[Show abstract][Hide abstract] ABSTRACT: Objectives. B cells are central to the pathology of ANCA-associated vasculitis (AAV), a disease characterized by autoantibodies and effectively treated by rituximab. In addition to promoting inflammation, a subset of B cells act to suppress harmful autoimmune responses (Breg). The balance of effector and regulatory B cell subsets in AAV is not known. This study was conducted to assess the relative frequency of these subsets during different states of disease activity.Methods. B memory (Bmem), naive (Bnaive) and regulatory (Breg) subsets were defined by their relative expression of CD24 and CD38. Function was assessed by cytokine production and suppressive action on CD4(+) Th1 activation evaluated in a co-culture system.Results. Compared with healthy controls, the frequency of Breg (CD24(hi)CD38(hi)) was significantly reduced during disease remission in both proteinase 3 (PR3)- and MPO-ANCA patients and during acute disease in PR3-ANCA patients, while the frequency of memory cells (CD24(hi)CD38(lo)) was reduced during active disease and restored during remission. Breg cell frequency showed a positive correlation, while Bmem had an inverse correlation with IL-10 production in vitro. B and T cell co-cultures revealed that memory and naive B cell subsets augmented Th1 activation in vitro, which was prevented by Breg, and this pattern did not differ between remission AAV patients and controls.Conclusion. In remission there is a numerical, but not functional, deficiency in Breg and preservation of Bmem associated with reduced IL-10 production and increased Th1 activation in vitro. This imbalance may contribute to the high rate of relapse observed in AAV, especially in PR3-ANCA patients.
[Show abstract][Hide abstract] ABSTRACT: Objectives. Rituximab is effective in inducing remission in ANCA-associated vasculitis (AAV), with randomized evidence to support its use as four infusions of 375 mg/m(2) (the conventional lymphoma dosing schedule). As B cell depletion (BCD) appears to occur very rapidly after the first dose, we questioned the need for repeat dosing and adopted a standard single-dose protocol of 375 mg/m(2) to treat active AAV.Methods. All consecutive cases with newly diagnosed or relapsing AAV for whom conventional immunosuppression was contraindicated or ineffective were enrolled. All were rituximab naive. Circulating CD19(+) B cells and clinical and serological markers of disease activity were recorded at regular intervals. Complete remission (CR) was defined as the absence of clinical features of AAV with a prednisolone dose <10 mg/day.Results. Nineteen patients were included, 17 (89%) with generalized disease and 2 (11%) with severe disease (creatinine level >500 μM). Eight (42%) were on additional immunosuppression at the time of rituximab treatment. Satisfactory BCD (<0.005 cells/μl) was achieved in 89% of patients after a median of 13 days. Three-month BCD probability was 89%. Median time to CR following a single dose of rituximab was 38 days and the 3-month probability of CR was 80%. Median time to B cell repopulation was 9.2 months and to disease relapse/redose was 27 months. Use of this single-dose protocol saved an estimated £4533/patient (US$7103; €5276) compared with a 4 × 375 mg/m(2) dosing schedule.Conclusion. Our single-centre experience suggests that a single dose of rituximab of 375 mg/m(2) is a reasonable and more cost-effective therapy for inducing remission in patients with AAV.
[Show abstract][Hide abstract] ABSTRACT: Soluble Flt1 (sFlt1) is a potent inhibitor of vascular endothelial growth factor, secreted mainly by the placenta, endothelial cells and monocytes. Increased sFlt1 serum levels correlate with endothelial dysfunction and cardiovascular complications in dialysis patients. However, the impact of dialysis by itself on sFlt1 serum levels remains unknown.
We assessed sFlt1 kinetics during dialysis and the impact of different dialysis techniques [high-flux haemodialysis (HD), haemodiafiltration (HDF)] and heparinization procedures on sFlt1 serum levels in 48 patients on regular dialysis.
sFlt1 serum levels increased as early as 1 min after the start of dialysis and peaked at 15 min before returning to baseline at 4 h [mean peak level 2551 pg/mL, versus 102 before dialysis (P < 0.0001)]. sFlt1 kinetics were similar with two different dialysis membranes. In contrast, when unfractionated heparin (UH) and low-molecular-weight heparin (LMWH) were omitted during dialysis (HD or pre-dilution HDF), no significant increase in sFlt1 levels occurred. Conversely, delayed administration of LMWH (after 30 min of a heparin-free HD) induced a sharp increase in sFlt1. Similarly, when UH and LMWH were omitted and citrate-based dialysate or a heparin-coated membrane was used, sFlt1 levels remained unchanged. When heparinization procedures were the same, no difference in sFlt1 levels was noted between HD and HDF. In vitro, UH and LMWH failed to induce sFlt1 release by monocytes from controls or HD patients. These findings suggest that priming of monocytes on the extracorporeal circuit is required for heparin-induced sFlt1 release or that endothelial cells contribute to this increase.
