ANCA-associated Systemic Vasculitis Research Articles

Coexpression of CD177 and membrane proteinase 3 on neutrophils in antineutrophil cytoplasmic autoantibody–associated systemic vasculitis: Anti–proteinase 3–mediated neutrophil activation is independent of the role of CD177‐expressing neutrophils

Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay. Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

Retinoid X receptor beta polymorphisms do not explain functional differences in vitamins D and A response in Antineutrophil cytoplasmic antibody associated vasculitis patients

It has been suggested that the retinoid X receptor beta (RXRB) gene is a risk factor for Wegener's granulomatosis. We addressed if there is a functional difference in the response to retinoic acid (RA) and vitamin D in Antineutrophil cytoplasmic antibody (ANCA) associated systemic vasculitis (AASV) patients and if this was associated with RXRB genotypes. TNFα and IL-10 production were measured in whole blood assay from AASV patients (n = 51) and healthy controls (HC, n = 67). One micromolar of 1,25-(OH)2 D3, 9-cis RA (9c-RA) or all-trans RA (ATRA) was added to the assay. Genotyping was performed for exons 7 and 2 of the RXRB gene and for a microsatellite in vicinity of the RXRB gene. Lipopolysaccharide (LPS) mediated TNFα production and IL-10 were significantly lower in patients. Addition of 1,25-(OH)2 D3, ATRA or 9c-RA, blunted TNFα production, more pronounced in patients. Although all three compounds inhibited IL-10 production significantly in HC, only 1,25-(OH)2 D3 was found to be effective in patients. Allele distribution of the RXRB microsatellite differed significantly between patients and HC. This was not found for the SNP in exons 2 and 7. Genotype of the latter correlated with the ability of 1,25-(OH)2 D3 and ATRA to inhibit IL-10 production. We provide immunological evidence for a functional difference in vitamins D and A responsiveness in AASV patients. Since the inhibition of TNFα was more effective in patients, vitamin D supplementation might be an additional therapeutical approach.

Successful Renal Transplantation in a Patient With Antineutrophil Cytoplasmic Autoantibody-Associated Microscopic Polyangiitis: Effect of Early Plasma Exchange and Mycophenolate Mofetil

Antineutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitis readily relapses after renal transplantation; the recurrence often causes graft losts. We have reported herein a 53-year-old cadaveric renal allograft recipient. His original disease was ANCA-associated microscopic polyangiitis (MPA), which was proven by renal biopsy. On postoperative day 4, the patient developed dyspnea, hemoptysis, and gross hematuria; the serum p-ANCA titer increased from 1:40 to 1:80. The patient was immediately treated with 5 sessions of plasma exchange. Five days later, the hemoptysis and gross hematuria both disappeared. Serum p-ANCA titer recovered to 1:40. The patient showed stable graft function for 2 months, until mycophenolate mofetil (MMF) was converted to mizoribine (150 mg/d). Seven days after the conversion, the serum creatinine increased from 1.27 to 1.7 mg/dL. Serum p-ANCA increased to 1:160. When mizoribine was converted to MMF, 2 days later the renal function and p-ANCA titer recovered. This case report suggested that early use of plasma exchange combined with MMF was effective to prevent the relapse of ANCA-associated systemic vasculitis after transplantation, and may be helpful to improve transplant outcomes.

