Faculty Bibliography

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non-sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

OBJECTIVE: The Manhattan Lupus Surveillance Program (MLSP) is a population-based registry designed to determine the prevalence of systemic lupus erythematosus (SLE) in 2007 and the incidence from 2007 to 2009 among residents of New York County (Manhattan), New York, and to characterize cases by race/ethnicity, including Asians and Hispanics, for whom data are lacking. METHODS: We identified possible SLE cases from hospital records, rheumatologist records, and administrative databases. Cases were defined according to the American College of Rheumatology (ACR) classification criteria, the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria, or the treating rheumatologist's diagnosis. Rates among Manhattan residents were age-standardized, and capture-recapture analyses were conducted to assess case underascertainment. RESULTS: By the ACR definition, the age-standardized prevalence and incidence rates of SLE were 62.2 and 4.6 per 100,000 person-years, respectively. Rates were approximately 9 times higher in women than in men for prevalence (107.4 versus 12.5) and incidence (7.9 versus 1.0). Compared with non-Hispanic white women (64.3), prevalence was higher among non-Hispanic black (210.9), Hispanic (138.3), and non-Hispanic Asian (91.2) women. Incidence rates were higher among non-Hispanic black women (15.7) compared with non-Hispanic Asian (6.6), Hispanic (6.5), and non-Hispanic white (6.5) women. Capture-recapture adjustment increased the prevalence and incidence rates (75.9 and 6.0, respectively). Alternate SLE definitions without capture-recapture adjustment revealed higher age-standardized prevalence and incidence rates (73.8 and 6.2, respectively, by the SLICC definition and 72.6 and 5.0 by the rheumatologist definition) than the ACR definition, with similar patterns by sex and race/ethnicity. CONCLUSION: The MLSP confirms findings from other registries on disparities by sex and race/ethnicity, provides new estimates among Asians and Hispanics, and provides estimates using the SLICC criteria.

Patients with systemic lupus erythematosus (SLE) often require immunosuppression to induce remission of active disease exacerbations. Over the past two decades, treatment modalities for this condition have emerged leading to improved morbidity from disease related outcomes. However, as a result, infection risks and patterns have changed, leading to higher rates of opportunistic infections among this population. We report four cases of cytomegalovirus (CMV) in patients with SLE who received immunosuppressive therapy, including pulse steroids, antimetabolites such as mycophenolate mofetil, and alkylating agents such as cyclophosphamide. We propose that given the rise in prevalence of CMV, there is a need for appropriate screening for this opportunistic pathogen and studies to determine the risks and benefits of prophylactic or preemptive treatment for this virus.

Background/Objective: Thrombocytopenia is a common feature of both SLE and antiphospholipid syndrome (APS) and in former most frequently results from antiplatelet antibodies (ie AITP) or antiphospholipid antibodies (aPL). Patients with AITP paradoxically have an increased risk of thrombosis and it has been speculated that this can result from co-presence of aPL. Primary APS is associated with both thrombosis and thrombocytopenia. We assessed whether there was an association between a history of thrombocytopenia and prevalence of a thrombotic event in a large, multiethnic cohort of SLE patients. Methods: We analyzed the NYU SLE SAMPLE Registry, consisting of patients fulfilling ACR and/or SLICC criteria for SLE. We identified 105 patients whose SLE criteria included the presence of one or more of the following APLS antibodies: lupus anticoagulant; IgG or IgM anti-beta2-glycoprotein-I; IgG or IgM anticardiolipin antibodies, and determined whether these patients had thrombocytopenia among their SLE classification criteria (<100,000/muL). We reviewed each patient's medical record to identify the prevalence of thrombosis (defined as DVT, PE, CVA, arterial occlusion with gangrene or amputation), and/or obstetric events. We compared the prevalence of thrombotic events in SLE patients with and without history of thrombocytopenia. Results The NYU SLE SAMPLE currently includes 612 patients (90% female, mean age 43.0+/-0.9 years, and 10% men, mean age 41.0+/-0.3 years). 54% of subjects were white, 31% African American, and 15% Asian. 30% were Hispanic White, and 5% Hispanic Black. 17% had aPL, of whom 89% were female and 11% male (mean age 43.0+/-0.2 years, 56% Caucasian, 33% African American, and 11% Asian). 24% were Hispanic white and 4% Hispanic Black. The total numbers of patients with thrombotic events were 45 (43%), with 5/21 (23%) in the SLE patients with aPL and prior history of thrombocytopenia and 40/ 84 (47%) (p=0.042) in the patients without thrombocytopenia. The most common thrombotic event was DVT followed by CVA. Conclusion The prevalence of aPL in the NYU SLE registry was 17%, and adverse thrombotic events were less common in the patients with prior history of thrombocytopenia (5/21, 23%) as compared to those without (40/84, 47%). This unexpected finding could be explained by protective benefit of antiplatelet antibodies when co-occur with aPL in SLE or less thrombogenic aPL in SLE when produces thrombocytopenia. Additionally, our data suggest that there might be different consequences of aPL between primary APS patients and SLE patients with aPL

