Our previous data [1],[2],[3] showed that renal artery stenosis (RAS) is more prevalent in antiphospholipid syndrome (APS) (26%) compared to the general hypertensive population (8%),and anticoagulation with INR≥3 was associated with initial reduction of chronic kidney disease (CKD) and hypertension.
Objectives
To characterise the long-term outcomes and associations with anticoagulant treatment for patients with renal artery stenosis and antiphospholipid syndrome.
Methods
We identified 37 patients with RAS and APS fulfilling Sapporo criteria [4]: anticardiolipin IgG/IgM titer>40 units or >99th percentile (or +lupus anticoagulant) on ≥2 occasions ≥6 weeks apart AND vascular thrombosis (or pregnancy morbidity). RAS was diagnosed by magnetic resonance angiography (MRA).
Results
15 patients had APS alone and 22 APS associated with autoimmune conditions (13 lupus, 5 ANCA vasculitis, 4 mixed). Median age at RAS diagnosis was 48 years, 31/37 (83.8%) were female and median follow-up was 10.4 years. 25/37 (67.6%) had previous thrombosis. 7/37 (18.9%) had bilateral RAS, 3 artery occlusion. 6/37 (16.2%) had concurrent coeliac stenosis. Recanalization of RAS occurred after hydroxychloroquine in 3/37 and 9/37 (24.3%) underwent angioplasty +/− stenting. MRA was repeated in 11/37 (29.7%) after 2 years. 23/37 (62.2%) were anticoagulated, with 9/37 (24.3%) on antiplatelet therapy. 13/37 (35.1%) received hydroxychloroquine, 10/22 (45.5%) immunosuppressives and 18/37 (48.6%) antihypertensives. 9/37 (24.3%) died after a median of 10 years since RAS diagnosis. 21/37 (56.8%) developed CKD: 6 endstage renal failure (ESRD) and 15 with median eGFR 39 mls/min.
Conclusions
The majority of patients with RAS and APS were female, developed CKD and did not benefit from renal angioplasty. Anticoagulation was not associated with longterm reduction of ESRD or death, suggesting a non-thrombotic pathogenic process underlying RAS, such as intimal hyperplasia. Treatment of associated vascular risk factors and autoimmune disease is paramount. Anticardiolipin antibodies and renal MRA are useful screening tests for lupus patients with difficult blood pressure control.
References
S R Sangle, D P D9Cruz et al. Ann Rheum Dis 2003;62:999–1002. S R Sangle, D P D9Cruz et al. Rheumatology 2005;44:372–37 Jordan NP, Chaib A, Sangle S, D9Cruz DP. Arthritis Care and Research) 2013 Sipek-Dolnicar A, Hojnik M, Bozic B, Vizjak A. Clin Exp Rheumatol. 2002; 20:335–42.
Both TLR7 and NF-κB hyperactivity are known to contribute to pathogenesis in Systemic Lupus Erythematosus (SLE), driving a pro-interferon response, autoreactive B cell expansion and autoantibody production. UBE2L3 is an SLE susceptibility gene which drives plasmablast/plasma cell expansion in SLE, but its role in TLR7 signalling has not been elucidated. We aimed to investigate the role of UBE2L3 in TLR7-mediated NF-κB activation, and the effect of UBE2L3 inhibition by Dimethyl Fumarate (DMF) on SLE B cell differentiation in vitro. Our data demonstrate that UBE2L3 is critical for activation of NF-κB downstream of TLR7 stimulation, via interaction with LUBAC. DMF, which directly inhibits UBE2L3, significantly inhibited TLR7-induced NF-κB activation, differentiation of memory B cells and plasmablasts, and autoantibody secretion in SLE. DMF also downregulated interferon signature genes and plasma cell transcriptional programmes. These results demonstrate that UBE2L3 inhibition could potentially be used as a therapy in SLE through repurposing of DMF, thus preventing TLR7-driven autoreactive B cell maturation.