A case of ANCA-associated systemic vasculitis induced by atorvastatin

We present a 45-year-old male patient who presented to Accident and Emergency department with a 6-week history of pain and stiffness involving his bilateral legs. Both calves were markedly tender, and he was not able to bear weight. He also complained of numbness involving his left big toe for a few days, which later spread to involve his arms, and tinnitus and hearing loss in his left ear. There were no respiratory, gastrointestinal or urinary symptoms. He had a background history of hypercholesterolemia and was treated with atorvastatin 10 mg for 6 months. His initial investigations showed markedly increased inflammatory markers, and serum antineutrophil cytoplasmic antibody (ANCA) was markedly positive at a titre of 1:160 (P-ANCA). Electromyography and muscle biopsy showed myopathic features. A diagnosis of drug-induced ANCA-associated vasculitis (on the basis of mononeuritis multiplex, sensorineural hearing loss and markedly increased anti-myeloperoxidase (MPO) ANCA) and statin-induced distal myopathy was made. He was treated with three 500 mg doses of methylprednisolone, followed by slowly tapering dose of oral corticosteroids from 30 mg once daily (OD). He was also started on azathioprin (2.5 mg/kg). He had a dramatic improvement of his myalgia, hearing loss and sensory symptoms and went into complete clinical remission. His inflammatory markers rapidly returned to normal, and MPO-ANCA normalised within 3 months of starting immunosuppressive therapy and remained negative on further testing. He is currently on a tapering regimen of corticosteroids (7 mg OD), and after weaning him off corticosteroids, we plan to slowly taper his azathioprin.

Treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis with high-dose intravenous immunoglobulin.

In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30 g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.

Therapie von Vaskulitiden und Vaskulopathien

Treatment and course of leukocytoclastic immune-complex vasculitis (LcV) depend on absence or presence of IgA in immune complexes [Henoch-Schoenlein-Purpura (PSH)]. LcV due to IgG- or IgM-containing immune complexes has a better prognosis. If triggers cannot be detected or avoided, symptomatic treatments are usually sufficient due to a usually favourable course. When hemorrhagic blisters suggest incipient skin necrosis corticosteroids are indicated. For chronic or relapsing LcV we suggest colchicine or dapsone. In adults with PSH and severe glomerulonephritis there is insufficient evidence for the efficacy of glucocorticoids; but e.g. ACE inhibitors can be helpful depending on symptoms. In cryoglobulinemic vasculitis underlying diseases (often plasmocytoma or hepatitis C) should be treated, sometimes supplemented by plasmapheresis. Dapsone or colchicine are usually started for urticarial vasculitis. ANCA-associated systemic vasculitis requires rapid and aggressive induction therapy, usually with glucocorticoids and cyclophosphamide. In classic polyarteriitis nodosa glucocorticoids improve prognosis, in polyarteriitis nodosa cutanea colchicine or dapsone are more appropriate. Giant cell arteriitis requires rapid therapy with glucocorticoids. For livedo vasculopathy antithrombotic measures are required with low molecular heparin or antagonists to vitamin K, for maintenance dipyridamol und aspirin.

Das Churg-Strauss-Syndrom

Churg-Strauss syndrome is a systemic ANCA-associated vasculitis arising almost exclusively in patients with a pre-existent asthma. Common clinical manifestations are marked blood eosinophilia, asthma, chronic sinusitis, cardiomyopathy, pulmonary infiltrates, gastrointestinal complaints and a multiplex neuropathy. The morphological substrate is an eosinophilic necrotizing vasculitis. Other eosinophilic disorders such as parasitic diseases, allergies and idiopathic hyper-eosinophilic syndrome have to be excluded. The mainstay of therapy is high-dose corticosteroids with the addition of cytotoxic drugs in patients with poor prognosis.

ANCA-associated systemic vasculitides

ANCA-associated systemic vasculitides

Randomized Trial of Plasma Exchange or High-Dosage Methylprednisolone as Adjunctive Therapy for Severe Renal Vasculitis

