Risk of relapse of ANCA-associated vasculitis among patients homozygous for the proteinase 3 gene Val119Ile polymorphism.

BACKGROUND: The frequency of proteinase 3 gene (PRTN3) polymorphisms in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not fully characterised. We hypothesise that the presence of a PRTN3 gene polymorphism (single nucleotide polymorphism (SNP) rs351111) is relevant for clinical outcomes. METHODS: DNA variant calling for SNP rs351111 (chr.19:844020, c.355G>A) in PRTN3 gene assessed the allelic frequency in patients with PR3-AAV included in the Rituximab in ANCA-Associated Vasculitis trial. This was followed by RNA-seq variant calling to characterise the mRNA expression. We compared clinical outcomes between patients homozygous for PRTN3-Ile119 or PRTN3-Val119. RESULTS: Whole blood samples for DNA calling were available in 188 patients. 75 patients with PR3-AAV had the allelic variant: 62 heterozygous PRTN3-Val119Ile and 13 homozygous for PRTN3-Ile119. RNA-seq was available for 89 patients and mRNA corresponding to the allelic variant was found in 32 patients with PR3-AAV: 25 heterozygous PRTN3-Val119Ile and 7 homozygous for PRTN3-Ile119. The agreement between the DNA calling results and mRNA expression of the 86 patients analysed by both methods was 100%. We compared the clinical outcomes of 64 patients with PR3-AAV: 51 homozygous for PRTN3-Val119 and 13 homozygous for PRTN3-Ile119. The frequency of severe flares at 18 months in homozygous PRTN3-Ile119 was significantly higher when compared with homozygous PRTN3-Val119 (46.2% vs 19.6%, p=0.048). Multivariate analysis identified homozygous PR3-Ile119 as main predictor of severe relapse (HR 4.67, 95% CI 1.16 to 18.86, p=0.030). CONCLUSION: In patients with PR3-AAV, homozygosity for PRTN3-Val119Ile polymorphism appears associated with higher frequency of severe relapse. Further studies are necessary to better understand the association of this observation with the risk of severe relapse.

Activation of a Latent Epitope Causing Differential Binding of Antineutrophil Cytoplasmic Antibodies to Proteinase 3.

OBJECTIVE: Proteinase 3 (PR3) is the major antigen for antineutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3. This study was undertaken to study the effect of mutations on PR3 antigenicity. METHODS: The recombinant PR3 variants, iPR3 (clinically used to detect PR3-ANCAs) and iHm5 (containing 3 point mutations in epitopes 1 and 5 generated for epitope mapping studies) immunoassays and serum samples from patients enrolled in ANCA-associated vasculitis (AAV) trials were used to screen for differential PR3-ANCA binding. A patient-derived monoclonal ANCA 518 (moANCA518) that selectively binds to iHm5 within the mutation-free epitope 3 and is distant from the point mutations of iHm5 was used as a gauge for remote epitope activation. Selective binding was determined using inhibition experiments. RESULTS: Rather than reduced binding of PR3-ANCAs to iHm5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5 compared to iPR3. This differential binding of PR3-ANCA to iHm5 is similar to the selective moANCA518 binding to iHm5. Binding of iPR3 to monoclonal antibody MCPR3-2 also induced recognition by moANCA518. CONCLUSION: The preferential binding of PR3-ANCAs from patients, such as the selective binding of moANCA518 to iHm5, is conferred by increased antigenicity of epitope 3 on iHm5. This can also be induced on iPR3 when captured by monoclonal antibody MCPR2. This previously unrecognized characteristic of PR3-ANCA interactions with its target antigen has implications for studying antibody-mediated autoimmune diseases, understanding variable performance characteristics of immunoassays, and design of potential novel treatment approaches.

