No association of G-463A myeloperoxidase gene polymorphism with MPO-ANCA-associated vasculitis.

BACKGROUND: The activation of neutrophils and monocytes by ANCA, resulting in the release of reactive oxygen species and proteases like myeloperoxidase (MPO), is essential to the pathogenesis of ANCA-associated vasculitis. As the A allele of the G-463A MPO gene polymorphism is associated with diminished activity of MPO, it is conceivable that the presence of this allele protects against MPO-ANCA-associated vasculitis. METHODS: Allelic frequencies of the G-463A polymorphism were studied in 119 ANCA-associated vasculitis patients, 48 with MPO-ANCA and 71 with proteinase 3 (PR3)-ANCA. RESULTS: Allelic frequencies of MPO G-463A promoter polymorphism did not differ between MPO-ANCA- and PR3-ANCA-associated vasculitis patients. Moreover, allelic distribution was similar to that of the normal population. CONCLUSIONS: The data suggest that G-463A polymorphism does not seem to contribute to either MPO-ANCA- or PR3-ANCA-associated vasculitis formation.     

Elevated soluble Flt1 inhibits endothelial repair in PR3-ANCA-associated vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis exhibits endothelial damage, but the capacity for vessel repair in this disorder is not well understood. Here, we observed a marked increase in serum levels of soluble Flt1 (sFlt1), a potent inhibitor of vascular endothelial growth factor, in patients with active ANCA-associated vasculitis compared with patients during remission and other controls. Serum levels of sFlt1 correlated with C5a, an anaphylatoxin released after complement activation. Serum from patients with acute ANCA-associated vasculitis disrupted blood flow in the chicken chorioallantoic membrane assay, suggesting an antiangiogenic effect. Preincubation with excess human vascular endothelial growth factor prevented this effect. Anti-proteinase-3 (PR3) mAb and serum containing PR3-ANCA from patients with active vasculitis both induced a significant and sustained release of sFlt1 from monocytes, whereas anti-myeloperoxidase (MPO) mAb or polyclonal antibodies did not. However, the serum containing polyclonal PR3-ANCA did not induce release of sFlt1 from cultured human umbilical vein endothelial cells. In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair.     

Antineutrophil cytoplasmic antibodies to proteinase 3 in Wegener's granulomatosis: epitope analysis using synthetic peptides

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity nor to functional effects of these antibodies in vitro, suggesting differences in epitope specificity. To define relevant epitopes for PR3-ANCA, sera of WG patients were analyzed on their reactivity to linear peptides of PR3. METHODS: Fifty linear peptides of 15 amino acids in length with an overlap of 10 aa spanning the entire PR3 sequence were synthesized. Sera of 27 WG patients with active disease and 27 age- and sex-matched healthy controls, eight anti-PR3 monoclonal antibodies (mAbs), and a rabbit anti-PR3 serum were tested by enzyme-linked immunosorbent assay for reactivity to PR3 peptides. RESULTS: Rabbit anti-PR3 serum recognized three distinct peptide areas, whereas none of the anti-PR3 mAbs bound PR3 peptides. Sera of both WG patients and healthy controls recognized a restricted number of PR3 peptides. Four of these peptide areas were recognized significantly more strongly by WG sera than by control sera. Sera drawn at the initial presentation of WG mainly recognized these peptides. Two of the recognized peptide areas were located near the active center of PR3. CONCLUSION: A restricted number of epitope areas of PR3 are recognized both by WG patient sera and control sera. Four peptide areas were bound stronger by sera of WG patients at initial presentation than by healthy controls.     

Renal survival and prognostic factors in patients with PR3-ANCA associated vasculitis with renal involvement