The spontaneously hypertensive rat (SHR) has increased peripheral cardiac sympathetic activity compared to Wistar Kyoto (WKY) controls. However, it is not clear whether this precedes the development of hypertension. We therefore compared peripheral cardiac sympathetic responsiveness in the SHR to the WKY at 4 weeks of age. Ventricular weight/body weight ratios and mean arterial pressure measured via the left carotid artery demonstrated that SHRs (n=8) were without left ventricular hypertrophy and were normotensive at this age compared to WKYs (n=7). In isolated cultured neurons from stellate ganglia, the magnitude of the calcium transient in response to high extracellular potassium depolarization was significantly (p<0.05) larger in SHRs (n=10) compared to WKYs (n=8). There was also a significantly greater release of [3H]‐norepinephrine (NE) to field stimulation (5Hz) of right atria (n=7 for both groups) and higher plasma levels of the sympathetic co‐transmitter neuropeptide Y (NPY) (n=12 for both groups). In an isolated organ bath atrial preparation with intact right stellate ganglion there was a greater heart rate response to right stellate stimulation at 5 and 7Hz in SHRs (n=9) compared to WKYs (n=7). Increased cardiac sympathetic neurotransmission may therefore represent an early marker in the development of hypertension driven by larger calcium transients. Supported by the British Heart Foundation
Polymorphisms of Ikaros (IKZF1) and Aiolos (IKZF3), transcription factors essential for the maturation, differentiation and survival of B cells, have been linked to systemic autoimmunity. The Cereblon modulator iberdomide, known to induce their degradation, is being explored as a therapeutic option in Systemic Lupus Erythematosus (SLE). However, the involvement of Ikaros and Aiolos in other autoimmune diseases is poorly known, and their effects on B cell activation and differentiation in the context of autoimmunity have been only partially described.
Objectives
To evaluate the expression of Ikaros and Aiolos in the salivary glands (SGs) of patients with Sjögren's syndrome (SS) and the effects of Iberdomide on the TLR7-mediated activation of B cells from patients with Sjögren's.
Methods
Immunohistochemical staining for Ikaros and Aiolos was performed on SG biopsies of patients with SS (n=23), classified into Ectopic Lymphoid Structures (ELS) positive (n=12) and negative (n=11) according to the presence of infiltrating B cells (CD20) and T cells (CD3), assessed by immunohistochemistry. SGs of patients with non specific chronic sialoadenitis (NSCS) (n=12) were used as controls. Digital image analysis and automated cell count (QuPath software) were used to calculate the density of Ikaros and Aiolos positive cells, which were correlated with histological and serological markers (SG semiquantitative scores for CD3+ T cells, CD20+ B cells, CD138+ plasmacells;Immunoglobulins; C3,C4) and disease activity (ESSDAI). CD19+ B cells were isolated from the peripheral blood of patients with Sjögren's (n=7) and triggered with the TLR7 ligand Resiquimod, with or without iberdomide (100 nM). After 5 days, cells were analysed by FACS and IgG and IgM in the supernatants were measured by ELISA.
Results
The density of Ikaros and Aiolos positive cells was significantly higher in the SGs of ELS positive SS patients compared to NSCS. As shown in Table 1, the density of Ikaros and Aiolos positive cells in the SG of SS patients significantly correlated with B and T cell semiquantitative scores and the number of lymphocytic foci. Ikaros positive cell density also correlated with IgG levels and disease activity measured by ESSDAI. Accordingly, SS patients with high expression of Ikaros (>10 Ikaros+ cells/mm2, n=11/23) had significantly higher ESSDAI (p=0.023) and higher prevalence of ANA positivity (p=0.019). In vitro, iberdomide significantly inhibited the TLR7-mediated production of IgG from Sjögren's B cells (p=0.03) and significantly reduced the number of CD27+CD38+ plasmablasts (p=0.03).
Conclusion
We here show that the transcription factors Ikaros and Aiolos are expressed in the salivary glands of patients with Sjögren's syndrome with ELS. The association of Ikaros with disease severity and autoantibody positivity and the inhibition of B cell activation by iberdomide point to the relevance of these transcription factors in Sjögren's, thus suggesting their potential use as therapeutic targets in patients with Sjögren's and other B cell-mediated autoimmune diseases.
Acknowledgement
This study was supported by research funding from Celgene Corporation.
