Faculty Bibliography

Background The impact of renal injury in lupus nephritis 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. Single-cell transcriptional states as performed in this study may provide a framework for understanding how in vivo biological function emerges from complex cell ensembles, thus allowing for a clearer understanding of potential mutual pathways. Methods Patients with proteinuria and known ISN/RPS Class and controls were recruited to discovery 1 and 2 cohorts. Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of non-lesional non sun-exposed skin from the buttocks. The libraries were prepared on the Fluidigm C1 platform (discovery 1) and 10X Genomics platform (discovery 2) along with Illumina HiSeq 2500 sequencing. Results Sorting based on COL1A1, COL1A2, COL3A1, MFAP5 and MFAP4 expression yielded 12 fibroblasts from 3 patients. The 1 Class II subject yielded 5 single-cell transcriptomes. The other 2 subjects (1 Class IV,V; 1 Class III,V) yielded 7 single-cell transcriptomes. 22 transcriptomes were derived from 3 controls. The aggregate data were used to determine the top upregulated genes in cases versus controls, most of which belonged to the interferon-stimulated gene category and the extracellular matrix category (DAVID databases). Fewer cells were obtained using Fluidigm C1 (36 single-cell) than 10X Genomics (7280 single-cell). For the latter, the major biopsy classes were represented (Class III, III/IV, III/V, V and no LN). We applied graph-based clustering and identified 12 major clusters of cells from the patient skin as visualized by t-distributed stochastic neighbor embedding (t-SNE; figure 1). Differential gene expression analysis guided by established lineage markers revealed three keratinocyte clusters (KC1- KC3), two fibroblast clusters (FB1, FB2), smooth muscle cells (SMC), two endothelial cell clusters (VEC, LEC), melanocytes (MEL), sweat gland cells (SG), macrophages/dendritic cells (MAC-DC) and T cells (TC). Ranked by abundance, patient skin exhibited KC>FB>EC>MAC-DC>SMC>TC>SG> MEL. Conclusions Single-cell RNAseq is both feasible and informative in cell-specific transcriptome analysis of fresh non-lesional non-sun exposed LN skin biopsies. 10X genomics significantly increases cell numbers and facilitates identification of major cell clusters compared to Fluidigm C1. The expression of fibroblasts and genes reflective of fibrotic and interferonrelated pathways support the application of this approach to study readily accessible tissue for biomarkers related to disease progression. (Figure Presented)

Background Autoantibody and laboratory testing is essential for SLE diagnosis. ANA by indirect immunofluorescence (ANA IIF) remains the gold standard to screen for lupus and studies demonstrate preclinical phase during which autoantibodies accumulate. Prevalence of ANA IIF alone without more specific autoantibodies and the accompanying clinical phenotype of these patients uncertain. Methods Queried 602 patients in the NYU Lupus Registry with 4 or more ACR or SLICC criteria as adjudicated by the authors for prevalence of ANA IIF, dsDNA, Sm, Ro, La, RNP, aPL, C3, C4. Compared clinical features of isolated ANA (ANA IIF alone) with the ANA IIF plus one or more lupus associated abnormalities (ANA IIF +). Results 590/602 (98%) ANA IIF positive. 548/590 (93%) patients at least one of associated tests compared to only 42/ 590 (7%) ANA IIF alone. SLE nephritis significantly more prevalent in ANA IIF+254/548 (46%), compared to 13/42 (31%) recorded with renal criteria ANA IIF alone. ANA IIF +, 158/254 (62%) biopsy proven nephritis (LN), rather than relying on proteinuria for diagnosis, compared to 5/13 (38%) of ANA IIF alone biopsy proven LN. Remaining 8 ANA IIF alone, uPCR exceeded 0.5 g in 1 of 44 (2%) encounters. Low incidence of proteinuria explained by complete renal response or prior proteinuria misconstrued as evidence of LN. In comparison, uPCR >0.5 g was present in 694 of 1157 (60%) encounters in ANA IIF +, casting doubt on validity of LN diagnosis in 8 ANA IIF alone without biopsy. Leucopenia, lymphocytopenia, AITP, AIHA statistically less ANA IIF alone compared to ANA IIF + ; 24% vs 36%, 29% vs 40%, 7% vs 15% and 0% vs 7%, respectively. 42 patients with ANA IIF alone prevalence of potentially misattributed (e.g. not result of IMID) clinical criteria such as photosensitivity (64%) and malar rash (60%) greater compared to ANA IIF +, 38% and 44%, respectively. Prevalence oral ulcers, DLE, arthritis, serositis, seizures and psychosis equivalent in both. Conclusions ANA IIF alone rare and patients infrequently develop nephritis, leucopenia, lymphocytopenia, AITP, AIHA. In patients ANA IIF alone attribution of ACR/SLICC clinical criteria needs to be point of emphasis and unless biopsy proven, alternative explanation for proteinuria should be considered. Data argues inclusion criteria for clinical trials, rather than allowing ANA IIF alone or dsDNA, may need to require ANA IIF and at least one of the following (dsDNA, Sm, Ro, La, aPL, or hypocomplementemia) to avoid enrolling patients that do not have SLE