Systemic vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA) is the most frequent cause of rapidly progressive glomerulonephritis. Renal failure at presentation carries an increased risk for ESRD and death despite immunosuppressive therapy. This study investigated whether the addition of plasma exchange was more effective than intravenous methylprednisolone in the achievement of renal recovery in those who presented with a serum creatinine >500 micromol/L (5.8 mg/dl). A total of 137 patients with a new diagnosis of ANCA-associated systemic vasculitis confirmed by renal biopsy and serum creatinine >500 micromol/L (5.8 mg/dl) were randomly assigned to receive seven plasma exchanges (n = 70) or 3000 mg of intravenous methylprednisolone (n = 67). Both groups received oral cyclophosphamide and oral prednisolone. The primary end point was dialysis independence at 3 mo. Secondary end points included renal and patient survival at 1 yr and severe adverse event rates. At 3 mo, 33 (49%) of 67 after intravenous methylprednisolone compared with 48 (69%) or 70 after plasma exchange were alive and independent of dialysis (95% confidence interval for the difference 18 to 35%; P = 0.02). As compared with intravenous methylprednisolone, plasma exchange was associated with a reduction in risk for progression to ESRD of 24% (95% confidence interval 6.1 to 41%), from 43 to 19%, at 12 mo. Patient survival and severe adverse event rates at 1 yr were 51 (76%) of 67 and 32 of 67 (48%) in the intravenous methylprednisolone group and 51 (73%) of 70 and 35 of (50%) 70 in the plasma exchange group, respectively. Plasma exchange increased the rate of renal recovery in ANCA-associated systemic vasculitis that presented with renal failure when compared with intravenous methylprednisolone. Patient survival and severe adverse event rates were similar in both groups.

Serum Procalcitonin Levels in Febrile Patients with Systemic Autoimmune Diseases

Procalcitonin (PCT), the precursor of the hormone calcitonin, is a new parameter of inflammation. This acutephase hormokine seems to be more accurate for differentiating between bacterial infections and viral or non-infective causes of inflammation than other serological markers. PCT, which is induced in invasive bacterial infections, is routinely assayed in some intensive care, surgery, haematological units and emergency rooms to help determine the cause of a systemic inflammatory response syndrome (SIRS). Patients with systemic autoimmune diseases have a higher risk of developing severe systemic bacterial infections due to the disease itself and to the immunosuppressive therapy received. Distinguishing between infection and a disease flare is sometimes difficult. Strategies to reach a precise diagnosis are decisive to establish early, adequate patient management. Studies in febrile patients with systemic autoimmune diseases (systemic lupus erythematosus, antineutrophil cytoplasmic antibody-associated systemic vasculitis, and rheumatoid arthritis) suggest that elevated PCT concentrations offer good sensitivity and specificity for diagnosing systemic bacterial infection. Nevertheless, there is little published information on PCT levels in other autoimmune diseases and inflammatory processes. In this review we summarise the current evidence regarding the pathophysiological basis of systemic PCT production in sepsis and the results from current case series on the response of this peptide to infectious and non-infectious SIRS in systemic autoimmune diseases.

ANCA-associated vasculitis: Diagnosis, clinical characteristics and treatment

The primary systemic vasculitides are a group of diseases characterized by an inflammatory process of the vessel walls and classified according to the smallest vessels involved. Small vessel vasculitides comprise the largest subgroup divided into diseases with a pauci-immune vasculitis and ANCA and diseases with deposition of immunoglobulin without ANCA. ANCA-associated systemic vasculitides include Wegener's granulomatosis, microscopic polyangiitis comprising renal-limited vasculitis and Churg-Strauss syndrome. Diagnosis is based on clinical manifestation, ANCA-testing and histology. Beside the role of ANCA as a diagnostic marker many studies and animal models have focused on the pathogenic role. The treatment of ANCA-associated vasculitis has changed from a standardized "Fauci-protocol" to an individualized less toxic strategy taking into consideration disease severity) organ manifestation, age of the patient and individual risk factors (e.g. increased bone marrow toxicity in patients with renal insufficiency). For remission induction patients are sub-grouped according to limited or generalized disease with moderate or severe renal involvement. Thus cyclophosphamide is only used in patients with generalized disease or - regarding Churg-Strauss-syndrome - patients with risk factors. For maintenance of remission azathioprine should be used in most of the patients.