The association of serum interleukin-6 levels with clinical outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To investigate serum IL-6 (sIL-6) levels during active disease, complete remission (CR), and relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and to explore the association of changes in sIL-6 with clinical outcomes. METHODS: sIL-6 levels were measured at baseline and longitudinally over 18 months, in 78 patients with AAV enrolled in a randomized controlled trial comparing treatment with either rituximab (RTX) or cyclophosphamide (CYC)/azathioprine (AZA). Outcome variables included baseline clinical features, ANCA specificity, disease activity (active disease versus CR), time to relapse events, B cell repopulation, and ANCA titer increases. RESULTS: At baseline, sIL6 levels were detectable in 81% of patients; 73% (n = 57) of subjects were proteinase 3 (PR3)-ANCA positive, sIL-6 levels were higher in subjects with PR3-ANCAs and positively correlated with their levels (rs = 0.36,p < 0.01), but not with levels of myeloperoxidase (MPO)-ANCA (rs = -0.17,p = 0.47). Higher baseline sIL-6 levels were associated with PR3-ANCA positivity, fever, pulmonary nodules/cavities, conductive deafness, and absence of urinary red blood cell casts (p < 0.05). Baseline sIL6 levels did not predict CR at month 6 (p = 0.71), and the median sIL-6 level declined from baseline with induction therapy, regardless of CR achievement. An increase in sIL-6 during CR was a predictor for subsequent severe relapse in RTX-treated patients (hazard ratio (HR):7.24,p = 0.01), but not in CYC/AZA-treated patients (HR:0.62,p = 0.50). In contrast, a sIL-6 increase did not predict B cell repopulation or ANCA titer increase in either treatment arm (p > 0.05). CONCLUSION: At baseline, sIL-6 concentrations correlate with PR3-ANCA titers and are associated with specific clinical manifestations of AAV. Baseline sIL6 concentrations do not predict CR at 6 months, but the increase in sIL-6 concentrations during CR is associated with subsequent severe relapse among RTX-treated patients. Further investigation into the mechanistic role of IL6 in AAV might lead to identifying this pathway as a potential therapeutic target in this disease.

Association of Pulmonary Hemorrhage, Positive Proteinase 3, and Urinary Red Blood Cell Casts With Venous Thromboembolism in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To assess the frequency of venous thromboembolism (VTE) events in the Rituximab in Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (RAVE) trial and identify novel potential risk factors. METHODS: VTE events in 197 patients enrolled in the RAVE trial were analyzed. Baseline demographic and clinical characteristics were recorded, and univariate and multivariate analyses were performed to identify factors associated with VTE in ANCA-associated vasculitis (AAV). RESULTS: VTE occurred in 16 patients (8.1%) with an overall average time to event of 1.5 months (range 1.0-2.75). In univariate analyses with calculation of hazard ratios (HRs) and 95% confidence intervals (95% CIs), heart involvement (HR 17.408 [95% CI 2.247-134.842]; P = 0.006), positive proteinase 3 (PR3)-ANCA (HR 7.731 [95% CI 1.021-58.545]; P = 0.048), pulmonary hemorrhage (HR 3.889 [95% CI 1.448-10.448]; P = 0.008), and the presence of red blood cell casts (HR 15.617 [95% CI 3.491-69.854]; P < 0.001) were associated with the onset of VTE. In multivariate models adjusted for age and sex, the significant associations between VTE events and heart involvement (HR 21.836 [95% CI 2.566-185.805]; P = 0.005), PR3-ANCA (HR 9.12 [95% CI 1.158-71.839]; P = 0.036), pulmonary hemorrhage (HR 3.91 [95% CI 1.453-10.522]; P = 0.007), and urinary red blood cell casts (HR 16.455 [95% CI 3.607-75.075]; P < 0.001) remained. CONCLUSION: Patients diagnosed as having AAV with pulmonary hemorrhage, positive PR3-ANCA, heart involvement, and the presence of red blood cell casts are at an increased risk to develop VTE. Further studies are needed to confirm and expand these findings and to explore the mechanisms of hypercoagulability in these patients with the aim of informing potential targets for therapeutic intervention.

