXA and MRI in an SLE cohort. Methods : DEXAs were performed on 31 women with SLE and 3T MRI of the non-dominant hip were performed on 29 of these cases. Results were associated with multiple demographic, clinical and laboratory measures. MRI parameters measured included trabecular plate width (PW), trabecular plate to rod ratio (PRR), plate volume fraction (PVF), rod volume fraction (RVF), trabecular bone thickness (Tb.Th), trabecular spacing (Tb.Sp) and trabecular network area (TNA). DEXA BMD was measured, and osteoporosis (OP) was defined as hip, spine or femoral neck Z score < -2.0 in premenopausal women, and T score < -2.5 in others, and low bone density (LBD) as Z score < -2.0 in premenopausal women and T score < -1.0 in others. Results : By DEXA, 8/31 (25.8%) had OP and 12 (38.7%) had LBD. History of lymphopenia (75.0% vs. 31.8%, p=0.049) and lower concurrent HCQ dose (340 vs. 400 mg, p=0.006) associated with DEXA OP, while older age (48.3 vs. 36.3 y, p=0.024) associated with LBD. Higher ESR was inversely correlated with favorable bone structure (PW r(22) = -.49, p=0.025, PRR rs = -.51, p=0.018, PVF rs = -.51, p=0.018, RVF rs = .51, p=0.018, Tb.Th rs = -.58, p=0.005, Tb.Sp rs = .44, p=0.046, TNA rs = -.50, p=0.022). Higher CRP was likewise inversely correlated with favorable bone structure (PW r(20) = -.61, p=0.004, PRR rs = -.57, p=0.009, PVF rs = -.57, p=0.009, RVF rs =.57, p=0.009, Tb.Th rs = -.56, p=.011, Tb.Sp rs =.67, p=0.001, TNA rs = -.64, p=0.002). A history of lupus nephritis was associated with unfavorable bone structure (PW 705.3 vs. 833.3 mum, p=0.048, PRR 6.6 vs. 8.1, p=0.024, PVF 0.83 vs. 0.89, p=0.024, RVF 0.17 vs. 0.11, p=0.024, Tb.Th 178.1 vs. 193.4 mm, p=0.012, Tb.Sp 358.6 vs. 296.5 mm, p=0.056, TNA 0.41 vs. 0.54 (1/mm), p=0.009). ESR, CRP and history of lupus nephritis were not significantly associated with DEXA hip BMD, OP or LBD. MRI parameters for favorable bone structure were inversely correlated with DEXA hip BMD (PW r(28) = -.47, p=0.011, Tb.Th rs = -.53, p=0.003) and BMI (PW r(28) = -.54, p=0.003, TbTh rs = -.72, p< 0.001, TNA rs = -.44, p=0.017). Conclusion : Higher ESR and CRP and a history of lupus nephritis associated with MRI parameters of unfavorable bone structure, but did not associate with DEXA abnormalities in SLE patients. MRI may be a more sensitive tool than DEXA to measure inflammatory effects on bone and potentially cumulative dose of steroid exposure. There were inverse correlations of MRI parameters with traditional osteoporosis risk factors and BMD measures on DEXA, and it is possible that each tool evaluates different aspects of bone health. Further evaluation of MRI screening for fracture risk in SLE and GC exposed individuals is warranted to better quantify risk and guide treatment

Background/Purpose : The impact of glucocorticoid (GC) use on major organ damage in SLE patients has not been formally studied by amalgamating the relevant data published in the literature over the past 40 years. We aimed to study the association between GC use and the occurrence of major organ damage in SLE patients by performing meta-analyses of observational studies published between 1970 and December 2018. Methods : Literature search on PubMed (from 1966 to December 2018) for prevalence and longitudinal studies which reported GC exposure (proportion of GC users in the cohort [%GC use] and/or GC use in defined doses) and the occurrence (prevalence/incidence) of major organ damage in SLE patients using the keywords cataract, cerebrovascular (CVA), stroke, cardiovascular (CVS), angina, myocardial infarction (MI), coronary artery bypass, osteoporosis, avascular necrosis (AVN) and osteonecrosis in respective combinations with lupus was conducted. Studies with sample size < 50 and observation duration < 12 months were excluded. The logit of the proportion of patients with disease damage was modelled as a random effect in the meta-analysis, which was employed to study the association between the proportion of patients with organ damage and variables of GC use (mean daily [mg/day] and cumulative [gm] prednisone [PDN] doses and %GC use). A 2-stage estimation of the random-effects logistic regression models was used with restricted maximum likelihood estimation. Univariate associations between organ damage and moderators were examined for statistical significance, and variables related to GC use were adjusted for SLE disease duration in multivariate models if their univariate P values were < 0.2. Results : Out of 8,882 publications screened, 212 articles involving 205,619 SLE patients were eligible for the metaanalyses (Figure 1), of which 97 were prevalence and 115 were longitudinal studies. Univariate analyses of prevalence studies revealed that mean daily PDN dose (odds ratio [OR]=1.10, p=0.007) and lower proportion of female in the cohort (OR=0.002, p=0.002) were associated with the prevalence of overall CVS