OBJECTIVE:Extant epidemiologic data of primary Sjögren's Syndrome (pSS) remains limited, particularly for racial/ethnic populations in the United States (US). The Manhattan Lupus Surveillance Program (MLSP), a population-based retrospective registry of cases with Systemic Lupus Erythematosus and related diseases including pSS in Manhattan, was used to provide estimates of the incidence and prevalence of pSS across major racial/ethnic populations. METHODS:MLSP cases were identified from hospitals, rheumatologists, and population databases. Three case definitions were used for pSS: physician diagnosis, rheumatologist diagnosis, and modified pSS criteria. Rates among Manhattan residents were age-adjusted, and capture-recapture analyses were conducted to assess case under-ascertainment. RESULTS:By physician diagnosis, age-adjusted overall incidence and prevalence rates of pSS among adult Manhattan residents were 3.5 and 13.1 per 100,000 person-years. Capture-recapture adjustment increased incidence and prevalence rates (4.1 and 14.2). Based on physician diagnosis, incidence and prevalence rates were approximately 6 times higher among women than men (p<0.01). Incidence of pSS was statistically higher among non-Latina Asian (10.5) and non-Latina White women (6.2) compared with Latina women (3.2). Incidence was also higher among non-Latina Asian women compared with non-Latina Black women (3.3). Prevalence of pSS did not differ by race/ethnicity. Similar trends were observed when more restrictive case definitions were applied. CONCLUSION/CONCLUSIONS:Data from the MLSP revealed disparities in pSS incidence and prevalence by sex among Manhattan residents and differences in pSS incidence by race/ethnicity among women. These data also provided epidemiologic estimates for the major racial/ethnic populations in the US.

Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus (LE) and other autoimmune and dermatologic conditions. Photosensitivity is associated with increased keratinocyte apoptosis, but mechanisms that modulate keratinocyte apoptosis that are dysfunctional in photosensitivity are poorly understood. Here, we identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. LCs express EGFR ligands and ADAM17, the metalloprotease that activates EGFR ligands. LC ADAM17 is required for skin protection and is activated by UVR, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic LE (SLE) models have reduced LC ADAM17, and topical EGFR ligand reduces photosensitivity. Human SLE skin shows reduced EGFR activation. Together, our data identify a new tissue protective function for LCs, delineate a novel mechanism that limits skin injury, and suggest EGFR stimulation as a treatment for photosensitivity and possibly other dermatologic conditions.
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Background There are no longitudinal studies regarding thelong term cardiac health of children with cardiac manifestations of neonatal lupus (NL). This study was performed toevaluate risk factors for morbidity and provide evidence-basedguidance regarding the course of cardiac NL.Methods Echocardiograms throughout life were evaluated in240 individuals born with cardiac NL from the ResearchRegistry for Neonatal Lupus: 142 were available from ages 0 1 years, 174 from ages 1 17 years, and 65>17 years. A composite adverse outcome defined as qualitatively decreased leftventricular (LV) function or concurrent use of cardiac medications was assessed. Aortic dilation (root or ascendingaorta z-score >2.0) was also recorded. Analyses were performed to associate the composite adverse outcome and aorticdilation with maternal medications, pacing, and fetal diseasestatus, including a severity score based on mortality risk factors such as lower fetal heart rate and extranodal disease.Results The composite adverse outcome for cardiac dysfunctionwas identified in 21.1% of echos in children ages 0 1, 13.2% ages1 17% and 29.2% ages>17. In 89 children in which echos wereavailable at ages 0 1 and 1 17, 6/16 with dysfunction at ages 0 1were also affected at ages 1 17, while 10 reverted to normal.Among those without dysfunction at age 0 1, 8/90 developednew worsening of cardiac function during age 1 17. In 35 caseswith echos at ages 1 17 and >17, 3/3 cases with dysfunction atage 1 17 were also affected at >17, and 2/32 developed new dysfunction in adulthood. Cardiac dysfunction was significantly associated with number of years paced at all ages (p<0.001, 0.001,<0.001). A lower fetal ventricular heart rate at the first time ofheart block detection was associated with cardiac dysfunction age0 1 and >17 (p=0.048, 0.005 respectively) and lowest heart ratein utero associated with dysfunction at age <1 and 1 17(p<0.001, 0.015). Fetal extranodal cardiac disease was associatedwith dysfunction in ages1 17 and >17 (p=0.026, 0.023). Higherfetal severity score associated with postnatal dysfunction in ages0 1 and 1 17 groups (p=0.013, 0.001). Aortic dilation waspresent in 13.4% at ages 0 1% and 14.9% at ages 1 17, butat >17, dilation only occurred in 9.2%. There was no associationof postnatal cardiac dysfunction or aortic dilation with maternalmedication use, maternal rheumatic disease, fetal age at heartblock detection or gestational age of birth.Conclusions Cardiac dysfunction in the first year normalizesby later childhood in the majority of cases, possibly due tothe short term effects of cardiac pacing or resolution ofinflammation with the clearance of maternal autoantibodies.However, new onset dysfunction can occur after the first yearof life. Aortic dilation can continue for longer periods, butmay decrease in frequency with age. Nevertheless, cardiac dysfunction is present in roughly 30%, and in adulthood thereare associations with fetal extranodal disease and heart rate atdetection. Patients who develop morbidity in utero may havesubclinical damage or be more susceptible to future insultsthat manifest later in life, which can be exacerbated by prolonged pacing. Close monitoring and aggressive treatment ofearly extranodal disease in cardiac NL may have long termbenefit in preventing subsequent morbidity