Brief Report: Circulating Cytokine Profiles and Antineutrophil Cytoplasmic Antibody Specificity in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To evaluate circulating cytokine profiles in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV), classified by antineutrophil cytoplasmic antibody (ANCA) specificity (proteinase 3 ANCA [PR3-ANCA] versus myeloperoxidase ANCA [MPO-ANCA]) or by clinical diagnosis (granulomatosis with polyangiitis [GPA] versus microscopic polyangiitis [MPA]). METHODS: A panel of 29 cytokines was tested in 186 patients with active AAV at inclusion into the Rituximab in AAV trial. Cytokine concentrations were compared between groups within each classification system. Multivariable analyses adjusted for age, sex, and renal insufficiency were performed, with each biomarker as a dependent variable and ANCA specificity and clinical diagnosis as explanatory variables of interest. RESULTS: Levels of 9 circulating cytokines (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-15, IL-18, CXCL8/IL-8, CCL-17/thymus and activation-regulated chemokine [TARC], IL-18 binding protein [IL-18 BP], soluble IL-2 receptor α [sIL-2Rα], and nerve growth factor ß [NGFß]) were significantly higher in PR3-AAV than MPO-AAV, 4 cytokines (sIL6R, soluble tumor necrosis factor receptor type II [sTNFRII], neutrophil gelatinase-associated lipocalin [NGAL], and soluble intercellular adhesion molecule 1 [sICAM-1]) were higher in MPO-AAV than in PR3-AAV, 6 cytokines (IL-6, GM-CSF, IL-15, IL-18, sIL-2Rα, and NGFß) were higher in GPA than in MPA, and 3 cytokines (osteopontin, sTNFRII, and NGAL) were higher in MPA than in GPA (all P < 0.05). For nearly all cytokines, the difference between PR3-AAV and MPO-AAV was larger than that between GPA and MPA. The multivariate analysis showed that 8 cytokines (IL-15, IL-8, IL-18 BP, NGF-ß, sICAM-1, TARC, osteopontin, and kidney injury molecule 1 (P < 0.05) distinguished patients with AAV better (lower P values and larger effect sizes) when grouped by ANCA specificity than by clinical diagnosis. CONCLUSION: Distinct cytokine profiles were identified for PR3-AAV versus MPO-AAV and for GPA versus MPA. Differences in these circulating immune mediators are more strongly associated with ANCA specificity than with clinical diagnosis, suggesting that heterogeneity in the AAV subtypes extends beyond clinical phenotypes.

The Utility of Urinalysis in Determining the Risk of Renal Relapse in ANCA-Associated Vasculitis.

BACKGROUND AND OBJECTIVES: The significance of persistent hematuria or proteinuria in patients with ANCA-associated vasculitis who are otherwise in clinical remission is unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A post hoc analysis was conducted using participants enrolled in two randomized, placebo-controlled clinical trials who had active GN due to ANCA-associated vasculitis, had positive ANCA, and achieved remission by month 6. Dipstick and microscopic urinalyses were performed at each visit. Persistent hematuria or proteinuria for at least 6 months and the cumulative duration of hematuria were examined. Renal relapse was defined as new or worsening red blood cell casts and/or worsening kidney function according to the Birmingham Vasculitis Activity Score for Granulomatosis with Polyangiitis. RESULTS: There were 149 patients included in this study: 42% had persistent hematuria, and 43% had persistent proteinuria beyond 6 months. Persistent hematuria was associated with a significantly higher risk of relapse, even after adjusting for potential confounders (subdistribution hazard ratio, 3.99; 95% confidence interval, 1.20 to 13.25; P=0.02); persistent proteinuria was not associated with renal relapse (subdistribution hazard ratio, 1.44; 95% confidence interval, 0.47 to 4.42; P=0.53). Furthermore, greater cumulative duration of hematuria was significantly associated with a higher risk of renal relapse (adjusted subdistribution hazard ratio, 1.08 per each month; 95% confidence interval, 1.03 to 1.12; P<0.01). The median time to renal relapse was 22 months. CONCLUSIONS: In patients with ANCA-associated vasculitis and kidney involvement who achieve remission after induction therapy, the presence of persistent hematuria, but not proteinuria, is a significant predictor of future renal relapse.

Interstitial Immunostaining and Renal Outcomes in Antineutrophil Cytoplasmic Antibody-Associated Glomerulonephritis.