Our results indicate that heparin-based HD induces a major sFlt1 release, which may exacerbate the anti-angiogenic state and thus endothelial dysfunction, commonly found in dialysis patients.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Lupus nephritis (LN) is a serious complication of systemic lupus erythematosus (SLE). All current treatment regimens include oral steroids, which are associated with severe adverse events and long-term damage. We have piloted a steroid-avoiding protocol (rituxilup) for the treatment of biopsy-proven active International Society of Nephrology/Renal Pathology Society (ISN/RPS) class III, IV, or class V LN. METHODS: We report the findings from the first 50 consecutive patients, treated with 2 doses of rituximab (1 g) and methyl prednisolone (500 mg) on days 1 and 15, and maintenance treatment of mycophenolate mofetil. Patients on maintenance steroids or with life-threatening SLE or requiring dialysis were excluded. Renal remission was defined as serum creatinine no greater than 15% above baseline; complete biochemical remission (CR) was defined as urine protein : creatinine ratio (PCR)<50 mg/mmol or partial remission (PR) if PCR>50 mg/mmol but non-nephrotic and >50% reduction. RESULTS: A total of 45 (90%) patients achieved CR or PR by a median time of 37 weeks (range 4-200). Overall, 72% (n=36) achieved CR (median time 36 weeks (11-58)) and a further 18% (n=9) achieved persistent PR (median time 32 weeks (19-58)). By 52 weeks, CR and PR had been achieved in 52% (n=26) and 34% (n=17) respectively. In all, 12 relapses occurred in 11 patients, at a median time of 65.1 weeks (20-112) from remission. A total of 6/50 patients had systemic flares. Of the 45 responders, only 2 required >2 weeks of oral steroids. Adverse events were infrequent; 18% were admitted, 10% for an infective episode. CONCLUSIONS: The rituxilup cohort demonstrates that oral steroids can be safely avoided in the treatment of LN. If findings are confirmed, it could mark a step change in the approach to the treatment of LN.
Annals of the rheumatic diseases 06/2013; · 8.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14(+) monocytes or CD16(+) neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis.Kidney International advance online publication, 20 February 2013; doi:10.1038/ki.2013.2.
Kidney International 02/2013; · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND OBJECTIVES: Induction therapy with oral cyclophosphamide (CYP) has been a mainstay of treatment in patients with severe renal failure secondary to ANCA-associated vasculitis (AAV). Recent evidence proposes using pulsed intravenous CYP in less severe disease to minimize adverse events. It is unclear if this can be translated to those with dialysis-dependent renal insufficiency. DESIGN, SETTING, PARTICIPANTS, & METHODS: All AAV patients presenting between 2005 and 2010 requiring dialysis at presentation were retrospectively analyzed. Patients were treated with plasma exchange, corticosteroids, and intravenous CYP. Rate of dialysis independence at 3 and 12 months and adverse effects were assessed and compared with the outcome of the plasmapheresis, prednisolone, and oral CYP arm of the randomized MEPEX (methylprednisolone versus plasma exchange) trial. RESULTS: Forty-one patients were included. At 3 months, 3 (7.3%) patients had died on dialysis, 12 (29.3%) remained dialysis dependent, and 26 (63.4%) were dialysis independent (creatinine, 2.5 mg/dl; GFR, 26 ml/min per 1.73 m(2)). Four patients subsequently reached ESRD at a median time of 83 days. Thirty-seven (90%) patients reached 1 year follow-up, 13 (35%) remained dialysis dependent, and 24 (65%) had independent renal function. Eleven patients (27%) had episodes of leukopenia (white cell count <4×10(9)/L) during CYP therapy and 17 (41%) experienced infectious complications. This compares favorably with the dialysis-dependent cohort treated with plasmapheresis in the MEPEX study in which 51% were alive with independent renal function at 1 year. CONCLUSIONS: Intravenous CYP used with corticosteroids and plasmapheresis may be an effective alternative to oral CYP in patients with dialysis-dependent AAV.
Clinical Journal of the American Society of Nephrology 11/2012; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis exhibits endothelial damage, but the capacity for vessel repair in this disorder is not well understood. Here, we observed a marked increase in serum levels of soluble Flt1 (sFlt1), a potent inhibitor of vascular endothelial growth factor, in patients with active ANCA-associated vasculitis compared with patients during remission and other controls. Serum levels of sFlt1 correlated with C5a, an anaphylatoxin released after complement activation. Serum from patients with acute ANCA-associated vasculitis disrupted blood flow in the chicken chorioallantoic membrane assay, suggesting an antiangiogenic effect. Preincubation with excess human vascular endothelial growth factor prevented this effect. Anti-proteinase-3 (PR3) mAb and serum containing PR3-ANCA from patients with active vasculitis both induced a significant and sustained release of sFlt1 from monocytes, whereas anti-myeloperoxidase (MPO) mAb or polyclonal antibodies did not. However, the serum containing polyclonal PR3-ANCA did not induce release of sFlt1 from cultured human umbilical vein endothelial cells. In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair.
Journal of the American Society of Nephrology 01/2012; 23(1):155-64. · 8.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS).
Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry.
Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1-3 months of treatment with rituximab.
ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.
