Faculty Bibliography

OBJECTIVE:This work aims at evaluating longitudinally titers of antibodies against β2-glycoprotein I (β2GPI) and domain 1 (anti-D1), identifying predictors of the variation of anti-D1 and anti-β2GPI antibody titers and clarifying whether antibody titer fluctuations predict thrombosis in a large international cohort of patients persistently positive for antiphospholipid antibodies (aPL), the "APS ACTION Registry". METHODS:Patients with available blood samples from at least 4 time points were included. Anti-β2GPI and anti-D1 IgG were tested by chemiluminescence (BioFlash, INOVA Diagnostics). RESULTS:In a cohort of 230 patients, anti-D1 and anti-β2GPI titers decreased significantly over time (p<0.0001 and p=0.010, respectively). After adjustment for age, gender, and number of positive aPL tests, the fluctuation of anti-D1 and anti-β2GPI titers was associated with treatment with hydroxychloroquine (HCQ) at each time-point. Treatment with HCQ, but not immunosuppressors, was associated with 1.3-fold and 1.4-fold decrease in anti-D1 and anti-β2GPI titers, respectively. Incident vascular events were associated with 1.9-fold and 2.1-fold increase of anti-D1 and anti-β2GPI titers, respectively. Anti-D1 and anti-β2GPI titers at the time of thrombosis were lower compared to the other time-points: 1.6-fold decrease in anti-D1 titers and 2-fold decrease in anti-β2GPI titers conferred an OR for incident thrombosis of 6.0 (95%CI 0.62-59.3) and 9.4 (95%CI 1.1-80.2), respectively. CONCLUSIONS:Treatment with HCQ and incident vascular events significant predicted anti-D1 and anti-β2GPI titer fluctuation over time. Both anti-D1 and anti-β2GPI titers drop around the time of thrombosis, with potential clinical relevance. This article is protected by copyright. All rights reserved.

OBJECTIVE:Platelets are mediators of inflammation with immune effector cell properties, and have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). This study investigated the role of platelet associated lectin galactoside-binding soluble 3 binding protein (LGALS3BP) as a mediator of inflammation in SLE, and a potential biomarker associated with clinical phenotypes. METHODS:We performed RNA sequencing on platelets of patients with SLE (n=54) and age, sex, and race-matched controls (n=18) and measured LGALS3BP in platelet releasate and in circulating serum. We investigated the association between levels of LGALS3BP with the prevalence, disease severity, and clinical phenotpyes of SLE, and studied platelet-mediated effects on myeloid inflammation. RESULTS:). Platelet-released LGALS3BP was highly correlated with circulating LGALS3BP (R = 0.69, p < 0.0001). Circulating LGALS3BP correlated with the SLE disease activity index (R = 0.32, p = 0.0006). Specifically, circulating LGALS3BP was higher in SLE patients with lupus nephritis than those with inactive disease (4.0 μg/mL vs 2.3 μg/mL, P < 0.001). IFN-α induced LGALS3BP transcription and translation in a megakaryoblastic cell line (MEG-01) cells in a dose-dependent manner. Recombinant LGALS3BP and platelet releasates from SLE patients enhanced pro-inflammatory cytokine production by macrophages. CONCLUSIONS:These data support that platelets act as potent effector cells contributing to the pathogenesis of SLE by secreting proinflammatory LGALS3BP, which also represents a novel biomarker of SLE clinical activity.

OBJECTIVE:Using the Manhattan Lupus Surveillance Program (MLSP), a multi-racial/ethnic population-based registry, we compared three commonly used classification criteria for Systemic Lupus Erythematosus (SLE) to identify unique cases and determine the incidence and prevalence of SLE using the EULAR/ACR criteria. METHODS:SLE cases were defined as fulfilling 1997 ACR, SLICC, or EULAR/ACR classification criteria. We quantified the number of cases uniquely associated with each and the number fulfilling all three. Prevalence and incidence using the EULAR/ACR classification criteria and associated 95% confidence intervals (CI) were calculated. RESULTS:1,497 cases fulfilled at least one of the three classification criteria, with 1,008 (67.3%) meeting all three classifications, 138 (9.2%) fulfilling only SLICC criteria, 35 (2.3%) fulfilling only ACR criteria and 34 (2.3%) uniquely fulfilling EULAR/ACR criteria. Patients solely satisfying EULAR/ACR criteria had fewer than four manifestations. The majority classified only by the ACR criteria did not meet any of the defined immunologic criteria. Patients fulfilling only SLICC criteria did so based on the presence of features unique to this system. Using the EULAR/ACR classification criteria, age-adjusted overall prevalence and incidence rates of SLE in Manhattan were 59.6 (95%CI:55.9-63.4) and 4.9 (95%CI 4.3-5.5) per 100,000 population, with age-adjusted prevalence and incidence rates highest among non-Hispanic Black females. CONCLUSION/CONCLUSIONS:Applying the three commonly used classification criteria to a population-based registry identified patients with SLE fulfilling only one validated definition. The most recently developed EULAR/ACR classification criteria revealed similar prevalence and incidence estimates to those previously established for the ACR and SLICC classification schemes.