BACKGROUND: Immunopathologic features predict renal function at baseline and follow-up in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (GN). The interstitial infiltrate consists predominantly of T lymphocytes, but their pathophysiologic significance is unclear, especially in light of the success of B-cell-directed therapy. METHODS: Renal biopsies from 33 patients treated with cyclophosphamide (CYC; n = 17) or rituximab (RTX; n = 16) in the RTX in ANCA-associated vasculitis (RAVE) trial were classified according to the new ANCA GN classification. T- and B-cell infiltration in the interstitium was assessed by immunostaining for CD3 and CD20. Correlations of clinical and histologic parameters with renal function at set time points were examined. RESULTS: The mean (SD) baseline estimated glomerular filtration rate was 36 (20) mL/min/1.73 m2. ANCA GN class distribution was 46% focal, 33% mixed, 12% sclerotic and 9% crescentic. The interstitial infiltrate consisted of >50% CD3 positive cells in 69% of biopsies, but >50% CD20 positive cells only in 8% of biopsies. In a multiple linear regression model, only baseline glomerular filtration rate (GFR) correlated with GFR at 6, 12, and 18 months. Interstitial B- and T-cell infiltrates had no significant impact on long-term prognosis, independent of the treatment limb. A differential effect was noted only at 6 months, where a dense CD3 positive infiltrate predicted lower GFR in the RTX group and a CD20 positive infiltrate predicted higher GFR in the CYC group. CONCLUSIONS: In ANCA-associated GN, the interstitial infiltrate contains mainly T lymphocytes. However, it is neither reflecting baseline renal function nor predictive of response to treatment, regardless of the immunosuppression regimen employed.

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides.

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the genetic make-up of S. aureus isolates from PR3-ANCA-positive GPA patients with that of isolates from patients suffering from myeloperoxidase (MPO)-ANCA-positive AAV, and isolates from healthy controls. Based on a DNA microarray-based approach, we show that not only PR3-ANCA-positive GPA patients, but also MPO-ANCA-positive AAV patients mainly carried S. aureus types that are prevalent in the general population. Nonetheless, our data suggests that MPO-ANCA-associated S. aureus isolates may be distinct from healthy control- and PR3-ANCA-associated isolates. Furthermore, several genetic loci of S. aureus are associated with either PR3-ANCA- or MPO-ANCA-positive AAV, indicating a possible role for pore-forming toxins, such as leukocidins, in PR3-ANCA-positive GPA. Contrary to previous studies, no association between AAV and superantigens was detected. Our findings also show that a lowered humoral immune response to S. aureus is common for PR3-ANCA- and MPO-ANCA-positive AAV. Altogether, our observations imply that the presence or absence of particular virulence genes of S. aureus isolates from AAV patients contributes to disease progression and/or relapse.

Position paper: Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

Are cytokines and chemokines suitable biomarkers for Takayasu arteritis?

There is a growing need for disease related biomarkers in Takayasu arteritis (TA).The assessment of pro-inflammatory cytokines and chemokines in TA may provide a better understanding of its pathophysiology, and circulating levels of these mediators may act as biomarkers of disease activity. Serum level of interleukin 6 (IL-6) is a potential biomarker for TA, which is mostly associated with TA status and disease activity. Associations between TA and serum/plasma levels of other cytokines are less clear. mRNA expression of IL-4 and tumor necrosis factor α (TNFα) are constitutively increased in peripheral blood mononuclear cells (PBMC) from TA patients and the expression of both cytokines increases even more after PBMC stimulation in vitro, while the expression of IL-10 mRNA decreases. In addition, circulating T cells from TA patients produce increased levels of both Th1- and Th17-related cytokines upon in vitro stimulation. In the aorta from TA patients, an increased expression of interferon γ (IFNγ), IL-6, IL-12 and IL-17 has been described. Regarding circulating chemokines in TA, serum/plasma levels of IL-8 (CXCL8), CCL2 and CCL5 were shown to be elevated in TA patients compared with healthy controls as well as in TA patients with active disease compared with those in remission. Serum IL-6 seems to be the best biomarker for disease state and disease activity in TA and increased Th1 and Th17 responses are predominant in the pathophysiology of TA.

M2 macrophage is the predominant phenotype in airways inflammatory lesions in patients with granulomatosis with polyangiitis.