events. Mean daily PDN dose (OR=1.52, p< 0.001) and %GC use (OR=2,255.2, p< 0.001) were associated with the prevalence of AVN. A significant association between cumulative PDN dose and prevalence of CVA was found after multivariate adjustment for SLE disease duration (OR=1.07, p=0.017). In longitudinal studies, a significant association was identified between cumulative PDN dose and incidence of cataracts after adjustment for SLE disease duration (OR=1.04, p=0.013). While the incidence of MI in SLE patients has dropped over the past 40 years (OR=0.94, p=0.002), it was associated with % GC use after adjustment for SLE disease duration (OR=8.18, p=0.012). Interestingly, significant univariate associations were found between antimalarial use and lower prevalence of MI (OR=0.05, p=0.002) and lower incidence of CVA (OR=0.20, p=0.032). Conclusion : Independent of SLE disease duration, cumulative PDN dose was associated with higher prevalence of CVA and incidence of cataracts, and higher incidence of MI was associated with overall GC use

Background/Purpose : SLE diagnostic criteria are important for reliable epidemiologic data. The prevalence of SLE in West Africa is falsely low due to barriers including limited access to both resource and labor-intensive diagnostic testing. Recently, the ACR and EULAR have proposed a weighted classification tool which is thought to improve diagnostic sensitivity and specificity compared to the established ACR and SLICC criteria. Here we aim to investigate the performance of each classification criteria in two West African cohorts--Korle bu Teaching Hospital, Accra Ghana (GH); and Lagos University Teaching Hospital, Lagos, Nigeria (N)--compared to an NYU Langone-African American (AA) cohort. Methods : We collected data on a total of 355 SLE patients: AA: n=151, GH: n=110, and N: n=94, diagnosed by expert clinicians. Clinical information including demographics, SLE criteria, SLEDAI scores, SLICC damage indexes, vital signs, and laboratory values as available was obtained at the initial patient encounter. Longitudinal data was collected over the course of at least 1 year at 6 month intervals during routine clinical visits. Where necessary, clinical charts were retrospectively reviewed, and the proportion of patients in each cohort meeting ACR, SLICC and the ACR/EULAR classification criteria was calculated. Results : The demographics per cohort were as follows: Age (in yrs): AA=43.1, GH=32.4yrs, N=35.5; percent female: AA=90, GH=100, N=97; Mean SLE disease duration (yrs): AA=14.3, GH=2.2, N=4.4. In each cohort, the percentage of patients meeting ACR, SLICC, and ACR/EULAR criteria were AA=96%, 96%, and 95%; GH=85%, 84%, 62%; N=90%, 87%, 61%. This discrepancy was largely due to missing laboratory data particularly with regard to immunologic and hematologic studies. ANA was missing in 0% of the AA cohort, 26% of the GH cohort, and 33% of the N cohort respectively. Compared to the GH and N cohorts, the reference AA cohort was more likely to meet ACR, SLICC, and ACR/EULAR criteria with likelihood ratios (LR) of GH=10.2 p< 0.001 and N=3.0 p=0.08; GH=11.5, p< 0.001 and N=6.3, p=0.01; and GH=46.1 P< 0.001 and N=44.9, p< 0.001 respectively. On average, the mean number of ACR/EULAR points by cohort was AA: 26.1+/-11.8, GH: 21.3+/-8.1, and N: 19.0+/-6.2. While the ANA entry criteria greatly diminished the new ACR/EULAR diagnostic utility in the GH and N cohorts, the weighted point system performed better than either of the ACR or SLICC criteria with 96% of the AA cohort, 92%of the GH, and 95% of the N cohort meeting criteria (LR: AA vs GH=1.9, p=0.2; AA vs N=0.23, p=0.6). Conclusion : Due to a relative lack of resources, supportive laboratory assays including an ANA may be more difficult to attain in developing nations. SLE is a clinical syndrome that may be efficiently diagnosed using the new weighted ACR/EULAR criteria. The entry criteria of ANA 1:80 greatly diminished the diagnostic utility of this classification system in the Ghanaian and Nigerian cohorts compared to the African American cohort. Clinical trials should consider offering wide ANA testing to cohorts in the developing world

Background/Purpose: Both plasmacytoid dendritic cells (pDCs) and switched memory B cells (SMBCs) are considered to be key effector cells in systemic lupus erythematosus. It seems likely that within these classical cell lineages, additional diversity of function will exist that will contribute to disease pathogenesis. To explore this question, we performed single-cell RNA sequencing in pDCs and SMBCs from SLE patients and controls to assess gene expression patterns and cellular sub-groupings within these lineages. Methods : pDCs and SMBCs from SLE patients (n=10) and Healthy controls (n=5) were purified by magnetic separation. For deep sequencing, we used the Fluidigm C1 HT system with 800 capture site chips to capture single cells. Single cell