Pathological severity determines the renal recovery for anti-myeloperoxidase antibody-associated vasculitis requiring dialysis at disease onset: a retrospective study.

BACKGROUND: Many patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) need dialysis at disease onset due to severe kidney injury. Determining whether they can become dialysis independent is an important clinical assessment. METHODS: Forty kidney biopsy-proved myeloperoxidase (MPO)-ANCA associated AAV patients who required dialysis at disease onset were enrolled. Relationships between laboratory and pathological characteristics and prognoses were analyzed. RESULTS: Twenty-five patients obtained dialysis independence within 3 months, while the other 15 patients remained dialysis dependent. No sclerotic class was identified among the 40 patients. Only two biopsies exhibited focal class diagnoses and both these patients recovered their renal function. The renal recovery rate of the 20 patients with mixed class was significantly lower than that of the 18 patients with crescentic class (40.0% vs. 83.3%, p = 0.006). Receiver operating characteristics (ROC) curves showed fibrous crescent+global glomerulosclerosis greater than 32.6% was a strong predictor of dialysis dependence with a sensitivity of 93.3% and specificity of 88.0%. When the percentage of fibrous crescent+global glomerulosclerosis exceeded 47.9%, dialysis independence was not possible. Correlation analysis indicated that platelet counts were negatively correlated with the percentage of fibrous crescent+global glomerulosclerosis (R = -0.448, p = 0.004). Most patients with increased platelets (84.62%) obtained renal recovery. Compared with methylprednisolone pulse therapy, plasma exchange accelerated renal recovery (29.4 ± 15.6 vs. 41.4 ± 11.7 days, p = 0.039). CONCLUSIONS: For MPO-ANCA AAV who required dialysis at disease onset, crescentic and mixed classes accounted for the majority of patients in our cohort. The renal outcome of mixed class patients was worse than that of crescentic class. A high proportion of fibrous crescent+global glomerulosclerosis is a predictor of dialysis dependence. Increased platelet count is associated with active and reversible renal lesions.

Matrix Metalloproteinases and Their Tissue Inhibitors: an Evaluation of Novel Biomarkers in ANCA-Associated Vasculitis.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may play an important role in both inflammation with subsequent fibrosis and in repair and healing in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We evaluated the circulating levels of MMPs, including pregnancy-associated plasma protein A (PAPP-A), and TIMPs in patients with AAV. PAPP-A, MMP-2, MMP-3, MMP-7, MMP-9, TIMP-1, TIMP-2 and selected parameters were measured in 100 AAV patients (36 patients with active disease and 64 patients in remission) and 34 healthy subjects. The levels of MMP-2, MMP-3, MMP-7, MMP-9, TIMP-1, TIMP-2, and PAPP-A in AAV were all found to be different to those of the controls. The MMP-7 and PAPP-A concentrations were increased in active disease in comparison to the controls (MMP-7: 13 ±.7 vs. 2 ± 0.6 ng/ml, PAPP-A: 14 ± 18 vs. 6.8 ± 2.6 ng/ml, both P < 0.005). The MMP-2 and TIMP-2 levels were increased in remission when compared to the controls (MMP-2: 242 ± 50 ng/ml vs. 212 ± 26 ng /ml, TIMP-2: 82 ± 14 ng/ml vs. 68 ± 93 ng/ml) and to the active AAV (MMP-2: 242 ± 50 vs. 219 ± 54 ng/ml, TIMP-2: 82 ± 14 ng/ml vs. 73 ± 15 ng/ml, all P < 0.005). MMP-3, MMP-7, TIMP-1, and PAPP-A correlated with serum creatinine. The serum levels of MMPs, TIMPs and PAPP-A are all altered in AAV. MMP-2, MMP-7 and TIMP-2 appear to be promising markers in distinguishing active AAV from remission. MMP-3, MMP-7, TIMP-1, and PAPP-A are associated with kidney function in AAV. Further studies are needed to delineate the exact roles of circulating MMPs, TIMPs and PAPP-A in patients with AAV.

High anti-neutrophil cytoplasmic antibody titers are associated with the requirement of permanent dialysis in patients with myeloperoxidase-ANCA-associated vasculitis.