BACKGROUND: Macrophages may present two distinct phenotypes indicated as M1 and M2 under different stimuli. M1 and M2 macrophages have divergent functions that range from enhancement of inflammation for M1 to tissue repair and remodeling for M2 macrophages. The objective of this study was to evaluate the distribution of M1 and M2 macrophage phenotypes in biopsies from the airways of patients with active granulomatosis with polyangiitis (GPA) and to analyze their associations with T and B cells in those biopsies, and with nasal carriage of Staphylococcus aureus, disease parameters and therapy. METHODS: Consecutive GPA patients (n = 35) with active airway disease, who underwent respiratory tract biopsy were included. Immunohistochemical evaluation was performed to assess the distribution of macrophages and T and B cells using the markers CD68, CD3 and CD20, respectively. CD86 was used as the M1 marker and CD163 as the M2 marker while Tbet and GATA-3 were used as Th1 and Th2 markers, respectively. At the time of the biopsy patients were assessed for nasal carriage of Staphylococcus aureus and treatment. RESULTS: Percentages of macrophages and T cells were significantly higher than those of B cells in lesional tissue from the respiratory tract in GPA. M2 macrophages and Th2 cells were more frequent than M1 macrophages (p = 0.0007) and Th1 cells (p < 0.0001), respectively. Percentages of T cells were higher in nose biopsies than in biopsies from other sites (p = 0.021); macrophages and CD163+ macrophages were more predominant in biopsy sites other than the nose (p = 0.039 and p = 0.012, respectively). Carriage of Staphylococcus aureus was associated with higher T cell scores (p = 0.014). The frequency of macrophages, especially M2 macrophages, was higher in GPA patients treated with immunosuppressive agents (p = 0.010); daily prednisolone dose was positively correlated with all macrophage markers. However, in multivariate analysis no independent associations were found between disease parameters and therapy with macrophage markers or T cells. CONCLUSION: In GPA, M2 is the predominant macrophage phenotype in the respiratory tract. Although some associations were observed between macrophages and T cells with therapy and nasal carriage of Staphylococcus aureus, they were not independently significant in multivariate analysis.

The Pharmacogenomic Association of Fcγ Receptors and Cytochrome P450 Enzymes With Response to Rituximab or Cyclophosphamide Treatment in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: The Rituximab in ANCA-Associated Vasculitis (RAVE) trial compared rituximab to cyclophosphamide as induction therapy for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. We undertook the current study to determine whether known single-nucleotide polymorphisms (SNPs) for Fcγ receptors (FcγR) or cytochrome P450 (CYP) enzymes were associated with the response to treatment with rituximab and cyclophosphamide, respectively. METHODS: Functional SNPs for FcγR (FcγRIIa 519G>A, FcγRIIb 695T>C, FcγRIIIa 559T>G) and CYP enzymes (CYP2B6 1459C>T, CYP2C19 681G>A) were analyzed by direct sequencing of polymerase chain reaction-amplified genomic DNA. Each SNP was tested as a predictor of complete remission at 6 months or remission with continued prednisone administration using logistic regression and including the covariates of baseline Birmingham Vasculitis Activity Score for Wegener's Granulomatosis, ANCA type, and new versus relapsing disease. The associations of these SNPs with the secondary outcomes of time to complete remission, time to relapse, or time to B cell reconstitution were analyzed by Cox proportional hazard tests. RESULTS: No significant associations were identified between complete remission and any FcγR genotype in the rituximab group or any CYP genotype in the cyclophosphamide group. However, when the treatment groups were combined, an association was found between the 519AA genotype of FcγRIIa and complete remission (P = 0.01). The 519AA genotype predicted complete remission (P = 0.006) and a shorter time to complete remission (P < 0.001). CONCLUSION: The finding that the homozygous FcγRIIa 519AA variant was associated with complete response and a shorter time to complete response in the RAVE trial, independent of treatment type, implies that FcγRIIa may be broadly involved in disease pathogenesis and response to therapy.