capture was verified by direct visualization using the Array Scan system, allowing us to remove empty wells and wells with multiple cells. After quality control and adaptor trimming, the data was analyzed using SeqGeq software. pDCs and SMBCs were clustered using UMAP and pseudo-time analysis was performed using the Monocle program. Type I IFN activity in SLE plasma was measured using reporter cell assay. Results : A total of 2774 pDCs and 2578 SMBCs from SLE and healthy controls passed the quality control and were used for further analysis. In pDCs, we observed unique clusters for patients with high interferon, low interferon, and controls, indicating that the IFN response is a major determinant of overall gene expression patterns in SLE patient pDCs. IFN signature in pDCs correlated with circulating type I IFN activity in the SLE patients measured at the same time. Other genes upregulated in pDCs included the type I interferon regulator AXL and MACC1. The SMBCs were heterogeneous in patients and controls, and in contrast to the pDCs, the overall clustering pattern was independent of the IFN score. SMBC clusters were predominantly defined by genes indicating cellular activation or proliferation such as HLA-DRs and CREB1, or genes associated with nucleic acid processing such as DNASE1 and SNORD3B-1. Conclusion : We find distinct clusters of cells defined transcriptionally within the pDC and SMBC lineages, and the transcripts which define these subgroups differ between cell lineages. Type I IFN induced transcripts are important to pDC diversity, while in SMBCs transcripts related to cellular activation and nucleic acid processing are critical markers of transcriptional heterogeneity

Background/Purpose : Despite the strong association of maternal anti-Ro60 autoantibodies in the development of SS, Neonatal Lupus (NL) and scLE, understanding causality is challenging given that the intracellular location of the target RNP antigen only becomes accessible to extracellular autoantibodies during cell death. While binding Y RNA is a property of Ro60 in dying cells, Ro60 contributes to events essential to proliferation including the degradation of non-coding RNAs. To define potentially targetable molecular events associated with Ro60 accessibility and their effects on proliferation, we evaluated two candidate chaperone Ro60 binding proteins, ZBP1 (also known as IGF2BP1, which has two RNA recognition domains and plays a role in nuclear export of protein) and PTBP1 (which binds RNA in heterogeneous nuclear complexes). Methods : Short hairpin (sh)RNA knockdown (KD) of both ZPB1 and PTBP1 was accomplished by using MISSION pLKO.1 derived lentiviral vehicles for targeted shRNA to yield each of the specific depletions in human fetal fibroblasts. Affinity purified anti-Ro60 antibodies (AP60) isolated from the serum of 2 mothers of children with cardiac NL and control IgG isolated from a healthy donor were used to evaluate Ro60 surface translocation in intact and apoptotic cells by flow cytometry. Fibroblast proliferation was determined using 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Results : KD of targeted transcripts were confirmed by qPCR and western blot. Permeabilized KD and wildtype fi-broblasts demonstrated equivalent intracellular expression of Ro60. As expected, flow cytometry with AP60 revealed no surface staining of non-permeabilized wildtype or KD fibroblasts. The next set of experiments addressed binding of AP60 to polyHEMA-apoptotic fibroblasts. Staining with annexin V (a proxy of apoptosis) was uniformly consistent between the wildtype and KD cells. As expected, compared to non-apoptotic fibroblasts, AP60 but not control donor IgG readily bound the surface of wildtype apoptotic fibroblasts, supporting that Ro60 was indeed translocated during apoptosis (MFI of 97 + 5 vs 211 + 14, respectively; P< 0.05; N=3). In contrast, binding of AP60 was significantly attenuated in the ZPB1 KD fibroblasts (81.5 + 7; N = 3) vs anti-Ro binding of apoptotic wildtype fibroblasts(P< 0.05). However, the binding of AP60 was equivalent in the apoptotic PTBP1 KD fibroblasts (MFI of 203 + 6, N= 3, P=NS) compared to apoptotic wildtype fibroblasts. With regard to cell proliferation, the percent positive EdU cells of wildtype fetal fibroblasts, ZBP1 KD fibroblasts, and PTBP1 KD fibroblasts were 56%, 2% and 45%, respectively, a result suggesting that the loss of ZBP1 restrains cell cycle progression. Conclusion : ZBP1 represents a novel and required chaperone for the translocation of Ro60 to the cell surface during apoptosis in addition to contributing to cell proliferation. Given that Ro60 antigen accessibility is essential, not only to the generation of anti-Ro60 responses but also to the formation of surface immune complexes and subsequent tissue injury, ZBP1 may represent a newly targetable candidate to forestall both the initiation and sequelae of autoimmunity