BACKGROUND/PURPOSE: A reliable noninvasive prognostic factor of ANCA-associated vasculitis (AAV) is still lacking, but little research has focused on the value of MPO-ANCA titers in patients with active vasculitis. This study explored the prognostic significance of MPO-ANCA titer in active AAV patients. METHODS: Ninety-seven inpatients diagnosed with MPO-ANCA associated vasculitis at Linkou Chang Gung Memorial hospital and Keelung Chang Gung Memorial hospital from January 2005 to December 2016 were enrolled. Serum ANCA titers and basic characteristics of these patients at diagnosis were collected completely Medical records since AAV diagnosis were reviewed to evaluate two years renal and patient outcome. RESULTS: The patients were divided into the two groups according to the median ANCA titers, the more than four times of the normal cut-off value group (high titer group) and the less ANCA titer group (low titer group). The high titer group had significant poor initial renal function (eGFR 16.7 vs 40.7 mL/min/1.73 m2, P = 0.006), and significantly lower two-year renal survival (Log rank P < 0.001). Whereas patient survival (Log rank P = 0.894) was not different The Cox regression models revealed that baseline Birmingham Vasculitis Activity Score, eGFR and a 4-fold increase in ANCA titer were associated with the requirement of permanent dialysis. In the subgroup analysis, the ANCA titer was still an important risk factor for renal outcomes (P = 0.036) in patients with better initial renal function (eGFR≧15 mL/min). CONCLUSION: This study demonstrated that higher MPO-ANCA titers at diagnosis was associated with poor initial renal function and 2-year renal outcomes.

Impact of MPO-ANCA-mediated oxidative imbalance on renal vasculitis.

Glomerulonephritis is a severe complication of microscopic polyangiitis (MPA), a small-vessel vasculitis associated with anti-myeloperoxidase antibodies (MPO-ANCA). We previously showed the pathogenic effects of MPO-ANCA that activate MPO to trigger an oxidative burst mainly through HOCl production, contributing to endothelial injury and lung fibrosis. The aim of this study was to investigate the relationship between MPO-induced oxidative stress, anti-oxidant defenses and renal histological lesions in MPA patients. We therefore analyzed histological data from a prospective cohort of ANCA-associated glomerulonephritis. Serum-mediated HOCl production, advanced oxidation protein products (AOPP), and thiol concentration in sera were determined. From 38 patients included, histological classification noted 50% focal glomerulonephritis, 15.8% crescentic-glomerulonephritis, and 34.2% mixed-glomerulonephritis. MPA patients' sera displayed higher HOCl production by MPO ( P < 0.001), higher AOPP ( P < 0.001) and thiol ( P < 0.01) levels, compared with healthy subjects. The presence of cellular crescents was associated with higher serum-mediated HOCl production ( P = 0.049) and lower thiol levels ( P = 0.022) at disease onset. Higher thiol concentrations were associated with focal glomerulonephritis ( P = 0.042), less interstitial fibrosis ( P = 0.039) and hyalinosis ( P = 0.066). In remission, HOCl production was decreased ( P < 0.01), and thiol concentration remained high ( P = 0.39). Our findings suggest that HOCl production by activated MPO could contribute to the very early stage of glomerulonephritis, whereas thiol may exert a protective effect against the development of renal vasculitis and glomerulosclerosis. This study highlights the importance of oxidative defenses to counteract the process of MPO-ANCA associated glomerulonephritis.

Myeloperoxidase influences the complement regulatory activity of complement factor H.

Objective: The interaction between neutrophils and activation of alternative complement pathway plays a critical role in the pathogenesis of ANCA-associated vasculitis (AAV). MPO, which can be released from ANCA-stimulated neutrophils, was recently demonstrated to be capable of activating the alternative complement pathway. Here we aimed to investigate the interaction between MPO and factor H (FH), a key regulator of the alternative pathway, and its effect on the functional activities of FH. Methods: Detection of FH and MPO on neutrophil extracellular traps (NETs) induced by serum from AAV patients and in kidney biopsies of AAV patients was performed by immunostaining. In vitro binding between MPO and FH was examined by ELISA and surface plasmon resonance. The influence of MPO on the complement regulatory activity of FH was further assessed. Results: FH deposited and co-localized with MPO in NETs. In kidney biopsies from AAV patients, MPO was closely adjacent to FH in glomerular capillaries. We demonstrated that MPO binds to FH with an apparent nanomolar affinity and identified short consensus repeats 1-4 of FH as the major binding sites. In terms of functional analysis, MPO inhibited the interaction between FH and C3b and the decay-accelerating activity of FH. The fluid phase and surface cofactor activities of FH upon C3b inactivation were inhibited by MPO. Conclusion: Our findings indicate that MPO binds to FH and influences the complement regulatory activity of FH. MPO-FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway.