Background Lupus nephritis (LN) is characterized by considerable variability in its clinical manifestations and histopathological findings. Understanding the cellular and molecular mechanisms underlying this heterogeneity is key for the development of personalized treatments for LN. Methods Droplet-based single-cell RNA-sequencing was applied to the analysis of dissociated kidney samples, collected from 155 LN patients with active kidney disease and 30 living donor controls as part of the Accelerating Medicines Partnership (AMP) in SLE consortium -a large-scale, multi-center study. 73,440 immune cells passing quality control were identified, spanning 134 cell subsets, representing various populations of tissue-resident and infiltrating leukocytes, as well as the activation states these cells assume as part of their diseaserelated activation and differentiation (figure 1). Principal component analysis (PCA) was used to characterize the variability in cell subset frequencies across the LN patients. Relationships between the resulting principal components (PCs) and the demographic, clinical and histopathological features of the patients were then assessed. Results The main source of variability in immune cell subset frequencies, as represented by the first PC (PC1), reflected the balance between lymphocytes and monocytes/macrophages. Subsequent PCs represented the balance between B cells and T cells (PC2); the levels of cytotoxic T lymphocytes and NK cells, as compared to plasma cells (PC3); and the degree of macrophage differentiation to an alternatively activated phagocytic profile (PC4). PC1 was significantly correlated with the Chronicity index, such that patients with a higher percentage of lymphocytes compared to monocytes/macrophages had a higher Chronicity score (rho = -0.422, p-value < 0.001; figure 2A). A high degree of macrophage differentiation, as represented by PC4, was associated with a high Activity score (rho = 0.387, p-value < 0.001; figure 2B), and, in addition, with proliferative or mixed histology class, compared to pure membranous nephritis (p-value = 0.001, Kruskal-Wallis test). The ratio of B cells to T cells, as represented by PC2, demonstrated a positive correlation with the Activity index (rho = 0.311, p-value < 0.001). We further identified a significant correlation of PC1 with age; specifically, older patients had a higher relative frequency of lymphocytes compared to monocytes/macrophages (rho = -0.239, p-value = 0.003). Our analysis indicated that these relations are not driven by demographic, clinical and technical sources of variation in our data, including race, ethnicity, the mixture of different nephritic classes, and the inclusion of both first and later biopsies. Conclusion Our work identifies distinct leukocyte populations active in different LN patients and, possibly, different stages of disease, and points to potential therapeutic targets, that must be validated in mechanistic studies. This approach may pave the way to personalized treatment of LN

BACKGROUND:Complement activation has been associated with adverse pregnancy outcomes (APO) in SLE. Pregnant women with SLE were studied to evaluate whether complement dysregulation within the first two pregnancy trimesters predicts APO. METHODS:Pregnant women fulfilled classification criteria for SLE. APO included neonatal death, preterm delivery before 36 weeks and small for gestational age newborn. Pre-eclampsia was also evaluated. Erythrocyte complement receptor 1 (ECR1) and erythrocyte-bound C4d (EC4d) were measured by flow cytometry. Complement proteins C3 and C4 were measured by immunoturbidimetry and anti-double-stranded DNA by ELISA in serum. Statistical analysis consisted of t-test, confusion matrix-derived diagnostic analysis, and multivariate logistic regression. RESULTS:Fifty-one women had 57 pregnancies and 169 visits during the study. Baseline visits occurred mainly in the first (n=32) and second trimester (n=21). Fourteen (24.6%) pregnancies resulted in 21 APO with preterm delivery being the most common (n=10). ECR1 <5.5 net mean fluorescence intensity in the first trimester predicted APO with a diagnostic OR (DOR) of 18.33 (95% CI: 2.39 to 140.4; t-test p=0.04). Other individual biomarkers did not reach statistical significance. To estimate the likelihood of APO, we developed an algorithm that included the week of pregnancy, ECR1 and EC4d. From this algorithm, a Pregnancy Adversity Index (PAI) was calculated, and a PAI >0 indicated an elevated likelihood of pregnancy complications (DOR: 20.0 (95% CI: 3.64 to 109.97)). CONCLUSIONS:Low levels of ECR1 in early or mid-pregnancy are predictive of an APO. Incorporating the weeks of gestation and both ECR1 and EC4d generated a PAI, which further predicted serious pregnancy complications.