BACKGROUND: Severe renal disease is a feature of anti-neutrophil cytoplasmic antibodies (ANCA)-associated small-vessel vasculitis. We evaluated patient and renal survival and prognostic factors in patients with PR3-ANCA associated vasculitis with renal involvement at diagnosis during long-term follow-up. METHODS: Eighty-five patients were diagnosed between 1982 and 1996 and followed until 2001 allowing >or=5 years of follow-up. All patients were treated with prednisolone and cyclophosphamide. Univariate and multivariate analyses with patient and renal survival as dependent variables were performed. RESULTS: Of the 85 patients in this study, 17 (20%) died within one year after diagnosis. Of the 25 patients (29%) who were dialysis dependent at diagnosis, two remained dependent and two again became dialysis dependent after less than one year; nine died early without renal recovery. Risk factors for death occurring within one year in univariate analysis (RR, 95% CI) were age>65 years (6.5, 1.6-13.7) and dialysis dependency at diagnosis (3.6, 1.0-13). Twenty patients died beyond one year during the long-term follow-up. Male gender (4.7, 1.6-10) and developing dialysis dependency during follow-up (4.1, 1.4-12) were associated with poor outcome. Risk factor for renal failure within one year was dialysis dependency at diagnosis (29, 3.6-229). Of 64 patients dialysis independent one year after diagnosis, 12 patients became dialysis dependent during follow-up. A renal relapse was strongly associated with development of renal failure in long-term follow-up (17, 3.5-81). CONCLUSIONS: Early death and failure to recover renal function in PR3-ANCA associated vasculitis is associated with age> 65 years and dialysis dependency at diagnosis. Long-term renal survival is determined by renal relapses during follow-up only. Slow, progressive renal failure without relapses is rarely observed in this group.     

Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3-ANCA-associated vasculitis

Wegener granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA) with specificity for proteinase 3 (PR3). Relapses of WG are frequently preceded by a rise of autoantibody titer and PR3-ANCA are able to activate primed neutrophils in vitro. Except being stored intracellularly and translocated to the cell surface upon neutrophil stimulation, PR3 can also be detected on the surface of non-stimulated neutrophils (membrane PR3 or mPR3), with an interindividual variability in percentages of mPR3(-)-positive cells and level of mPR3 expression. This study began with the hypothesis that the presence of PR3 on the surface of non-stimulated neutrophils enables interaction with PR3-ANCA and influences clinical manifestations of the disease. It analyzed mPR3 expression on neutrophils of 89 WG patients in complete remission and 72 healthy controls to evaluate whether the presence of PR3 on the surface of resting neutrophils is related to clinical manifestations of WG and/or to the susceptibility to develop relapses. The number of patients with a bimodal mPR3 expression on resting neutrophils did not differ between patients and controls. However, in WG patients, an increased percentage of mPR3(+) neutrophils and an elevated level of mPR3 expression compared with healthy individuals (P = 0.037) were found. Within the group of WG patients, an elevated level of mPR3 expression was significantly associated with an increased risk for relapse (P = 0.021) and with an increased relapse rate (P = 0.011), but not with the disease extent or particular manifestations at diagnosis or at relapse. These data support the hypothesis that PR3 expression on the membrane of neutrophils plays a role in the pathophysiology of PR3-ANCA associated vasculitis.     

Serial ANCA titers: useful tool for prevention of relapses in ANCA-associated vasculitis

BACKGROUND: The value of measuring serial antineutrophil cytoplasmic autoantibody (ANCA) titers in guiding therapy among patients with ANCA-associated vasculitis is controversial. METHODS: We measured serial titers of proteinase 3 (PR3)- and myeloperoxidase (MPO)-ANCA by antigen-specific enzyme-linked immunosorbent assays (ELISAs) in 48 patients with ANCA-associated vasculitis who were followed up during remission at the Massachusetts General Hospital from 1990 through 2000 (mean follow-up, 46.2 months). We retrospectively assessed disease activity by Birmingham Vasculitis Activity Score (BVAS). RESULTS: We found 21 episodes of fourfold or greater ANCA titer rises in 17 patients who were in complete remission (BVAS=0). Among eight patients who had 10 such titer rises and were not given increased immunosuppression, (group I), all suffered relapses after each episode (mean interval, 5.8 months), whereas among 11 patients, each with one titer rise, who received preemptive increased immunosuppression, (group II), only two relapses occurred, at 3 and 6 months. The difference in the cumulative incidence of relapses in a 1-year period between the two groups was 82% (P=0.0002). Changes in ANCA titers were also used to help guide therapy in the other 31 patients in the study; patients with slight titer rises often received incremental increases in immunosuppression, whereas those with falling titers received incremental decreases. The overall outcome in the entire group was favorable; 46 patients were alive at the end of the study; two died of unrelated diseases. CONCLUSION: Serial measurements of PR3- and MPO-ANCA titers in patients with ANCA-associated vasculitis during remission can help predict relapses, and preemptive increases in immunosuppression following fourfold titer rises reduces the risk of relapses. Moreover, adjustment of immunosuppression based on lesser titer changes appears to result in a favorable outcome.     

