Faculty Bibliography

INTRODUCTION/BACKGROUND:Treatment failures for lupus nephritis (LN) are high with 10%-30% of patients progressing to end-stage renal disease (ESRD) within 10 years. Interstitial fibrosis/tubular atrophy (IFTA) is a predictor of progression to ESRD. Prior studies suggest that tubulointerstitial injury secondary to proteinuria in LN is mediated by complement activation in the tubules, specifically through the membrane attack complex (MAC). This study aimed to investigate the associations between tubular MAC deposition with IFTA and proteinuria. METHODS:In this cross-sectional study, LN kidney biopsies were assessed for MAC deposition by staining for Complement C9, a component of the MAC. Chromogenic immunohistochemistry was performed on paraffin-embedded human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). Tubular C9 staining intensity was analysed as present versus absent. IFTA was defined as minimal (<10%), mild (10%-24%), moderate (25%-50%) and severe (>50%). RESULTS:Renal biopsies from 30 patients with LN were studied. There were 24 (80%) female sex, mean age (SD) was 33 (12) years old and 23 (77%) had pure/mixed proliferative LN. Tubular C9 staining was present in 7 (23%) biopsies. 27 patients had minimal-to-mild IFTA and 3 patients had moderate IFTA. Among the C9 + patients, 3 (43%) had moderate IFTA as compared with none in the C9- group, p=0.009. C9 + patients had higher median (IQR) proteinuria as compared with C9- patients: 6.2 g (3.3-13.1) vs 2.4 g (1.3-4.6), p=0.001 at the time of biopsy. There was no difference in estimated glomerular filtration rate (eGFR) between the C9 + and C9- groups. CONCLUSION/CONCLUSIONS:This study demonstrated that tubular MAC deposition is associated with higher degree of IFTA and proteinuria, which are predictors of progression to ESRD. These results suggest that tubular MAC deposition may be useful in classification of LN. Understanding the role of complement in tubulointerstitial injury will also identify new avenues for LN treatment.

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

Coronavirus disease 2019 (COVID-19) is associated with a high rate of thrombosis. Prolonged activated partial thromboplastin times (aPTT) and antiphospholipid antibodies (aPL) are reported in COVID-19 patients. The majority of publications have not reported whether patients develop clinically relevant persistent aPL, and the clinical significance of new aPL-positivity in COVID-19 is currently unknown. However, the reports of aPL-positivity in COVID-19 raised the question whether common mechanisms exist in the pathogenesis of COVID-19 and antiphospholipid syndrome (APS). In both conditions, thrombotic microangiopathy resulting in microvascular injury and thrombosis is hypothesized to occur through multiple pathways, including endothelial damage, complement activation, and release of neutrophil extracellular traps (NETosis). APS-ACTION, an international APS research network, created a COVID-19 working group that reviewed common mechanisms, positive aPL tests in COVID-19 patients, and implications of COVID-19 infection for patients with known aPL positivity or APS, with the goals of proposing guidance for clinical management and monitoring of aPL-positive COVID-19 patients. This guidance also serves as a call and focus for clinical and basic scientific research.