Background/Objective: Few studies have analyzed the risk factors for thrombosis in SLE patients with antiphospholipid antibodies and most had small sample sizes and homogenous patient populations. We examined whether a history of nephritis is a risk factor for thrombosis using a large multi-ethnic SLE cohort. Methods: The NYU SLE SAMPLE Biorepository and Registry was initiated in September 2013 and includes 612 patients fulfilling ACR and/or SLICC criteria for SLE. Within SAMPLE, we identified patients with a positive test for antiphospholipid antibodies (aPL) (lupus anticoagulant, IgG or IgM anti-beta2-glycoprotein-I antibodies, IgG or IgM anticardiolipin antibodies) and determined if these patients had also ever fulfilled nephritis criteria. We reviewed each patient's medical record for presence/absence of thrombosis (DVT, PE, CVA, arterial occlusion with gangrene or amputation), and obstetric events as well as the noncriteria manifestations thrombocytopenia or valvulitis. We compared the prevalence of APLS manifestations in SLE patients with and without history of lupus nephritis. Results: Of the initial 612 SLE patients, 90% were female (mean age 43.0+/-0.9 years), and 10% were male (mean age 41.0+/-0.3 years). 54% were white, 31% African American, and 15% Asian; 30% were Hispanic white, and 5% Hispanic Black. 105 of the 612 patients had aPL, including 93 females and 12 males (mean age 43.0+/-0.2 years, 56% Caucasian, 33% African American, and 11% Asian). 24% were Hispanic white and 4% Hispanic Black. The total number of patients with thrombotic events was 45/105 (43%), including 26/43 (60%) in the nephritis subset and 19/62 (30%) (p: 0.04) among patients without prior renal involvement. The most common thrombotic event was DVT followed by CVA. Conclusion: The prevalence of aPL criteria in NYU SLE SAMPLE was 17%. Within this group, adverse events were more common among the patients with, versus without, prior history of renal involvement (60% vs 30%). It remains uncertain if association is explained by different accompanying antibodies that distinguish nephritis from non-nephritis SLE, e.g., dsDNA, complement, or drug treatment. This observation provides further support for universal use of hydroxychloroquine in SLE especially in setting of nephritis and suggests that event-free aPL positive patients with prior history of nephritis may be at increased risk for future thrombosis. Further studies are needed to determine whether such patients could benefit from prophylaxis with aspirin or other mild antithrombotic agents