Monocyte activation and relationship to anti-proteinase 3 in acute vasculitis.

BACKGROUND: Monocytes have been suggested to play a role in antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis, but their state of activation in vivo in patients is still unknown. METHODS: Twelve consecutive patients with acute anti-proteinase 3 (PR3)-positive vasculitis were included prospectively, and blood samples were drawn at diagnosis. As controls, peripheral blood was obtained from a group of patients with acute infection (n = 12) and from healthy controls (n = 12). Monocyte activation was estimated from the expression of adhesion molecules (CD62L and CD11b), production of oxygen radicals and serum concentrations of soluble inflammation markers and adhesion molecules [intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1)]. RESULTS: Compared with monocytes from healthy subjects, monocytes from patients with acute vasculitis expressed upregulated CD11b (P < 0.05) but had reduced production of oxygen radicals (P < 0.01). A high concentration of anti-PR3 correlated with decreased expression of CD62L (r = 0.71, P = 0.009) and increased expression of CD11b (r = 0.63, P = 0.02). The serum concentrations of soluble inflammation markers [soluble CD14, interleukin (IL)-6, tumour necrosis factor receptor 1 (TNFR1), IL-10 and IL-8] as well as soluble adhesion molecules (sVCAM-1 and sICAM-1) were increased. Monocytes in patients with acute vasculitis displayed a reduced production of oxygen free radicals (P < 0.01) but similar serum concentrations of soluble inflammation markers and adhesion molecules, as compared with the control group of patients with acute infection and negative PR3-ANCA. CONCLUSIONS: High anti-PR3 concentration in patients with acute vasculitis correlated with an activated adhesion molecule phenotype (CD62L(low)/CD11b(high)) in circulating monocytes, indicating a potential pathophysiological role for anti-PR3. An impaired production of oxygen radicals in monocytes in patients with vasculitis compared with those with acute infection may mirror the longer time interval from onset of first symptoms to admission, in patients with vasculitis.     

A Japanese case of proteinase 3 antineutrophil cytoplasmic autoantibody-associated pauci-immune-type crescentic glomerulonephritis without valvular endocarditis

A 74-year-old male without recent medical treatment visited our hospital complaining of fever and lack of appetite. Upon examination severe azotemia, proteinuria, and urinary occult blood were noted, and the patient was admitted. Results of a blood test showed that his proteinase 3 antineutrophil cytoplasmic autoantibody (PR3-ANCA) level was high. A transthoracic echocardiogram indicated normal cardiac function and no valvular regurgitation or stenosis. Necrotizing glomerulonephritis accompanied by cellular crescentic bodies, but not granuloma, was noted on renal biopsy. An immunofluorescence study demonstrated no immunofluorescence staining in the glomerulus or in the tubulointerstitial or vascular compartments. No lesion was present in the lung or upper respiratory tract. The patient was diagnosed with PR3-ANCA-associated pauci-immune-type crescentic glomerulonephritis and treated with steroids. This treatment resulted in rapid normalization of C-reactive protein, and the PR3-ANCA level slowly decreased and converted to negative. The renal function, however, did not improve, and maintenance dialysis was introduced. No pulmonary or upper airway lesion has developed during 18?months of follow-up. PR3-ANCA-positive crescentic glomerulonephritis accompanied by valvular endocarditis has been described by several reports in Japan; however, this case was not complicated by valvular endocarditis. To our knowledge, this is the 4th case report describing PR3-ANCA-associated crescentic glomerulonephritis in Japan.     

Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA

BACKGROUND: Autoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes. METHODS: This study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA. RESULTS: The rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA. CONCLUSIONS: This expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely.     