BACKGROUND:Antiphospholipid syndrome (APS) is characterized by episodes of thrombosis, obstetric morbidity or both, associated with persistently positive antiphospholipid antibodies (aPL). Studying the profile of a rare disease in an admixed population is important as it can provide new insights for understanding an autoimmune disease. In this sense of miscegenation, Brazil is characterized by one of the most heterogeneous populations in the world, which is the result of five centuries of interethnic crosses of people from three continents. The objective of this study was to compare the clinical and laboratory characteristics of Brazilian vs. non-Brazilian primary antiphospholipid syndrome (PAPS) patients. METHODS:We classified PAPS patients into 2 groups: Brazilian PAPS patients (BPAPS) and PAPS patients from other countries (non-BPAPS). They were compared regarding demographic characteristics, criteria and non-criteria APS manifestations, antiphospholipid antibody (aPL) profile, and the adjusted Global Antiphospholipid Syndrome Score (aGAPSS). RESULTS:We included 415 PAPS patients (88 [21%] BPAPS and 327 [79%] non-BPAPS). Brazilian patients were significantly younger, more frequently female, sedentary, obese, non-white, and had a higher frequency of livedo (25% vs. 10%, p < 0.001), cognitive dysfunction (21% vs. 8%, p = 0.001) and seizures (16% vs. 7%, p = 0.007), and a lower frequency of thrombocytopenia (9% vs. 18%, p = 0.037). Additionally, they were more frequently positive for lupus anticoagulant (87.5% vs. 74.6%, p = 0.01), and less frequently positive to anticardiolipin (46.6% vs. 73.7%, p < 0.001) and anti-ß2-glycoprotein-I (13.6% vs. 62.7%, p < 0.001) antibodies. Triple aPL positivity was also less frequent (8% vs. 41.6%, p < 0.001) in Brazilian patients. Median aGAPSS was lower in the Brazilian group (8 vs. 10, p < 0.0001). In the multivariate analysis, BPAPS patients still presented more frequently with livedo, cognitive dysfunction and sedentary lifestyle, and less frequently with thrombocytopenia and triple positivity to aPL. They were also less often white. CONCLUSIONS:Our study suggests a specific profile of PAPS in Brazil with higher frequency of selected non-criteria manifestations and lupus anticoagulant positivity. Lupus anticoagulant (not triple positivity) was the major aPL predictor of a classification criteria event.

Background/Purpose: Hydroxychloroquine (HCQ) is an antimalarial drug used in the treatment of systemic lupus erythematous (SLE). There is limited data assessing cardiac toxicity as arrhythmias in association with HCQ exposure based on dose prescribed or pharmacy records and none relying on measured drug levels. Some of the risk factors associated with conduction abnormalities in the setting of hydroxychloroquine use include presence of chronic kidney disease, older age, underlying cardiomyopathy, and the use of concomitant prolonging QTc agents. In a retrospective study of 194 SLE patients on HCQ, the authors found that there was no significant difference in mean QTc based on HCQ use. Additionally, patients with CKD were more likely to have prolonged QTc when compared to those without CKD, but there was no significant difference in mean QTc based on HCQ use as well in this subset. Severe prolongation of QTc was rare in all groups and no episodes of serious tachyarrhythmia or Torsade de Pointes were observed. The purpose of this study is to determine the relationship between whole blood HCQ levels and QTc intervals on simultaneous EKG performed during a routine visit.
Method(s): This prospective study was IRB approved and all patients provided consent. At the time of data lock, 84 patients fulfilled ACR/SLICC criteria for SLE. These patients were on HCQ for at least 3 months at doses used for standard of care treatment. Whole blood levels were drawn and EKGs were obtained during a routine outpatient faculty practice visit for patients consecutively seen between February 5 and May 10, 2021 with senior author. Statistical analyses was performed using one way ANOVA, Pearson's correlation coefficient and t-test.
Result(s): 84 patients, 93% female, 47% European, 35% African, 15% Asian, and 25% Hispanic were included (Table 1). HCQ levels were higher in patients on 400 mg, lower after 10 years of exposure, and unrelated to eGFR (Table 2). There was no correlation between blood HCQ levels and QTc intervals in the 84 patients (r=-0.017; p=0.87) (Fig 1a). Additionally, there was no correlation between blood HCQ levels and QTc intervals in patients on 200 mg or 400 mg of HCQ (r=0.113, p=0.61; r=-0.06, p=0.65) (Fig 1b-c). There was no correlation between blood HCQ levels and QTc intervals in patients who had chronic kidney disease (defined as eGFR < 60), (r=-0.482, p=0.09) or those with underlying cardiac abnormalities noted on transthoracic echocardiogram (r=-0.430, p=0.16) (Fig 1d-e). However, there was a positive correlation between blood HCQ levels and QTc intervals in patients who were on concomitant QTc prolonging agents, (r=0.795, p=0.005). but none in excess of 456 msec (Fig 1f).
Conclusion(s): Our study provides reassurance that hydroxychloroquine is not associated with QTc prolongation in patients with SLE and across different subsets of patients irrespective of blood level, dose prescribed, CKD or underlying cardiac abnormalities. There was a positive correlation between blood HCQ levels and QTc intervals in patients on concomitant QTc prolonging agents, but none were severely prolonged (eg > 500 msec). This is the first study relying on measured blood levels demonstrating the absence of consequential increase in QTc levels in HCQ treated SLE patients