Annals of the rheumatic diseases 08/2011; 70(12):2229-33. · 8.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lupus nephritis is a life-threatening complication of SLE. Treatment regimes include steroids and cyclophosphamide, both associated with significant morbidity. Newer regimes include mycophenolate mofetil (MMF). We report our outcomes in a prospectively monitored cohort of patients receiving our new standard treatment protocol, comprising rituximab induction therapy and MMF maintenance in patients already taking maintenance immunosuppression for SLE who developed lupus nephritis. We then attempted steroid reduction/withdrawal.
Patients with class III/IV/V lupus nephritis were included. All patients were on steroids prior to the development of lupus nephritis. Eighteen patients have reached at least 1 year follow-up. These patients received rituximab induction therapy and MMF maintenance therapy. Steroid reduction/withdrawal was guided by clinical response.
Fourteen of 18 (78%) patients achieved complete or partial remission with a sustained response of 12/18 (67%) at 1 year, with 2 patients having a relapse of proteinuria. Four patients did not respond. There was a significant decrease in proteinuria from a mean protein:creatinine ratio (PCR) of 325 mg/mmol at presentation to 132 mg/mmol at 1 year (P = 0.004). Serum albumin significantly increased from a mean of 29 g/L at presentation to 34 g/L at 1 year (P = 0.001). The complication rate was low with no severe infections. Following treatment with rituximab, 6 patients stopped prednisolone, 6 patients reduced their maintenance dose and 6 patients remained on the same dose (maximum 10 mg).
This data demonstrates the efficacy of a rituximab and MMF based regime in the treatment of lupus nephritis, allowing a reduction or total withdrawal of corticosteroids.
[Show abstract][Hide abstract] ABSTRACT: Large-bore dual lumen in-dwelling venous catheters are used in hemodialysis. These catheters are usually locked with heparin after the treatment. This study addressed the underappreciated postdialysis coagulopathy that can result. Thirty-six patients were included: 7 dialyzed through arterio-venous fistulae, 29 through in-dwelling venous catheters. The latter group was further subdivided according to whether they received heparin or heparin-free dialysis. To assess the heparin lock, a full-dose heparin lock as well as a much weaker heparin lock and a citrate lock were used. To assess the coagulopathy, blood was taken 1 hr after dialysis. The activated partial thromboplastin time (APTT) and anti-Xa level was measured. Additionally, 6 venous catheters were removed and the amount of fluid expelled upon locking with saline was measured. Clotting from the patient group with arterio-venous fistulae was normal following dialysis. The patients with in-dwelling venous catheters and heparin locks had significantly deranged clotting; 6 out of 10 patients had abnormal APTT results. All patients with catheters, heparin-free dialysis, and heparin locks had deranged clotting (7 out of 7). The rate decreased significantly when heparinized saline was used as a lock. A subset of patients had a citrate lock rather than a heparin lock; the clotting results normalized in all but one patient. An in vitro study demonstrated immediate leakage of fluid from the end of the ports upon locking. Significant postdialysis anticoagulation can occur after dialysis, which can be attributed to the heparin line locks. This risk is considerably reduced when a citrate lock is used instead.
Hemodialysis International 11/2007; 11(4):430-4. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Calprotectin, an endogenous toll-like receptor 4 (TLR 4) agonist that is expressed in neutrophils, monocytes, and infiltrating macrophages, promotes endothelial activation and transcription of pro-inflammatory cytokines. We investigated calprotectin in renal biopsy samples and serum of patients with anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) and in mice deficient in calprotectin (cal−/−), and we assessed the interaction of calprotectin with macrophages and endothelial cells in vitro.Methods
We examined renal biopsy samples with immunohistochemistry. Serum calprotectin levels were measured with ELISA, and cell surface expression with flow cytometry. Accelerated nephrotoxic nephritis experiments were performed on both wild-type (WT) and cal−/− mice. Macrophages were isolated from WT, TLR4−/−, and cal−/− mice, and kidney endothelial cells from WT mice, and stimulated with calprotectin and supernatants harvested. Phagocytosis with opsonised beads was compared between WT and cal−/− macrophages.FindingsPatients with active AAV glomerular lesions demonstrated the most calprotectin positivity in renal biopsy samples, sclerotic lesions the least (p<0·05), linking calprotectin with disease activity. Serum levels in patients were significantly higher than in controls. In limited systemic disease, calprotectin levels assessed at 1 and 6 months after treatment predicted relapse (sensitivity 78·6%, specificity 92·3%). Patients had persistently higher monocyte and neutrophil cell surface calprotectin expression than did healthy controls suggesting a persistently activated state. Cal−/− mice were protected from renal disease with less macrophage and T-cell infiltration, less thrombosis, and preserved renal function. Calprotectin stimulation of WT macrophages and endothelial cells increased production of tumour necrosis factor α, interleukin 6, and interleukin 8 (p<0·05), an effect abrogated in TLR4−/− and cal−/− macrophages. Additionally, cal−/− macrophages had decreased phagocytosis ability compared with WT (p<0·005). Together these data demonstrate a positive amplification of inflammation mediated by calprotectin.InterpretationSerum calprotectin is a potential biomarker in AAV, and may predict relapse. Calprotectin contributes to pathogenesis by promoting leukocyte and endothelial cell activation in a positive feedback loop.FundingUK Medical Research Council.