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 proteinase 3-anti-neutrophil cytoplasmic antibody (ANCA) level measured by the capture enzyme-linked immunosorbent assay method is associated with decreased patient survival in ANCA-associated vasculitis with renal involvement

Wegener granulomatosis (WG) and microscopic polyangiitis (MP), diseases associated with antineutrophil cytoplasmic antibodies (ANCA), had an extremely poor prognosis before the introduction of cyclophosphamide and corticosteroids for their treatment. However, there is still reduced patient survival, and some studies have documented severe side effects of the immunosuppressants used. This 10-yr follow-up study assessed 117 consecutive patients with WG or MP with biopsy-confirmed renal involvement. The cumulative relative patient survival was lower: 0.664 for women and 0.648 for men. The causes of death (n = 64) were in most cases registered as associated with the vasculitic disease. Analysis of possible predictive factors for patient survival by multiple Cox regression analysis revealed that a very high level of proteinase 3 (PR3)-ANCA measured by the capture ELISA method, a diagnosis of MP, and older age were factors predicting poorer patient survival. High levels of B-thrombocytes at time of diagnosis were associated with a better prognosis. For patients surviving the first year, remission-sustaining therapy with azathioprine for longer than 12 mo was associated with improved patient survival. Thirty-nine patients developed end-stage renal failure. Elevated serum creatinine at time of diagnosis and a very high level of PR3-ANCA by capture ELISA were factors predicting a higher risk for renal failure during follow-up. The epitope on PR3 assessed by capture ELISA needs to be further analyzed and explored: it seemed to implicate poorer patient and renal survival in WG or MP with renal involvement.     

抗髓过氧化物酶抗体阳性的韦格纳肉芽肿病的特点

目的 分析抗髓过氧化物酶(MPO)抗体阳性的韦格纳肉芽肿病(WG)患者的临床病理特点及其与传统的抗蛋白酶3(PR3)抗体阳性者之间的差异&#65377;方法 89例WG患者经本院肾内科确诊,分析其临床病理资料并对比抗MPO抗体阳性和抗PR3抗体阳性的两组患者之间的差异&#65377;结果 89例患者中54例抗MPO抗体阳性,34例抗PR3抗体阳性&#65377;抗MPO抗体阳性患者中男性所占的比例显著低于抗PR3抗体阳性者(男:女分别为23:31与24:10, P<0.05)&#65377;抗MPO抗体阳性的WG临床亦呈多器官受累的表现,其中关节痛&#65380;皮疹&#65380;眼&#65380;耳受累的发生率显著低于抗PR3抗体阳性者(分别为46.3%比70.6%,P < 0.05; 20.4%比44.1%,P < 0.05;27.8%比58.8%,P < 0.01和40.7%比67.6%,P < 0.05);伯明翰血管炎活动度积分(BVAS)显著低于抗PR3抗体阳性者(22.2±6.2比24.7±6.9, P < 0.05);而在确诊时Scr增高的发生率则显著高于抗PR3抗体阳性者(81.5%比61.8%, P < 0.05)&#65377;结论 在国人的WG患者中,抗MPO抗体阳性者可能不占少数,它与抗PR3抗体阳性者临床表现有所不同&#65377;     

Antiproteinase 3- and antimyeloperoxidase-associated vasculitis

Antiproteinase 3- and antimyeloperoxidase-associated vasculitis. Wegener's granulomatosis, microscopic polyangiitis, and idiopathic pauci-immune necrotizing crescentic glomerulonephritis (NCGN) are strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against either proteinase 3 (anti-PR3) or myeloperoxidase (anti-MPO). This has led some investigators to prefer combining these diseases under the common heading of ANCA-associated vasculitides. However, it is increasingly recognized that there are characteristic differences between patients with anti-PR3 and those with anti-MPO-associated vasculitis. This review focuses on the clinical, histopathologic, and possibly pathophysiologic differences between anti-PR3- and anti-MPO-associated vasculitis. Although there is considerable overlap, the anti-PR3- and anti-MPO-associated vasculitides are each characterized by particular clinical and histopathological findings. Extrarenal organ manifestations and respiratory tract granulomas occur more frequently in patients with anti-PR3 than in those with anti-MPO. Anti-PR3-positive patients with NCGN generally have a more dramatic deterioration of their renal function compared with anti-MPO-positive patients. The term "ANCA-associated vasculitis" is considered as a useful concept in the presence of systemic vasculitis. Likewise, in the presence of vasculitis, the terms "anti-PR3-associated vasculitis" and "anti-MPO-associated vasculitis" are useful concepts.     