CD8+ T Cells Effect Glomerular Injury in Experimental Anti-Myeloperoxidase GN.

Observations in patients with ANCA-associated vasculitis suggest that CD8+ T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8+ T cells mediate disease in experimental ANCA-associated vasculitis. CD8+ T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-γ, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8+ T cell MPO epitope (MPO431-439) and found that cotransfer of MPO431-439-specific CD8+ T cell clones exacerbated disease mediated by MPO-specific CD4+ cells in Rag1-/- mice. Transfer of MPO431-439-specific CD8+ cells without CD4+ cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8+ T cells, provide evidence that CD8+ cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8+, CD4+, and B cell epitopes.

Proteinase 3-ANCA Vasculitis versus Myeloperoxidase-ANCA Vasculitis.

In patients with GN or vasculitis, ANCAs are directed against proteinase 3 (PR3) or myeloperoxidase (MPO). The differences between PR3-ANCA-associated vasculitis (AAV) and MPO-AAV described in the past have been supplemented during the last decade. In this review, we discuss the differences between these two small-vessel vasculitides, focusing especially on possible etiologic and pathophysiologic differences. PR3-AAV is more common in northern parts of the world, whereas MPO-AAV is more common in southern regions of Europe, Asia, and the Pacific, with the exception of New Zealand and Australia. A genetic contribution has been extensively studied, and there is a high prevalence of the HLA-DPB1*04:01 allele in patients with PR3-AAV as opposed to patients with MPO-AAV and/or healthy controls. Histologically, MPO-AAV and PR3-AAV are similar but show qualitative differences when analyzed carefully. Clinically, both serotypes are difficult to distinguish, but quantitative differences are present. More organs are affected in PR3-AAV, whereas renal limited vasculitis occurs more often in patients with MPO-AAV. For future clinical trials, we advocate classifying patients by ANCA serotype as opposed to the traditional disease type classification.

High interpatient variability in response to mycophenolic acid maintenance therapy in patients with ANCA-associated vasculitis.

BACKGROUND: Mycophenolic acid (MPA) is used in the maintenance therapy of antineutrophil cytoplasm antibody-associated systemic vasculitis (AASV). MPA exerts its immunosuppression by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH), depleting activated lymphocytes of guanine nucleotides and retarding their proliferation. The purpose of our study was to examine the correlation between clinical outcome and pharmacokinetic-pharmacodynamic (PD) relationships of MPA in patients with AASV. METHODS: We studied 358 Caucasian control patients without any MPA therapy to examine basal IMPDH activity. Thirty Caucasian patients with AASV under maintenance therapy with mycophenolate mofetil (MMF) underwent therapeutic drug monitoring. RESULTS: We observed a high interindividual variability with regard to basal IMPDH activity in patients without any MPA treatment (0.8-35 nmol/mg protein/h). Patients were followed for a mean (±SD) period of 22 ± 8 months. During the observation period, seven patients had a relapse with an elevated Birmingham Vasculitis Activity Score of 9.2 ± 6. The basal IMPDH activity (Abasal) in patients who subsequently relapsed was raised at baseline, before receiving their first dose of MMF, and further increased at the time of relapse, when compared with stable patients. Patients with a relapse during the maintenance therapy had significantly higher levels of IMPDH activity [IMPDH enzyme activity curve (AEC) (0-12)] than stable patients (P = 0.001), indicating inadequate IMPDH suppression. MPA-AUC (0-12) was significantly decreased in relapse patients, in contrast to stable patients (P < 0.05). CONCLUSIONS: Due to the highly variable response to maintenance therapy with MPA, PD drug monitoring is a new tool for detecting inadequate immunosuppression in AASV patients.

Significance of serum peptidylarginine deiminase type 4 in ANCA-associated vasculitis.