Follow‐up of avidity and titre of anti‐myeloperoxidase antibodies in sera from patients with propylthiouracil‐induced vasculitis

Propylthiouracil (PTU) has been known to induce myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) positive vasculitis. Our previous study indicated that the increase of avidity of MPO-ANCA might be associated with the occurrence of clinical vasculitis in patients with PTU-induced ANCA. The current study aimed to follow-up the avidity and titre of anti-MPO antibodies in sequential sera from patients with PTU-induced ANCA-associated systemic vasculitis (AASV). Six patients with PTU-induced vasculitis were enrolled in the current study. Serial sera in both active phase and in remission were collected. MPO-ANCA avidity was assessed by antigen-inhibition enzyme-linked immunosorbent assays (ELISAs), and avidity constant (aK) was determined as the reciprocal value of the MPO molar concentration in the liquid phase resulting in 50% inhibition of anti-MPO antibody binding to MPO in solid phase ELISA. Titres of MPO-ANCA were determined by using serial serum dilutions in MPO-ELISA. After cessation of PTU and initiation of immunosuppressive therapy, the avidity and titre of MPO-ANCA decreased significantly during follow-up in sera from all the patients, and the avidity decreased much more quickly than the titres. Our study indicates that avidity of anti-MPO antibodies might be more closely associated with clinical vasculitis than titre.

Respiratory System Involvement??in Antineutrophil??Cytoplasmic-Associated Systemic Vasculitides

Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg- Strauss syndrome (CSS) are small-vessel vasculitides that, because of their frequent association with antineutrophil cytoplasmic antibodies (ANCA), are usually referred to as ANCA-associated systemic vasculitides (AASV). The diagnosis of AASV is made on the basis of clinical findings, biopsy of an involved organ and the presence of ANCA in the serum. Lung disease is a very common and important clinical feature of AASV. In WG, almost all patients have either upper airway or lower respiratory tract disease. Solitary or multiple nodules, frequently cavitated, and masses are the most common findings on chest radiography. Asthma is a cardinal symptom of CSS, often preceded by allergic rhinitis. Pulmonary transient and patchy alveolar infiltrates are the most common radiographic findings. In MPA, diffuse alveolar haemorrhage as a result of alveolar capillaritis is the most frequent manifestation of respiratory involvement, and is clinically expressed as haemoptysis, respiratory distress and anaemia. However, diffuse alveolar haemorrhage may also be subclinical and should be suspected when a chest radiograph demonstrates new unexplained bilateral alveolar infiltrates in the context of falling haemoglobin levels. Normal and high-resolution CT have a higher sensitivity than chest radiography for demonstrating airway, parenchymal and pleural lesions. However, many of these radiological findings are nonspecific and, therefore, their interpretation must take into account all clinical, laboratory and pathological data. Therapy of AASV is commonly divided into two phases: an initial 'remission induction' phase, in which more intensive immunosuppressant therapy is used to control disease activity, and a 'maintenance' phase, which uses less intensive therapy, for maintaining disease remission while lowering the risk of adverse effects of immunosuppressant drugs. In patients with AASV refractory to standard therapy with corticosteroids and oral cyclophosphamide, new therapeutic options are now available. Recurrence of pulmonary symptoms suggesting a flare indicates the need for a careful search for an opportunistic lung infection or iatrogenic pulmonary complications. In conclusion, involvement of the respiratory system is a very common and important organ manifestation of AASV. Respiratory system involvement comprises a wide spectrum of clinical features and radiological findings, and because of its frequency and prognostic significance, a complete assessment of the respiratory system should be included in the work-up of all patients with AASV.

Elevated Serum Neutrophil Elastase but Not Proteinase 3 Is Present in Patients with Myeloid Leukemia.