Background/Purpose: Data on fluctuation of antibodies against domain 1 (anti-D1) of beta2-glycoprotein I (beta2GPI) are scarce. Patients with antiphospholipid syndrome (APS) and all three criteria tests for antiphospholipid antibodies (aPL) display higher titers of anti-D1, which correlate with abeta2GPI levels. This project aims at evaluating anti-D1 titers over time in a large international cohort of persistently aPL positive patients.
Method(s): AntiPhospholipid Syndrome Alliance For Clinical Trials and InternatiOnal Networking (APS ACTION) Registry was created to study the course of persistently aPL-positive patients with or without autoimmune disorders over at least 10 years. Inclusion criteria are positive aPL by Updated Sapporo Criteria tested within one year prior to enrolment. Patients are followed up every 12+/-3 months with clinical data and blood collection. Patients with available blood samples from at least three time points were included in this analysis. Anti-beta2 GPI and anti-D1 IgG were tested by chemiluminescence (BioFlash, INOVA Diagnostics) at APS ACTION core laboratories. Positive results were defined as >20 CU. Clinical data were retrieved from APS ACTION online database. Anti-D1 titers within the same subject were compared by Friedman's test. The association between categorical and continuous variables was assessed by chi-squared and Spearman's tests.
Result(s): In this longitudinal study, 1942 samples from 515 patients were tested for anti-D1 and abeta2GPI IgG; 230 patients with anti-D1 tested at >=3 time points were included (Table). Patients with thrombotic APS had anti-D1 titers significantly higher than those without thrombosis (p=0.022). Among 135 patients with at least one anti-D1 positive result, anti-D1 titers varied significantly over time (Friedman statistics: 508.5, p< 0.0001; anti-D1 geometric mean [95%CI] at baseline 189.0 [115.9-308.3]; T1 132.3 [81.1-215.8]; T2 113.8 [69.8-185.5]; T3 109.2 [66.9-178.1]. Anti-D1 titers were significantly higher at baseline compared to T3 (p=0.029). Over time, anti-D1 titers significantly decreased in 107 patients, and increased in 28 (p< 0.0001). In 11.3% of patients, anti-D1 results changed from positive to neg-ative (n: 20), or negative to positive (n: 6). (Mc Nemar's chi2=6.5; p=0.011). Anti-beta2GPI titers correlated with anti-D1 titers and significantly reduced at T3 compared to baseline (abeta2GPI at baseline 187.1 [14.5-1586.5]; T1 150.8 [11.1-1379.2]; T2 124.9 [12.2-1304]; T3 117.6 [8.7-1136.6]; Friedman statistics=11.32, p=0.010).
Conclusion(s): Anti-D1 antibodies vary significantly overtime and approximately 10% may become negative during follow up. Our future analysis of the registry will demonstrate the clinical relevance of this variation, and the impact of treatment. (Figure Presented)