OBJECTIVE: To investigate the clinical significance of peptidylarginine deiminase type 4 (PAD4) in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). To make a primary observation on the relationship of chronic bronchitis and bronchiectasis (CB) with the pathogenesis of AAV by PAD4. METHODS: The sera from 13 patients with AAV, 13 patients with CB, 11 patients with rheumatoid arthritis (RA), 11 patients with primary chronic kidney disease (CKD) and 12 normal controls were collected. Serum PAD4 was detected using commercial ELISA kits. The serum levels of PAD4 were compared not only among the different groups but also between the activity and remission stage of the same disease. The associations between serum PAD4 and the Birmingham Vasculitis Activity Score (BVAS) of AAV were further investigated. RESULTS: (1) The serum levels of PAD4 in patients with AAV, RA and CB at activity stage were all higher than that in the normal controls (P<0.001, respectively, α'=0.007). The serum level of PAD4 in patients with CB at remission stage and that in CKD group were not found elevated compared with the normal controls (P=0.02, P=0.085, respectively, α'=0.007). (2) At activity stage, among the groups of simple AAV, AAV with a long history of CB and CB without AAV, no significant difference was detected. While at remission stage among the 3 groups, the serum level of PAD4 was at the lowest level in CB group without AAV. (3) The serum level of PAD4 in some patients with CB without AAV were found still higher at remission stage. (4) The serum level of PAD4 in AAV with renal damage at activity stage was positively correlated with BVAS (the activity score of AAV, r=0.71, P=0.02). CONCLUSION: PAD4 is involved in the pathogenesis of AAV. Whether some patients with CB might progress to AAV by the link with PAD4 still need further investigation.

Possible implication of disordered neutrophil extracellular traps in the pathogenesis of MPO-ANCA-associated vasculitis.

Neutrophil extracellular traps (NETs) are characterized by the presence of extracellular DNA fibers studded with antimicrobial proteins, including myeloperoxidase (MPO). Although NETs play an important role in the innate immune system, the scattered extracellular enzymes, such as MPO, pose risks to the host. Therefore, NETs are strictly regulated by DNase I in the serum, which prevents them from persisting. Recent studies have demonstrated that dysregulation of NETs could be involved in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. In this review, we interpret the association of disordered NETs with autoimmune diseases, especially propylthiouracil-induced MPO-ANCA-associated vasculitis.

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis.

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is an autoimmune disease in which the contributions of genetic, epigenetic and environmental factors to aetiology and pathogenesis are being unravelled. The ANCA immunoglobulin G targeting proteinase 3 and myeloperoxidase affects several neutrophil functions, usually to augment or dysregulate these, promoting a proinflammatory phenotype whereby neutrophils have enhanced capabilities of causing collateral damage to endothelial and other cells. In addition, B cells are intimately involved in pathogenesis as anti-B cell therapies are highly effective, but the manner of this involvement still needs to be delineated. Similarly, the T cell compartment is disturbed in ANCA vasculitis and numerous alterations in T cell subsets have been described, but recognition of a novel CD8(+) T cell transcription signature which can predict likelihood of relapse in ANCA vasculitis indicates that more needs to be learnt about the influence of T cells in the disease process. Finally, the role of the alternative complement pathway and the potential therapeutic value of its neutralization is under active investigation after compelling studies in murine models have demonstrated that C5 and factor-B knock-out mice are protected.

Effects of p38 mitogen-activated protein kinase inhibition on anti-neutrophil cytoplasmic autoantibody pathogenicity in vitro and in vivo.