Disclosure of Interests
Felice Rivellese: None declared, Sotiria Manou-Stathopoulou: None declared, Daniele Mauro: None declared, Elena Pontarini: None declared, Davide Lucchesi: None declared, MATTIA CONGIA: None declared, Peter Schafer Shareholder of: Celgene corporation, Employee of: Celgene Corporation, Nurhan Sutcliffe: None declared, Costantino Pitzalis Grant/research support from: Celgene, Myles Lewis Grant/research support from: Celgene, Michele Bombardieri Grant/research support from: Celgene, Consultant for: Medimmune
Objective To evaluate the effects of targeting Ikaros and Aiolos by cereblon modulator iberdomide on the activation and differentiation of B-cells from patients with systemic lupus erythematosus (SLE). Methods CD19 + B-cells isolated from the peripheral blood of patients with SLE (n=41) were cultured with TLR7 ligand resiquimod ±IFNα together with iberdomide or control from day 0 (n=16). Additionally, in vitro B-cell differentiation was induced by stimulation with IL-2/IL-10/IL-15/CD40L/resiquimod with iberdomide or control, given at day 0 or at day 4. At day 5, immunoglobulins were measured by ELISA and cells analysed by flow cytometry. RNA-Seq was performed on fluorescence-activated cell-sorted CD27 - IgD + naïve-B-cells and CD20 low CD27 + CD38 + plasmablasts to investigate the transcriptional consequences of iberdomide. Results Iberdomide significantly inhibited the TLR7 and IFNα-mediated production of immunoglobulins from SLE B-cells and the production of antinuclear antibodies as well as significantly reducing the number of CD27 + CD38 + plasmablasts (0.3±0.18, vehicle 1.01±0.56, p=0.011) and CD138 + plasma cells (0.12±0.06, vehicle 0.28±0.02, p=0.03). Additionally, treatment with iberdomide from day 0 significantly inhibited the differentiation of SLE B-cells into plasmablasts (6.4±13.5 vs vehicle 34.9±20.1, p=0.013) and antibody production. When given at later stages of differentiation, iberdomide did not affect the numbers of plasmablasts or the production of antibodies; however, it induced a significant modulation of gene expression involving IKZF1 and IKZF3 transcriptional programmes in both naïve B-cells and plasmablasts (400 and 461 differentially modulated genes, respectively, false discovery rate<0.05). Conclusion These results demonstrate the relevance of Ikaros and Aiolos as therapeutic targets in SLE due to their ability to modulate B cell activation and differentiation downstream of TLR7.
Abstract Background/Aims Neutropaenia occurs in 20-40% of patients with systemic lupus erythematosus (SLE) and is attributed to a combination of autoimmune and complement-mediated destruction of white cells, increased neutrophil apoptosis and medication side-effects. Commonly when patients become neutropaenic (<2x10^9/L), immunosuppressant doses are reduced or discontinued. However, studies have not consistently shown an increased risk of infection in SLE patients that suffer neutropaenia while taking immunosuppressants. This retrospective case note review aimed to examine the association between degree of neutropaenia and rates of infection in SLE patients. Methods Data was collected for 100 patients attending an SLE clinic in a tertiary London hospital between May 2019 and August 2020. Patients completed a questionnaire reporting the number and nature of infections they had suffered in the previous year, and whether antibiotics and/or hospitalisation was required. Clinical data was collected by retrospective review of electronic patient records including immunosuppressant therapy and neutrophil counts. Sustained neutropaenia was defined as a neutrophil count <2x10^9/L for >3 months, and severe neutropaenia defined as a neutrophil count of < 1x10^9/L). Results 95% of patients were female with a median age of 42 years (range 18-87). 55 (55%) of our patients were prescribed an immunosuppressant - 32 mycophenolate mofetil, 14 azathioprine, 4 rituximab, 4 methotrexate, 4 ciclosporin, 3 cyclophosphamide and 1 leflunomide. This cohort was more likely to develop neutropenia (n = 19, 35% vs n = 10, 22%, OR 1.8, p = 0.13), report one or more infections (n = 29, 53% vs n = 19, 42%), require antibiotics (n = 19, 35% vs n = 10, 22%) or inpatient treatment (13% vs 7%). However, immunosuppressed patients with neutropaenia >1x10^9/L (8/19) did not experience more infections than those without neutropaenia (21/36) (42% vs 58%, OR 0.5, p = 0.2). Chest, urine and upper respiratory tract were the most commonly reported infections. 29 (29%) patients had sustained neutropaenia, with only 2 (2%) patients experiencing severe neutropaenia - both latter patients experienced recurrent viral infections and were only taking hydroxychloroquine. Conclusion Immunosuppressive therapy was correlated with an increased prevalence in the absolute number of reported infections, antibiotic prescriptions and hospital admissions. Although there were more cases of infection and neutropenia in patients receiving immunosuppressants, rates of infection were not higher amongst neutropenic patients whose counts were above 1x10^9/L. Our data suggest that the increased risk of infection in our patients receiving immunosuppressants may be independent of the risk of neutropenia. Rates of infection, antibiotic use and hospital admission were not increased in patients with sustained neutropenia. There were few cases of severe neutropenia in our cohort, and in the absence of immunosuppressant use, this may have been driven by the SLE itself. Disclosure S. Manou-Stathopoulou: None. W. Hann: None. A. Pakozdi: None. R. Rajakariar: None. M. Lewis: None. A. Cove-Smith: None. D. Pyne: None.