Background/Purpose: Damage Index in APS (DIAPS) is a scoring system developed to assess long-term damage in thrombotic primary antiphospholipid syndrome (PAPS), which also correlates with impaired quality of life (EuroQoL) in Latin Americans. DIAPS is not validated in aPL-positive patients without thrombosis. Our primary objective was to quantify damage accrual measured by DIAPS in aPL-positive patients with or without a history of thrombosis in an international cohort. Secondly, we aimed to identify clinical and laboratory characteristics associated with damage in aPL-positive patients.
Method(s): In this cross-sectional study, we analyzed the baseline damage, measured by DIAPS, in APS ACTION Registry patients. The inclusion criteria were positive aPL according to Updated Sapporo Classification Criteria tested within one year prior to enrollment. We excluded patients with other autoimmune diseases. We analyzed the demographic, clinical, and laboratory characteristics of patients based on two subgroups: (1) thrombotic APS patients with high (DIAPS >=3) versus low damage (DIAPS < 3); and (2) non-thrombotic aPL-positive patients with damage (DIAPS >0) versus without damage (DIAPS=0). Chi-square, Fisher's exact test, Mann-Whitney U and Student t test were used when applicable. In the multivariate analysis, our model included age, gender, race and variables with p< 0.10 in the univariate analysis.
Result(s): Of the 826 aPL-positive patients included in the registry as of April 2020, 576 with no other systemic autoimmune diseases were included in the analysis (412 thrombotic and 164 non-thrombotic [108 aPL only and 56 obstetric]). Baseline demographic, clinical and laboratory characteristics are summarized in Table 1. For the thrombotic group, the most frequent domains contributing to damage were peripheral vascular (n=260, 63% -mainly deep vein thrombosis), neuropsychiatric (n=107, 30% -mainly ischemic stroke with sequelae) and cardiovascular (n=57, 14% -mainly heart valve disease). Older age, male gender, hypertension, hyperlipidemia and obesity were associated with high damage (Table 1). In the multivariate analysis, male gender (OR 1.73, 95%CI 1.10-2.71, p=.018) and hypertension (OR 1.90, 95%CI 1.21-2.99, p=.006) were independently correlated with high damage. For the non-thrombotic group, the most frequent domains contributing to damage were neuropsychiatric (n=25, 15% -mainly cognitive impairment) and cardiovascular (n=13, 8% -mainly heart valve disease). Hypertension and hyperlipidemia were independently associated with damage in the multivariate analysis (OR 2.72, 95%CI 1.09-6.80, p=.032 and OR 4.48, 95%CI 1.62-12.29, p=.004, respectively). There was no correlation between aPL profile (triple vs double vs single aPL) and damage in either group.
Conclusion(s): DIAPS was able to discriminate damage in a large multicenter cohort of aPL-positive patients. Traditional cardiovascular risk factors, namely older age, male gender, hypertension, hyperlipidemia and obesity, correlate with higher damage in thrombotic primary APS patients. Hypertension and hyperlipidemia also correlate with damage in aPL-positive patients without a history of thrombosis. (Figure Presented)

Background/Purpose: The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcgamma receptor type IIa (FcgammaRIIa)-R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcgammaRIIa genotypes and distinct clinical features.
Method(s): RNA-sequencing was done on platelets isolated from 51 patients fulfilling criteria for the classification of SLE based on recent EULAR/ACR definitions, and 18 healthy controls matched on age, sex, and race. Unsupervised clustering, differential gene expression, and gene set enrichment analysis (GSEA) were used to analyze differences between SLE patients and controls, and SLE subpopulations, based on SELENA SLEDAI Hybrid disease activity, specific organ manifestations, and FcgammaRIIa genotype. Weighted Gene Correlation Network Analysis (WGCNA) was performed to create a modular transcriptomic framework.
Result(s): Our cross-sectional SLE cohort (N=51, age = 41.1+/-12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, SLEDAI = 4.4+/-4.2) was comprised of patients consecutively enrolled excluding those on aspirin or anticoagulants. Compared to the 18 controls, there were 2290 (p.adj < 0.05) differentially expressed genes. ( Figure 1 A, B) GSEA revealed positive enrichment for pathways related to interferon response, TNFa signaling, and coagulation in SLE. ( Figure 1C) WGCNA was used to create a modular transcriptomic framework. ( Figure 2A ) Modules enriched for platelet activity, immune response, and WNT signaling were significantly increased in SLE versus controls. Moreover, modules enriched for interferon response and WNT signaling paralleled increases in disease activity. ( Figure 2B) When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. (Figure 2C) Analyzing the ratio of fold changes between SLE/Control vs SLE Proteinuria/SLE No Proteinuria, genes increased in SLE and those with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. (Figure 2D) The module enriched for FCR activation was decreased in SLE and was affected by the FcgammaRIIa genotype. (Figure 3A) FcgammaRIIa R131 and H131 patients showed significantly different platelet transcriptomes. (Figure 3B) The combination of SLE with an FcgammaRIIa R131 variant leads to a significant increase in the platelet activity module not seen in controls. (Figure 3C)
Conclusion(s): These analyses reveal that SLE patients have a significantly different platelet transcriptome from controls, different phenotypic presentations of SLE patients associate with distinct platelet transcriptomic signatures, and FCGR2a variants may differentially influence the role of platelets in the contribution to SLE disease activity