BACKGROUND/PURPOSE/OBJECTIVE:APS ACTION Registry was created to study the outcomes of patients with persistently positive antiphospholipid antibodies (aPL) with or without other systemic autoimmune disease (SAIDx). Given that immunosuppression (IS) is used for certain aPL manifestations, for example, thrombocytopenia (TP), our primary objective was to describe the indications for IS in aPL-positive patients without other SAIDx. Secondly, we report the type of IS used in patients with selected microvascular or non-thrombotic aPL manifestations. METHODS:An online database is used to collect clinical data. The inclusion criteria are positive aPL based on the laboratory section of the APS Classification Criteria, tested at least twice within one year prior to enrollment. Patients are followed every 12 ± 3 months. For this descriptive retrospective and prospective analysis, we included aPL-positive patients without other SAIDx and excluded those with new SAIDx classification during follow-up. For each patient, we retrieved clinical data at baseline and follow-up including selected aPL manifestations (diffuse alveolar hemorrhage [DAH], antiphospholipid-nephropathy [aPL-N], livedoid vasculopathy [LV]-related skin ulcers, TP, autoimmune hemolytic anemia [AIHA], cardiac valve disease [VD]), and IS medications. RESULTS:Of 899 patients enrolled, 537 were included in this analysis (mean age 45 ± 13 years, female 377 [70%], APS Classification in 438 [82%], and at least one selected microvascular or non-thrombotic aPL manifestation in 141 (26%)). Of 537 patients, 76 (14%) were reported to use IS (ever), and 41/76 (54%) received IS primarily for selected aPL manifestation. In six of 8 (75%) DAH patients, 6/19 (32%) aPL-N, 4/28 (14%) LV, 25/88 (28%) TP, 6/11 (55%) AIHA, and 1/43 (2%) VD, the IS (excluding corticosteroids/hydroxychloroquine) indication was specific for selected aPL manifestation. CONCLUSION/CONCLUSIONS:In our international cohort, 14% of aPL-positive patients without other SAIDx were reported to receive IS; the indication was at least one of the selected microvascular and/or non-thrombotic aPL-related manifestations in half. Thrombocytopenia was the most frequent among those selected aPL-related manifestations; however, approximately one-third received IS specifically for that indication. Diffuse alveolar hemorrhage was frequently treated with IS followed by AIHA and aPL-N. Systematic controlled studies are urgently needed to better define the role of IS in APS.

OBJECTIVES/OBJECTIVE:Hydroxychloroquine is recommended for all patients with systemic lupus erythematous, but reports of cardiac toxicity in SARS CoV-2 patients raised concerns. We aimed to study the relationship between hydroxychloroquine blood levels and QTc intervals. METHODS:Cohort 1 is a retrospective review of 90 SLE patients with data collected regarding demographics, QTc interval and chronic kidney disease (CKD). Cohort 2 is a prospective study of 84 SLE patients with data collected regarding demographics, dose of HCQ, duration of HCQ treatment, presence of echocardiographic abnormalities, and CKD simultaneous with whole blood HCQ levels measured by high performance liquid chromatography. Statistical analysis utilized one way ANOVA, Pearson's correlation coefficient and t test. RESULTS:In the retrospective cohort there was no significant difference in mean QTc based on 75 HCQ-treated (437.91 +/- 20.02) as compared with 15 untreated (434.6 +/- 27.49) patients. In patients with CKD mean QTc in HCQ users (448 +/- 23.37) as compared with non-users (444.5 +/- 24.61) was also with no significant difference. In the prospective cohort HCQ levels did not correlate with QTc interval (r = 0.017) and this applied regardless of dose prescribed (r = 0.113 for 400 mg and r = 0.06 for 200 mg), duration of exposure (p= 0.36 for 0-5, 5-10, or > 10 years), CKD (r = 0.482) or underlying cardiac abnormalities (r = 0.430). CONCLUSION/CONCLUSIONS:This is the first study relying on measured blood levels demonstrating the absence of clinically consequential increase in QTc levels in HCQ treated SLE patients.