A. Régent

Rituximab for induction and maintenance therapy of granulomatosis with polyangiitis: a single-centre cohort study on 114 patients

OBJECTIVES To assess efficacy and safety of rituximab (RTX) induction and maintenance therapy for granulomatosis with polyangiitis (GPA) in a single-centre cohort study. METHODS All patients with active GPA, not enrolled in trials, who received ⩾1 RTX infusion(s) for induction were included. At remission, protocolized maintenance RTX infusions were given every 6 months for 18 months. Kaplan-Meier curves were used to estimate survival rates. Univariable analyses identified factors associated with remission failure and relapse, and Cox models retained independent predictors of relapse. RESULTS One hundred and fourteen adults with relapsing (65%), refractory/grumbling (22%) or new-onset (13%) GPA received RTX for induction; 100 were given ⩾1 RTX maintenance infusion(s) and 90 received 500 mg every 6 months. Median daily prednisone induction dose was 30 mg; 76% of patients were still receiving a median daily prednisone dose of 5 mg at 2 years. Median follow-up was 3.6 years. Respective 2-year relapse-free survival and RTX retention rates were 85 and 78%. Serious infection and serious adverse event rates were 4.9 and 8.1 per 100 patient-years, respectively. Refractory/grumbling vs new-onset and/or relapsing GPA (P < 0.01 for each individually; P < 0.001 vs the latter two taken together), pachymeningitis (P < 0.05), pure granulomatous disease (P < 0.05) or estimated glomerular filtration rate ⩾60 ml/min (P < 0.01) were associated with remission failure. Multivariate analyses retained refractory/grumbling GPA (P = 0.05), subglottic stenosis (P < 0.005), ENT involvement (P = 0.01) and skin involvement (P < 0.0005) as independent predictors of relapse. CONCLUSION RTX induction and low-dose preemptive maintenance can effectively and safely induce sustained remission in GPA in a real-life setting.

Identification of target antigens of anti-endothelial cell antibodies in patients with ANCA-associated systemic vasculitis: A proteomic approach

Introduction.– Calprotectin (MRP8/14) is an endogenous TLR4 agonist, expressed in neutrophils, monocytes and infiltrating macrophages, promoting endothelial activation and transcription of proinflammatory cytokines. We have shown patients with active ANCA-associated vasculitis (AAV) have elevated cell surface and serum levels, and relapsers from the NORAM trial have higher early serum levels than non-relapsers. Calprotectin (+) macrophages are found in crescentic renal lesions but not sclerotic lesions, and calprotectin deficient mice (cal-/-) are protected in a nephrotoxic nephritis (NTN) animal model. We investigate the proinflammatory mechanisms of calprotectin on bone marrow derived macrophages (BMDMs), endothelial cells (EC) and mesangial cells (MC). Methods.– EC isolated from wild-type (WT) mice; BMDMs from WT, TLR4-/and cal-/mice; MC from WT and TLR4-/micewere stimulated with calprotectin. WT EC were co-cultured with WT BMDMs or cal-/BMDMs. Cytokines measured in supernatants by ELISA. The phagocytosis ability of WT and Cal-/BMDMs were compared using opsonised beads. Serum calprotectin levels were measured in WT mice during NTN. Results.– The calprotectin induced increase in IL-8, TNF-a, MCP-1 in BMDMs was abrogated in TLR4-/BMDM (P < 0.001), but no differences seen in MC. Cal-/vs WT BMDM stimulated with exogenous calprotectin demonstrate little pro-inflammatory activity and less TNFa, IL-6, IL-8 (P < 0.005). The increase in IL-6, IL-8 and MCP-1 following co-culture of EC and WT BMDM was absent with cal-/BMDM. Cal-/BMDMs demonstrate decreased phagocytosis (P < 0.005). WT mice have increased serum calprotectin (correlates with thrombosis). Conclusion.– Calprotectin has inflammatory effects mediated by TLR4 on BMDMs and a TLR4 independent effect on MC possibly through mesangial RAGE receptors, known to bind calprotectin. Cal-/BMDMs lack a pro-inflammatory effect, suggesting a role for calprotectin in amplifying endothelial and glomerular damage in AAV, and may be a potential therapeutic target.

FRI0369 Neurotrophins are involved in vascular remodelling of giant cell arteritis.

Background Giant cell arteritis (GCA) is characterized by intimal hyperplasia leading to ischemic manifestations involving extra-cranial branches of carotid arteries and aorta. The pathogenesisof GCA is poorly understood. Neurotrophins (NT) and their receptors (NTR), are protein factors for differentiation and survival of neurons. We distinguish the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5). NT produce their effects by interacting with receptors: the selective tropomyosin receptor kinase (Trk) (TrkA, TrkB and TrkC) and a non-selective receptor p75NTR. NT and NTR are also involved in the proliferation and migration of endothelial cells and in the migration of vascular smooth muscle cells (VSMC). Objectives We hypothetized that neurotrophins are involved in vascular remodelling of GCA. Methods We included consecutive patients who underwent temporal artery biopsy (TAB) for a suspicion of GCA. VSMC were cultured from TAB. We selected four patients with biopsy proven GCA and four patients with another diagnosis that GCA (controls). Protein expression of NT and NTR were determined by immunohistochemistry on paraffin-embedded temporal arteries. Immunostaining intensities were quantified. NT and NTR mRNA extracted from VSMC were measured by quantative real-time reverse transcription-PCR. Enzyme-linked immunosorbent assay Kit were used to measure concentrations of NGF and BDNF in serum. Cell proliferation assay was assessed by BrdU incorporation. All experiments were performed with primers and antibodies against NGF, BDNF, NT3, TrkA, TrkB, TrkC, and p75NTR. Exogenous BDNF was used for proliferation assay. Results Temporal arteries of GCA patients (n=22) and controls (n=20) showed immunoreactivity for NGF, BDNF, and TrkB, whereas only GCA patients showed immunoreactivity for NT3 and P75NTR. Immunostaining was more intense in GCA patients for NGF in the media (p=0.005) and adventitia (p=0.01) and for BDNF in the intima (p=0.04) and media (p=0.008). No significant difference was found for serum NGF and BDNF concentrations between GCA patients (n=28) and controls (n=48). An increase in VSMC expression of BDNF and TrkB transcripts was observed in GCA patients compared with controls with a significant difference for TrkB (Fold difference > 2). Exogenous BDNF appears to decrease proliferation of VSMC in GCA patients as compared to controls. Conclusions Our results suggest that NT and NTR are involved in the vascular remodelling of GCA particularly BDNF and his receptors. In VSMC, BDNF can bind either TrkB or P75NTR. TrkB signalling rather promotes cell survival whereas p75NTR leads rather to apoptosis. In GCA patients BDNF seems to bind rather to p75NTR. This pro-apoptotic pathway could be activated to supply ischemic effects in patients with GCA. Further experiments performed on larger number of VSMC samples will be needed to confirm these results. Disclosure of Interest: None Declared