Background/Objective: We and others have suggested that complement activation can serve as an initiating signal that increases the thrombosis risk in SLE patients with antiphospholipid antibodies (aPL). Generation of complement activation products can result in proinflammatory and/or prothrombotic responses providing a permissive environment for the pathological effects of antibodies to negatively charged phospholipid protein complexes. Methods: We analyzed the NYU SLE SAMPLE Biorepository initiated in September 2013, which consists of 599 patients fulfilling ACR and/or SLICC criteria for systemic lupus erythematosus. We identified 98 patients whose criteria included the presence of one or more for the following aPL: lupus anticoagulant, IgG or IgM anti-beta2- glycoprotein-I, or IgG or IgM anticardiolipin antibodies and determined if these patients received SLEDAI points for hypocomplementemia during any encounter. We then reviewed each patient's medical record to identify the prevalence of thrombosis defined as DVT, PE, CVA, arterial occlusion with gangrene or amputation, and obstetric events as well as noncriteria manifestation of thrombocytopenia or valvulitis. We then compared the prevalence of these APLS manifestations in the SLE patients with and without evidence of complement activation. Results: The NYU SLE SAMPLE biorepository includes 599 patients (90% female, mean age 43.0+/-.9, and 10 % men, mean age 41.0+/-.3, 54% Caucasian, 31% African American, 15% Asian, 30% Hispanic White, and 5% Hispanic Black). 98 of the 599 SLE patients had aPL, 86 female and 12 male (mean age 43.0+/-.2, 56% Caucasian, 33% African American, and 11% Asian). 24 % were Hispanic white and 4% Hispanic Black. The total number of patients with adverse events was 54 of 98 (55%) with 33/50 (66%) in the SLE patients with aPL and evidence of hypocomplementemia and 21/48 (43%) (p=0.04) in the patients without evidence of hypocomplementemia. The most common thrombotic event was DVT followed by CVA. Conclusions: The prevalence of aPL as a criteria in the NYU SLE registry is 98 of 599, and adverse events were more common in the patients with evidence of hypocomplementemia (33/50, 66%) as compared to the patients without complement activation (21/48, 43%). These findings can inform decisions regarding which patient subsets that may benefit from the prophylactic use of low-dose aspirin for primary prevention in asymptomatic lupus patients. Moreover, future clinical trials should be stratified on the basis of complement consumption to be sure that equal numbers of these patients appear in both the experimental and comparator treatment arms. Finally, future prospective studies should explore the interaction between complement activation products, platelets, neutrophils, mononuclear cells, endothelial cells, coagulation cascade in the adverse events that constitute antiphospholipid syndrome

Background/Objective: The spectrum of the vascular pathology affecting SLE patients with secondary APS includes vasculopathy with endothelial cell hyperplasia as in APS nephropathy. Previous studies established a role for endothelial cell activation via the mammalian target of rapamycin (mTORC) but were limited to primary APS patients and the IgG fractions. We extend this finding by studying SLE patients with secondary APS and demonstrate this activity resides in both IgG and IgA fractions. Methods: Endothelial cell phenotypes were assessed using an immunofluorescent assay to report the mTORC biomarker, phospho-S6 ribosomal protein (S6RP) using the human microvascular cell line (HMEC1) in the presence and absence of purified human IgG, IgGFab2, and IgA with or without LY294002, a mTORC inhibitor. We studied eight SLE patients with secondary APS (mean age 46.8+/-.3, 89 % female and 67% white), four disease controls that were either SLE without APS or asymptomatic anti-Ro positive, and three normal controls. Purified IgG and IgA fractions were used as follows: SLE patients four IgG and IgA, three IgG and I IgA, four disease controls two IgG and IgA and two IgG, and controls two IgG and IgA. HMEC1 were serum starved (2 hr), and preincubated with beta2 glycoprotein I (5 ug/mL, 1 hour) followed by a five min exposure to test conditions. Fixed cells were acetone permeabilized and stained using an anti-phospho - S6RP (anti-mouse IgG TRITC) and counterstained with Hoechst 33342. Immunofluorescence was reported using both intensity (1-3+) and a staining scale reflecting % positive cells (3 fields) with 1, <10%; 2, 10-30%; 3, 40-50%; and 4>50%. Results: The phenotype of the endothelial cells which were coincubated with the IgG fractions of 3 (of 8) patients were diffusely positive for phospho- S6RP which was substantially attenuated by co-incubation with LY294002 (1+, 1 % positive cells). In addition, treatment of endothelial cells with IgA fractions from the same three individuals resulted in an increase of phosphorylation of S6RP in microvascular endothelial cells, suggesting the importance of both IgG and IgA APS isotypes. For the total group of eight SLE patients with APS antibodies however there was no association with APS titers and the ability to elicit the biomarker in HMEC1. The expression of phospho- S6RP by HMEC1 were within the ranges that is reported for no treatment (1+, <3 to report % positive cells) for IgG fractions isolated from both disease and healthy controls, Fab2 from a disease control and a control IgA. Conclusions: We report the novel finding that both IgG and IgA fractions from SLE patients with secondary APS activate endothelial cells via the mTORC pathway as demonstrated by S6RP phosphorylation. Future studies will explore these findings using Western blot analysis and explore endothelial cell gene expression for ICAM-1, E-selectin, NOS-2 and tissue factor in an effort to determine potential role of this pathway in the full spectrum of vascular pathology that accompanies APS in SLE