Background Missing data due to drop-out and loss to followup is a common problem in SLE trials. The usual approachesfor handling this issue include analyzing only subjects withcomplete data (complete case analysis; CC), last observationcarried forward (LOCF), or imputing non-responses for missing outcomes (non-responder imputation; NRI). However, thevalidity of these methods depends on strong assumptionsabout the missing data mechanism. Multiple imputation (MI)is a flexible model-based technique that accounts for uncertainty in the imputation process by generating several possiblevalues for the missing data, resulting in multiple completedata sets. These are analyzed separately and results are combined. MI is being used more widely in different disease settings but has not been applied to analyze the primaryoutcome in a SLE trial. We explored the use of MI to addressmissing data in the composite outcome, SLE Responder Index(SRI)-5, using data from patients assigned to standard of care(SoC) in a 52 week trial.Methods Data on 279 SLE patients randomized to SoC for 52weeks who were receiving mycophenolate mofetil (MMF), azathioprine, or methotrexate at entry were obtained from theLupus Foundation of America-Collective Data Analysis Initiative database. Multiple imputation using chained equationswas applied to handle missing data in an analysis to evaluatedifferences in SRI-5 response rates at 52 weeks betweenpatients on MMF and the other immunosuppressants (nonMMF). Three different imputation models were consideredthat included various combinations of longitudinal measures ofdisease activity (both composite and individual measures) andpatient characteristics. Results were compared to estimatesusing the CC, LOCF, and NRI.Results Missing data rates were 32% in the MMF and 23%in the non-MMF groups. As expected, the NRI missing dataapproach yielded the lowest response rates; the smallest andleast significant estimates of between group differences wereobserved with LOCF (table 1). Group differences were magnified with all three MI models compared to results of othermethods. Imputing SRI-5 directly (MI-1) versus the individualcomponents (MI-2) yielded nearly identical results.Conclusions Given the limitations of conventional approachesfor handling missing data, the MI method should also be considered in SLE trials. However, results can vary depending onthe imputation model that is used, and the assumptionsrequired for validity of this and other missing data methodsmust be justified. Sensitivity analysis using different approachesis important to demonstrate robustness of results especiallywhen missing data rates are non-negligible

Background Although hydroxychloroquine (HCQ) is a mainstay of treatment for patients with Systemic Lupus Erythematosus (SLE), ocular toxicity can result from accumulatedexposure. The introduction of highly sensitive tools has engendered even more concern. As the longevity of patients withSLE improves, additional data will help physicians accuratelybalance the risk of ocular toxicity and the risk of diseaseflare, especially in older patients who have stable/quiescent disease. Accordingly, this study was initiated to examine thesafety of HCQ withdrawal in older SLE patients.Methods Data were obtained by retrospective chart review atthree lupus centers. Twenty-seven patients met the following inclusion criteria:-4 ACR criteria, disease duration-5 years, HCQuse of 200 400 mg per day-5 years, and discontinuation ofhydroxychloroquine at-age 55 years. The comparator groupcomprised 39 age, gender and racial/ethnic matched patients whoremained on HCQ. The primary outcome was a clinically meaningful flare within one year of HCQ withdrawal, defined asmoderate or severe, using a revised version of the SELENA-SLEDAI Flare composite that separates mild from moderate flares,evaluates each organ system separately, and incorporates increasesin corticosteroid dose and/or addition of immunosuppressiveagents. Mild flares were considered secondary outcomes.Results Demographics are provided in table 1. There was atrend toward longer disease duration in the HCQ withdrawalgroup but no difference in prevalence of prior lupus nephritisbetween the groups. The reasons for HCQ withdrawal weremaculopathy (n=13), presumed/biopsy proven cardiomyopathy(n=2), patient request (n=4), and miscellaneous other reasons(n=8). There was no difference in the primary or secondaryoutcomes between the groups (table 1). Two patients had amoderate flare after discontinuing HCQ, of whom one hadarthritis treated with methotrexate and one had thrombocytopenia (>30K) and proteinuria of 2 grams/d (baseline 700 mg).Three patients had severe flares while continuing HCQ, ofwhom two were hospitalized, one for seizures and one forpericarditis; the third had worsening nephritis (urinaryprotein >4 g/d, requiring treatment). Two patients had moderate flares while remaining on HCQ, one of whom had a rashand arthritis treated with tofacitinib and one a rash treatedwith prednisone.Conclusions In this retrospective study of older patients withSLE on long-term HCQ, withdrawal did not increase the riskof moderate or severe flares. These data provide reassuranceregarding the safety of withdrawing HCQ in stable older SLEpatients