Polymorphisms in IKZF1 (Ikaros) and IKZF3 (Aiolos), which are transcriptions factors essential for B cell activation, maturation and differentiation, have been associated with Systemic Lupus Erythematosus (SLE). However the functional role of IKZF1 and IKZF3 in the context of failed B cell checkpoints and aberrant plasmablast development in SLE has not been previously characterised. Iberdomide (a cereblon modulator) known to induce the degradation of transcription factors IKZF1 and IKZF3 is being explored as a therapeutic target for SLE. The aim of this study was to utilise iberdomide to evaluate the effect of inhibition of IKZF1 and IKZF3 on transcriptional programmes underlying B cell differentiation, gene expression and immunoglobulin production in SLE B cells.
Methods
CD19 +B cells were isolated from peripheral blood of 25 SLE patients and stimulated with IL-2, IL-10, IL-15, CD40L and TLR7 ligand Resiquimod for 5 days to induce plasmablast differentiation. In separate studies, B cells were treated from the outset with iberdomide (10 nM) or vehicle and subsequently differentiated, or differentiated plasmablasts (day 4) were treated with iberdomide or vehicle for 18 hour. Treated plasmablasts underwent fluorescence-activated cell sorting (FACS), and IgG/IgM secretion analysed with ELISA. FACS-sorted CD27-IgD+naïve B cells and CD20lowCD27+CD38+plasmablasts were subjected to bulk ultra-low input RNA-seq along with matched baseline B cells. Unsupervised clustering, differential gene expression and pathway analysis were performed on transcriptome data.
Results
Day 0 iberdomide (n=9), but not day 4 iberdomide (n=16), significantly reduced the CD20lowCD27+CD38+plasmablast numbers following cell culture (p=0.03). Similarly, Day 0 iberdomide significantly decreased supernatant IgG/IgM concentrations (p=0.050 and 0.017, respectively), but not day 4 iberdomide. RNA-seq of sorted naïve B cells and plasmablasts cultured with day 4 iberdomide demonstrated significant differential gene expression in both populations (400 and 461 differentially modulated genes in naïve B cells and plasmablasts, FDR-adjusted p<0.05). Pathway analysis showed that IKZF1/IKZF3 inhibition resulted in downregulation of JAK-STAT signalling downstream of IL12 (FDR=7.92E-04), IL12 signalling (FDR=0.0014), and p53 signalling regulation of cell death (FDR=0.0043) and showed a trend to upregulation of RUNX1 signalling and Rho GTPase cycle.
Conclusions
Iberdomide exposure significantly blocked SLE B cell differentiation into plasmablasts, but did not alter fully differentiated plasmablast viability, confirming the role of IKZF1 and IKZF3 in the process of B cell differentiation into aberrant plasmablasts in SLE. Our study demonstrates that IKZF1 and IKZF3 inhibition results in differential expression of key B cell development transcriptional gene modules in both SLE naïve B cells and plasmablasts.
Funding Source(s):
This study was supported by research funding from Celgene Corporation.
Arteriovenous fistulas are the best form of vascular access for haemodialysis. A radiological balloon angioplasty is the standard treatment for a clinically relevant stenosis, but the recurrence rate is high. Data on factors associated with recurrence are limited.A single centre, retrospective analysis was performed for 124 consecutive patients who had successful interventions for dysfunctional arteriovenous fistulae, to examine factors associated with post-intervention patency. Follow-up was at least 1 year for all patients. Variables associated with primary and cumulative patency were pre-specified and assessed using both un-adjusted (univariate) and adjusted Cox proportional hazards models. Analysis was repeated for a subgroup of 80 patients with a single lesion only in order to examine the potential effects of stenotic lesion characteristics on patency.Factors found to have a significant association with poorer outcomes (less time to loss of patency) included thrombosis at the time of intervention and a history of previous intervention. Fistula age (log days) was significantly associated with better outcomes (greater time to loss of patency). Non-white ethnicity, lesion length, and patient age were also significantly associated with accelerated loss of patency.The factors we have identified as linked to poor outcome may help to identify patients in whom a balloon angioplasty is unlikely to provide a durable outcome. This may prompt exploring alternative treatment or dialysis options at an early stage.
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