Effect of Disease Activity, Glucocorticoid Exposure, and Rituximab on Body Composition During Induction Treatment of Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: We investigated the relationships between glucocorticoid use, disease activity, and changes in body mass index (BMI) in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: We analyzed AAV patients enrolled in the Rituximab in AAV trial. Glucocorticoid use, BMI, and disease activity were measured regularly during the trial period. We performed mixed-effects regressions to examine the associations of time-dependent cumulative average glucocorticoid use and disease activity with changes in BMI over time, while adjusting for potential confounders. RESULTS: The mean ± SD baseline BMI of the 197 patients enrolled was 28.8 ± 6.3 kg/m2 . Patients with newly diagnosed AAV tended to have a lower mean ± SD BMI than those with relapsing disease (28.0 ± 5.7 kg/m2 versus 29.6 ± 6.8 kg/m2 ) and higher disease activity (mean ± SD Birmingham Vasculitis Activity Score for Wegener's Granulomatosis 8.7 ± 3.3 versus 7.4 ± 2.7). The most significant change in BMI occurred during the first 6 months of the trial (mean ± SD increase of 1.1 ± 2.2 kg/m2 ; P < 0.0001). Disease activity improvement, glucocorticoid exposure, and randomization to rituximab were each independently associated with an increase in BMI (P < 0.001 for all analyses). CONCLUSION: Our findings suggest that changes in BMI, as well as glucocorticoid exposure, are independently associated with improvements in disease activity in AAV. Rituximab may also have effects on BMI independent of its impact on disease activity.

Myeloperoxidase-Antineutrophil Cytoplasmic Antibody (ANCA)-Positive and ANCA-Negative Patients With Granulomatosis With Polyangiitis (Wegener's): Distinct Patient Subsets.

OBJECTIVE: To examine the relationship of antineutrophil cytoplasmic antibody (ANCA) type and ANCA-associated vasculitis (AAV) diagnosis with demographic features, disease manifestations, and clinical outcomes. We focused on patients who account for the differences between ANCA type and disease type classifications: anti-myeloperoxidase (MPO)-ANCA-positive and ANCA-negative patients with granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: We performed a pooled analysis of the Wegener's Granulomatosis Etanercept Trial and the Rituximab in ANCA-Associated Vasculitis trial comparing patients with MPO-ANCA-positive GPA and patients with ANCA-negative GPA to patients with proteinase 3 (PR3)-ANCA-positive GPA and patients with MPO-ANCA-positive microscopic polyangiitis (MPA). RESULTS: Of the 365 patients analyzed, 273 (75%) had PR3-ANCA-positive GPA, 33 (9%) had MPO-ANCA-positive GPA, 15 (4%) had ANCA-negative GPA, and 44 (12%) had MPO-ANCA-positive MPA. MPO-ANCA-positive GPA patients were younger at diagnosis compared to MPO-ANCA-positive MPA patients (53 versus 61 years; P = 0.02). Their disease manifestations and rates of relapse were similar to those of PR3-ANCA-positive GPA patients. Relapse was more frequent in MPO-ANCA-positive GPA patients than in patients with MPO-ANCA-positive MPA at trial entry as well as at 12 and 18 months. ANCA-negative patients with GPA had lower Birmingham Vasculitis Activity Score for Wegener's Granulomatosis scores at trial entry than PR3-ANCA-positive patients with GPA (4.5 versus 7.7; P < 0.01), primarily because of a lower prevalence of renal involvement. CONCLUSION: We were unable to demonstrate important clinical differences between MPO-ANCA-positive and PR3-ANCA-positive patients with GPA. The risk of relapse was associated more closely with disease type than with ANCA type in this patient cohort. These findings deserve consideration in the assessment of relapse risk in patients with AAV.

Pro: Cyclophosphamide in lupus nephritis.

Based on efficacy and toxicity considerations, both low-dose pulse cyclophosphamide as part of the Euro-Lupus Nephritis protocol and mycophenolate mofetil (MMF) with corticosteroids may be considered for induction of remission in patients with proliferative lupus nephritis. The long-term follow-up data available for low-dose pulse cyclophosphamide, the fact that compliance is guaranteed with this regimen and economic issues all favour the Euro-Lupus regimen in this author's opinion. For maintenance treatment, either azathioprine (AZA) or MMF may be used; AZA is preferred in case pregnancy is planned, while MMF is preferred when the disease relapses during use of AZA and, possibly, after successful induction of remission with MMF.