OBJECTIVE: To determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo. METHODS: The effects of the p38MAPK-specific inhibitor AR-447 were studied in vitro using neutrophil respiratory burst and degranulation assays, and in lipopolysaccharide (LPS)-stimulated human glomerular endothelial cells. In vivo, p38MAPK inhibition was investigated in a mouse anti-myeloperoxidase (MPO) IgG/LPS glomerulonephritis model. Mice were treated orally with AR-447 daily, starting before (pretreatment group) or 24 h after disease onset (treatment group), and killed after 1 or 7 day(s). RESULTS: In vitro, AR-447 diminished neutrophil respiratory burst and degranulation induced by patient-derived MPO-ANCA and proteinase 3 (Pr3)-ANCA. In glomerular endothelial cells, AR-447 reduced LPS-induced secretion of IL-6 and IL-8, but not of MCP-1. In mice, pretreatment with AR-447 reduced albuminuria 1 day after induction of glomerulonephritis. After 7 days, no effects on urinary abnormalities were observed upon AR-447 pretreatment or treatment. Also, glomerular neutrophil accumulation was not diminished. In contrast, glomerular macrophage accumulation and the formation of glomerular crescents was significantly reduced by AR-447 pretreatment (vehicle: 12.5 ± 5.6% crescentic glomeruli; AR-447: 7.7 ± 2.7%) and treatment (vehicle 14.6 ± 1.8%; AR-447 6.0 ± 3.4%) at 7 days. CONCLUSION: This study shows that p38MAPK inhibition markedly reduces ANCA-induced neutrophil activation in vitro. In vivo, p38MAPK inhibition partly reduced crescent formation when the drug was administered prior to disease induction and after disease onset, suggesting that besides p38MAPK activity other signalling pathways contribute to the disease activity.

Serum levels of BAFF and APRIL in myeloperoxidase anti-neutrophil cytoplasmic autoantibody-associated renal vasculitis: association with disease activity.

BACKGROUND: A proliferation-inducing ligand (APRIL) and the B cell activation factor belonging to the tumor necrosis factor family (BAFF) have proven to be key factors in the selection and survival of B cells, and a higher concentration of BAFF has been shown to contribute to autoreactive B cell survival and elevated autoantibody production. Here, serum BAFF and APRIL levels were investigated to analyze their association with disease activity in myeloperoxidase anti-neutrophil cytoplasmic autoantibody (MPO-ANCA)-associated renal vasculitis. METHODS: APRIL and BAFF levels in serum obtained from 37 patients with MPO-ANCA-associated vasculitis were measured by ELISA. Samples were taken from active vasculitis patients, inactive vasculitis patients and inactive vasculitis patients with infectious complications. RESULTS: Although there was no difference in serum APRIL among the active vasculitis, inactive vasculitis and infectious complication patients, serum BAFF was higher in active vasculitis patients than in inactive vasculitis, infectious complication and control patients (for all, p < 0.001). There was no significant correlation between serum APRIL and ANCA titers, but there was a significant correlation between serum BAFF and ANCA titers (r = 0.465, p < 0.001). CONCLUSION: Excessive BAFF production in MPO-ANCA-associated vasculitis may be one of the factors for autoimmune B cell tolerance, resulting in MPO-ANCA production.

Development of aortic valve stenosis in myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis with renal involvement.

INTRODUCTION: Degenerative aortic valve stenosis (AS) is a chronic progressive disease that resembles atherosclerosis development. Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is reportedly associated with accelerated atherosclerosis. This study aimed to examine the development of AS in patients with myeloperoxidase-AAV (MPO-AAV) with renal involvement at more than 1 year after the onset of vasculitis. METHODS: We performed a retrospective review of clinical records of MPO-AAV patients with renal involvement without AS at the onset of vasculitis who were treated in three hospitals and three dialysis clinics. RESULTS: The study included 97 MPO-AAV patients with renal involvement and 230 control patients with chronic kidney disease (CKD). Among them, 64 patients had AS. The prevalence rates of AS were 28.9% and 15.7% in MPO-AAV and control patients, respectively (p = 0.006). The multivariable logistic regression analysis showed that MPO-AAV, dialysis dependence, and hypertension were independently associated factors for AS. In MPO-AAV patients, systolic blood pressure was positively significantly associated with AS, whereas glucocorticoid dose of induction therapy was negatively significantly associated. The use of cyclophosphamide tended to be negatively associated with AS. The survival rate was significantly lower for patients with AS than for those without AS. CONCLUSIONS: The AS prevalence rate was significantly higher in MPO-AAV patients at more than 1 year after the onset of vasculitis than in control CKD patients. Therefore, regular monitoring of echocardiography during MPO-AAV treatment is suggested.

Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression.