Proteinase 3 (P3) and neutrophil elastase (NE) are abnormally expressed in the cytoplasm of myeloid leukemia, and we have shown that the HLA-A2-restricted peptide PR1 is processed and cross-presented from both proteins by antigen-presenting cells (APC) to cytotoxic T lymphocytes (CTL). PR1-specific CTL kills leukemia cells and contributes to cytogenetic remission in patients with chronic myelogenous leukemia. Both exogenous P3 and NE can bind to monocytes and P3 promotes DC maturation in vitro. Since high levels of circulating P3 were found in patients with the ANCA-associated systemic vasculitis Wegener's granulomatosis, modulation of DC by P3 has been implicated in reversal of tolerance. The role of extracellular P3 and NE in reversing tolerance in patients with myeloid leukemia is unknown. We used an ELISA assay to assess P3 and NE level in the sera of patients with AML and CML. Serum NE level was highly increased in all 15 AML patients studied (1045±149 ng/ml, n = 15) and in all 15 CML patients (1013±128, n = 15), compared to a control group of healthy donors (54±13 ng/ml, n = 4; p = 0.0038 and p = 0.0014, respectively). In contrast, serum P3 was increased in only 5 out of 18 (28%) AML patients (47 ± 18, n = 18 ng/ml) and in 8 out of 28 (29%) CML patients (95 ± 44 ng/ml, n = 28) compared to a group of 14 healthy donors (10 ± 2.2 ng/ml; p = 0.09 and p = 0.06, respectively). There was no relationship between elevated serum NE and P3 levels (r=0.16, n=35). P3 level in untreated patients and healthy donors did not change within at least 13 months, although P3 decreased after allogeneic BMT in 5 out of 5 patients (p < 0.05) with high serum levels of P3 pre-BMT. Because P3 is expressed by myeloid progenitor cells in the bone marrow, we studied the concentration of P3 in bone marrow-derived serum (BMS). To our surprise, BMS P3 concentration in patients with myeloid leukemia (23 ± 9.5 ng/ml, n = 9) was similar to that of a control group of patients with non-myeloid hematological malignancies (27 ± 11.8, n = 5) (p = 0.08), and BMS P3 concentration was similar to sera P3 concentration in the same patient (p = 0.52). Interestingly, BMS P3 level correlated with the level of BMS GM-CSF (quantified by ELISA) in a small group of patients with AML and CML (r = 0.94, n = 9), suggesting that P3 secretion by leukemia cells may be downstream of GM-CSF. We also analyzed membrane expression of P3 and NE in 5 AML patients using flow cytometry and found that P3, but not NE, was expressed on circulating cells in 3 of 5 patients. This unique expression of P3 and NE suggests that circulating NE might be the predominant source of cross-presented PR1 antigen by APC or myeloid leukemia cells.

Les nouveaux ANCA : leur mode d'emploi

Antineutrophil cytoplasmic autoantibodies (ANCA) are a useful diagnostic tool for ANCA-associated systemic small vessel vasculitis. For these patients, combining indirect immunofluorescence (IIF) with PR3 and MPO specific Elisa leads to the best diagnostic performance. However, ANCA targeting other autoantigens with positive IIF, anti-PR3 and/or anti-MPO positivity without IIF may be found and special measures must be taken to help and avoid clinical misinterpretation.

Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the − 564 A/G promotor polymorphism

Several findings link proteinase 3 (PR3) to small vessel vasculitis. Besides being a major target of anti-neutrophil cytoplasm antibodies (ANCA), previous findings have shown increased circulating levels of PR3 in vasculitis patients, increased levels of neutrophil membrane-PR3 (mPR3) expression and a skewed distribution of the - 564 A/G polymorphism in the promotor region of the PR3 gene. In this study we elucidate how these three findings relate to each other. The plasma concentration of PR3 was measured by enzyme-linked immunosorbent assay (ELISA), mPR3 expression by fluorescence activated cell sorter (FACS) and the gene polymorphism by real-time polymerase chain reaction (PCR). We compared results from 63 patients with ANCA-associated systemic vasculitis (AASV) with 107 healthy blood donors. In accordance with previous reports, AASV patients had increased plasma concentrations of PR3 compared to healthy controls (mean 224 microg/l versus 155 microg/l, P < 0.0001). They also showed an increased number of mPR3-positive neutrophils (60%versus 42%, P < 0.001). However, contrary to a previous report, we found no skewed distribution of the polymorphism in PR3 gene. There was a weak correlation between mPR3 mean fluorescence intensity (MFI) and plasma PR3 among healthy controls and myeloperoxidase-ANCA (MPO-ANCA)-positive patients (r = 0.24, P = 0.015 and r = 0.52, P = 0.011, respectively). In conclusion, increased plasma PR3 and high expression of mPR3 are associated with small vessel vasculitis, but neither of them is a consequence of the - 564 A/G polymorphism of the PR3 gene promotor.

Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody–stimulated neutrophils

To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil-endothelial cell interactions. Superoxide release from neutrophils binding to purified P-selectin or to tumor necrosis factor-activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme-linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically. ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P-selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil-endothelial cell coculture. There was enhanced release of vWF during activated neutrophil-endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate. Endothelial cells inhibit superoxide generation by fMLP and ANCA-activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.

Persistent T-cell activation and clinical correlations in patients with ANCA-associated systemic vasculitis

Although in antineutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AASV) patients, activation of T-cells has been described, persistence of these alterations has not been well characterized. This study was conducted to define persistent T-cell activation (PTA) in AASV patients and to assess whether this correlates with disease activity, disease severity, age or therapy. The expression of CD4, CD45RO, CD25, CD26, CD28, CCR7 and HLA-DR was examined longitudinally in 38 consecutive AASV patients. Clinical parameters were compared by univariate and multiple analysis and Kaplan-Meier curves for relapse-free survival were calculated. PTA could be defined as either of two activation phenotypes, i.e. a low percentage of CD4+ CD45RO- T-cells or a high percentage of CD25 in the naïve CD4+ population (n = 26), since only these phenotypes were stable over time and were not associated with active disease. In patients with PTA, major organ involvement was significantly more often found than in patients without PTA. Moreover, the cumulative cyclophosphamide dose (26.86 vs 8.53 P < 0.01) was significantly increased in these patients, suggesting that PTA was associated with disease severity. In general, patients with PTA were older than those without (62.92 +/- 9.4 years vs 48.42 +/- 16.9 years respectively, P < 0.01). PTA was independent of disease duration. Interestingly, patients with a low percentage of CD4+CD45RO- T-cells were significantly more often diagnosed as microscopic polyangiitis (P < 0.01). We identified two independent phenotypes of T-cell activation in AASV patients. These phenotypes are persistent and do not reflect disease activity. PTA predominantly occurs in patients with severe disease. This might explain the higher cumulative cyclophosphamide dose found in these patients.

The clinical features of anti-neutrophil cytoplasmic antibody-associated systemic vasculitis in Chinese children

Anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis is reported mainly in adults. Studies in children are limited. The current study retrospectively analyzed the clinical characteristics and pathology of ANCA-associated systemic vasculitis in children in our hospital during the past 7 years. Twenty-four pediatric patients were diagnosed as having ANCA-associated systemic vasculitis, including 19 patients with microscopic polyangiitis (MPA), one with Wegener's granulomatosis (WG), three with propylthiouracil (PTU)-induced ANCA-positive vasculitis and one with anti-glomerular basement membrane (GBM) disease. Of patients with primary ANCA-associated systemic vasculitis (MPA and WG), with an average age of 10.8+/-2.8 (6-14) years, 18 patients (90%) were female and two (10%) were male. Nineteen patients (95%) were p-ANCA/MPO-ANCA positive and one (5%) was c-ANCA/PR3-ANCA positive. The interval between onset and diagnosis was 8.5+/-24.3 (0.2-108) months. The majority of the patients (85%) had multi-organ involvement. All patients had clinical evidence of renal involvement and presented with hematuria and proteinuria. Of 20 patients, 16 (80%) also had acute renal failure, and five patients were dialysis dependent. Nine patients underwent renal biopsy and were diagnosed with necrotizing and crescentic glomerulonephritis. However, six biopsies showed immune complex deposition. All patients received immunosuppressive therapy including prednisone and cyclophosphamide, and ten patients also received intravenous administration of methylprednisone pulse therapy according to their clinical situation and renal pathology. Sixteen patients achieved clinical remission, and four patients presented as treatment failure. Patients were followed up for 12.3+/-5.1 months (median 12 months; range 1 to 91 months). Ten patients maintained their clinical remission, and ten progressed to renal failure requiring dialysis. Our study showed that the clinical features and pathology of primary ANCA-associated systemic vasculitis in children were similar to those of adults, but there were a predominance of female patients and late diagnoses. We suggest that early recognition and prompt aggressive treatment might improve outcome.

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, ∼20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.