Toll-like receptor 9 activation enhances B cell activating factor and interleukin-21 induced anti-proteinase 3 autoantibody production in vitro.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) is a relapsing small-vessel vasculitis characterized by circulating ANCA against PR3. The mechanisms that trigger PR3-ANCA production are unknown. The aim of this study was to determine whether endogenous factors [B cell activating factor (BAFF) and IL-21] and exogenous factors [oligodeoxynucleotides containing CpG motifs (CpG-ODN)] synergize in stimulating PR3-ANCA production in GPA patients. METHODS: Peripheral blood mononuclear cells from GPA patients and healthy controls (HCs) were cultured in the presence of BAFF and IL-21, with or without CpG-ODN, for 12 days. PR3-ANCA production in culture supernatants was quantified by Phadia EliA. Phenotypic characterization and the influence of CpG-ODN treatment on IL-21 receptor (IL-21R), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) expression on B cells was analysed by flow cytometry. RESULTS: Stimulation with BAFF and IL-21 significantly increased ANCA production in patient samples, which could be augmented further by addition of CpG-ODN. Stimulation with CpG-ODN increased the percentage of IL-21R(+) and TACI(+) B cells but did not affect BAFF-R expression. GPA patients had an increased percentage of circulating IL-21R(+) and a decreased percentage of TACI(+) circulating memory B cells when compared with HCs. Additionally, patients had decreased expression of BAFF-R on B cells, which was inversely correlated with BAFF concentrations in plasma. CONCLUSION: Our data demonstrate that endogenous and exogenous factors can synergize to promote PR3-ANCA production. Mechanistically, CpG-ODN up-regulated IL-21R and TACI expression on B cells, possibly sensitizing these cells for IL-21- and BAFF-mediated signals. Agents inhibiting Toll-like receptor 9, BAFF and IL-21 signalling pathways may serve as potential therapeutics for intervention in GPA patients.

High mobility group box 1 levels in large vessel vasculitis are not associated with disease activity but are influenced by age and statins.

INTRODUCTION: Takayasu arteritis (TA) and giant cell arteritis (GCA) are large vessel vasculitides (LVV) that usually present as granulomatous inflammation in arterial walls. High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released by dying or activated cells. This study aims to evaluate whether serum HMGB1 can be used as a biomarker in LVV. METHODS: Twenty-nine consecutive TA patients with 29 healthy controls (HC) were evaluated in a cross-sectional study. Eighteen consecutive GCA patients with 16 HC were evaluated at the onset of disease and some of them during follow-up. Serum HMGB1 levels were measured by enzyme-linked immunosorbent assay. RESULTS: In GCA patients at disease onset mean serum HMGB1 levels did not differ from HC (5.74 ± 4.19 ng/ml vs. 4.17 ± 3.14 ng/ml; p = 0.230). No differences in HMGB1 levels were found between GCA patients with and without polymyalgia rheumatica (p = 0.167), ischemic manifestations (p = 0.873), systemic manifestations (p = 0.474) or relapsing disease (p = 0.608). During follow-up, no significant fluctuations on serum HMGB1 levels were observed from baseline to 3 months (n = 13) (p = 0.075), 12 months (n = 6) (p = 0.093) and at the first relapse (n = 4) (p = 0.202). Serum HMGB1 levels did not differ between TA patients and HC [1.19 (0.45-2.10) ng/ml vs. 1.46 (0.89-3.34) ng/ml; p = 0.181] and no difference was found between TA patients with active disease and in remission [1.31 (0.63-2.16) ng/ml vs. 0.75 (0.39-2.05) ng/ml; p = 0.281]. HMGB1 levels were significantly lower in 16 TA patients on statins compared with 13 patients without statins [0.59 (0.29-1.46) ng/ml vs. 1.93 (0.88-3.34) ng/ml; p = 0.019]. Age was independently associated with higher HMGB1 levels regardless of LVV or control status. CONCLUSIONS: Patients with TA and GCA present similar serum HMGB1 levels compared with HC. Serum HMGB1 is not useful to discriminate between active disease and remission. In TA, use of statins was associated with lower HMGB1 levels. HMGB1 is not a biomarker for LVV.