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Expression of tumor necrosis factor receptors on granulocytes in patients with myeloperoxidase anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND/AIMS: To clarify the clinical significance of tumor necrosis factor (TNF) receptors in patients with myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, we evaluated the cell surface expression of TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). PATIENTS AND METHODS: 43 patients with MPO-ANCA-associated vasculitis, 16 patients with chronic renal failure, 10 patients with sepsis, 15 patients with systemic lupus erythematosus, and 18 healthy controls were enrolled in this study, and the surface expression levels of TNFR1, TNFR2, CD63, and CD64 on granulocytes were assessed. In 21 patients with MPO-ANCA-associated vasculitis, soluble TNFR1 (sTNFR1), soluble TNFR2(sTNFR2), and TNF-alpha in the serum were also measured. RESULTS: The surface expression levels of TNFR1 and TNFR2 on granulocytes were significantly higher in patients with MPO-ANCA-associated vasculitis than in the healthy controls, and positively correlated with the Birmingham Vasculitis Activity Score (BVAS). The levels of sTNFR1, sTNFR2, and TNF-alpha in the serum were also significantly higher in patients with MPO-ANCA-associated vasculitis than in the healthy controls. Serum levels of sTNFR1 and sTNFR2 correlated with serum creatinine, while the surface expression of TNFR1 and TNFR2 on the granulocytes did not. There was no significant correlation between the BVAS and CD63 or BVAS and CD64. CONCLUSION: The surface expression levels of TNFR1 and TNFR2 on granulocytes were upregulated in patients with MPO-ANCA-associated vasculitis and reflected disease activity.

Proteinase 3 (PR3)-antineutrophil cytoplasmic antibody (ANCA)-associated vasculitic neuropathy in diffuse cutaneous systemic sclerosis: a rare duo.

Systemic sclerosis (SSc) is characterised by non-inflammatory vasculopathy, autoimmunity and widespread fibrosis. While the presence of antineutrophil cytoplasmic antibodies (ANCAs) has been reported in SSc, their association with ANCA-associated vasculitis is exceedingly rare. Myeloperoxidase ANCA is more common than proteinase-3 ANCA, and glomerulonephritis is the most common clinical presentation of ANCA-associated vasculitis in SSc. ANCAs have been associated with the adverse disease outcomes in SSc, including higher mortality per recent reports. A 65-year-old man with diffuse cutaneous SSc for 6 years presented with new-onset peripheral neuropathy. Workup revealed a positive proteinase-3 and cytoplasmic ANCA, and histopathology confirmed an inflammatory vasculitic neuropathy. The patient was successfully treated with rituximab. Our case highlights the importance of checking ANCA in SSc at baseline, given the risk of disease-related complications, even years later. Tissue biopsy is often warranted for confirmation of vasculitis and prompt treatment can optimise long-term outcomes.

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

Thiopurine methyltransferase genotype and activity cannot predict outcomes of azathioprine maintenance therapy for antineutrophil cytoplasmic antibody associated vasculitis: A retrospective cohort study.

OBJECTIVE: Azathioprine is a widely used immunosuppressive drug. Genetic polymorphisms and activity of the enzyme thiopurine methyltransferase (TPMT) have been associated with azathioprine efficacy and toxicity in several populations. We investigated whether these associations also exist for ANCA associated vasculitis (AAV) patients, who receive azathioprine maintenance therapy after remission induction with cyclophosphamide. METHODS: 207 AAV patients treated with cyclophosphamide induction and azathioprine maintenance therapy were included and followed for 60 months. TPMT genotype and tertiles of TPMT activity were compared to relapse free survival and occurrence of adverse events, particularly leukopenia. Multivariable regression was performed to account for confounders. RESULTS: In univariable analysis, relapse free survival was not significantly associated with TPMT genotype (P = 0.41) or TPMT activity (P = 0.07), although it tended to be longer in lower tertiles of TPMT activity. There was no significant association of TPMT genotype and activity with occurrence of any adverse event. In multiple regression, leukocyte counts at the end of cyclophosphamide induction were related to risk of leukopenia during azathioprine therapy [P<0.001; OR 0.54 (95% CI 0.43-0.68)] and risk of relapse during follow-up [P = 0.001; HR 1.17 (95% CI 1.07-1.29)] irrespective of TMPT genotype or activity. CONCLUSION: TPMT genotype and activity were not independent predictors of relapse, and could not predict leukopenia or other adverse effects from azathioprine. Leukocyte counts after cyclophosphamide induction were related to both outcomes, implying a greater influence of cyclophosphamide response compared to azathioprine and TPMT in AAV patients.

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.