Low anti-staphylococcal IgG responses in granulomatosis with polyangiitis patients despite long-term Staphylococcus aureus exposure.

Chronic nasal carriage of the bacterium Staphylococcus aureus in patients with the autoimmune disease granulomatosis with polyangiitis (GPA) is a risk factor for disease relapse. To date, it was neither known whether GPA patients show similar humoral immune responses to S. aureus as healthy carriers, nor whether specific S. aureus types are associated with GPA. Therefore, this study was aimed at assessing humoral immune responses of GPA patients against S. aureus antigens in relation to the genetic diversity of their nasal S. aureus isolates. A retrospective cohort study was conducted, including 85 GPA patients and 18 healthy controls (HC). Humoral immune responses against S. aureus were investigated by determining serum IgG levels against 59 S. aureus antigens. Unexpectedly, patient sera contained lower anti-staphylococcal IgG levels than sera from HC, regardless of the patients' treatment, while total IgG levels were similar or higher. Furthermore, 210 S. aureus isolates obtained from GPA patients were characterized by different typing approaches. This showed that the S. aureus population of GPA patients is highly diverse and mirrors the general S. aureus population. Our combined findings imply that GPA patients are less capable of mounting a potentially protective antibody response to S. aureus than healthy individuals.

Rituximab versus cyclophosphamide for ANCA-associated vasculitis with renal involvement.

Rituximab (RTX) is non-inferior to cyclophosphamide (CYC) followed by azathioprine (AZA) for remission-induction in severe ANCA-associated vasculitis (AAV), but renal outcomes are unknown. This is a post hoc analysis of patients enrolled in the Rituximab for ANCA-Associated Vasculitis (RAVE) Trial who had renal involvement (biopsy proven pauci-immune GN, red blood cell casts in the urine, and/or a rise in serum creatinine concentration attributed to vasculitis). Remission-induction regimens were RTX at 375 mg/m(2) × 4 or CYC at 2 mg/kg/d. CYC was replaced by AZA (2 mg/kg/d) after 3-6 months. Both groups received glucocorticoids. Complete remission (CR) was defined as Birmingham Vasculitis Activity Score/Wegener's Granulomatosis (BVAS/WG)=0 off prednisone. Fifty-two percent (102 of 197) of the patients had renal involvement at entry. Of these patients, 51 were randomized to RTX, and 51 to CYC/AZA. Mean eGFR was lower in the RTX group (41 versus 50 ml/min per 1.73 m(2); P=0.05); 61% and 75% of patients treated with RTX and 63% and 76% of patients treated with CYC/AZA achieved CR by 6 and 18 months, respectively. No differences in remission rates or increases in eGFR at 18 months were evident when analysis was stratified by ANCA type, AAV diagnosis (granulomatosis with polyangiitis versus microscopic polyangiitis), or new diagnosis (versus relapsing disease) at entry. There were no differences between treatment groups in relapses at 6, 12, or 18 months. No differences in adverse events were observed. In conclusion, patients with AAV and renal involvement respond similarly to remission induction with RTX plus glucocorticoids or CYC plus glucocorticoids.

Advances in pathogenesis and treatment of ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed to proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are sensitive and specific markers for their associated diseases, granulomatosis with polyangiitis (GPA, formerly Wegener's granulomatosis) and microscopic polyangiitis (MPA), respectively. Clinical observations suggest but do not prove that ANCA are involved in the pathogenesis of GPA and MPA. In vivo and in vitro experimental data strongly suggest if not prove that MPO-ANCA underlie the pathological lesions seen in MPO-ANCA associated MPA. This is less clear for PR3-ANCA associated GPA in which, besides small-vessel vasculitis, granulomatous inflammation is apparent. Here, cellular immunity appears to play an additional role. Insight into the pathogenetic events involved in these diseases has resulted in new ways of treatment that target the specific pathways that underlie the development of the lesions.