人重组蛋白酶3的cDNA克隆化及其在真核细胞的表达

目的：应用真核表达系统表达人重组蛋白酶3（PR3）,并用于检测患者血清中的抗蛋白酶3抗体（anti-PR3),方法 利用聚合酶链反应（PCR）技术获得蛋白酶3的cDNA,并克隆至真核表达载体pcDI,将其瞬时转染至COS－7细胞,应用夹心ELISA不检测细胞上清中重组蛋白酶3（rPR3)的表达,并以表达上清作为抗原检测患者血清中的anti-PR3,同时转染CHO细胞,建立CHO／PR3稳定表达株,结果,转染后的COS－7细胞和CHO细胞均可以表达rPR3,且COS－7细胞表达的rPR3可以被多数anti-PR3阳性患者血清识别。结论：用真核细胞表达的rPR3具有抗原活性,为重组PR3替代天然PR3用于临床检测和实验研究提供了新的途径。     

A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis.

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.     

Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils

BACKGROUND: Recently, the in vivo pathogenic role of anti-neutrophil cytoplasm autoantibodies (ANCA) in ANCA-associated vasculitis has been challenged by Abdel-Salam et al. In their report, they observed that ANCA directed against proteinase 3 (PR3) cannot bind to their target autoantigen PR3 on circulating neutrophils (PMN). Here we present evidence that human PR3-ANCA do specifically bind to PMN that express PR3 on their membrane. METHODS: PMN were isolated from donors showing bimodal membrane PR3 expression on their PMN (N= 3). TNFalpha-primed PMN or PMA-stimulated PMN were incubated with serum or plasma from PR3-ANCA-positive patients with Wegener's granulomatosis (WG) (N= 8) or healthy controls (N= 8). Binding of IgG in serum or plasma samples to PMN was assessed by indirect immunofluorescence. RESULTS: Binding of IgG in undiluted plasma or serum from PR3-ANCA-positive WG-patients to PMN was significantly increased compared to plasma or serum from healthy controls. Dilution of plasma and serum showed concentration-dependent binding of IgG. Double staining for PR3 and IgG demonstrated that IgG in plasma or serum from PR3-ANCA-positive patients only bound to those PMN that expressed PR3, and not to PMN that lacked PR3 expression on their membrane. CONCLUSION: PR3-ANCA in undiluted serum or plasma from PR3-ANCA-positive WG patients bind to TNFalpha- primed and PMA-stimulated PMN that express PR3 on their membrane. Therefore, the hypothesis that PR3-ANCA can bind and activate primed PMN is still the most attractive explanation for the contribution of PR3-ANCA to the pathogenesis of Wegener's granulomatosis.     

Renal expression of ICAM-1 and VCAM-1 in ANCA-associated glomerulonephritis--are there differences among serologic subgroups?

BACKGROUND: Antineutrophil cytoplasmic autoantibodies (ANCA) play a role in the expression of adhesion molecules. Differences in ANCA test results in ANCA-associated vasculitis may provide differences in their renal expression. PATIENTS AND METHODS: We assessed the renal expression of ICAM-1 and VCAM-1 with monoclonal antibodies in 19 patients with ANCA-vasculitis: 7 microscopic polyangiitis, 5 Wegener's granulomatosis, 4 renal-limited vasculitis and 3 Churg-Strauss disease. Immunofluorescence and ELISA for myeloperoxidase (MPO) and proteinase 3 (PR3) were performed for ANCA-testing. 10 normal renal tissues were used as controls. RESULTS: The ANCA staining pattern was perinuclear in 14 patients, with MPO-ANCA 31 - 220 EU/ml, and cytoplasmic in 5, with PR3-ANCA 37 - 144 EU/ml. Abnormal tubular expression of ICAM-1 and VCAM-1 was seen in more than 80% of biopsies and abnormal expression of VCAM-1 in glomerular tuft was seen in 60%. Glomerular tuft stains of ++ or +++ for VCAM-1 were observed in 10% of renal biopsies from MPO-ANCA-GN patients, but in 60% of biopsies from PR3-ANCA-GN patients (Fi = 8.538, p = 0.03). IN CONCLUSION: De novo expression of VCAM-1 on glomerular tuft suggests that the endothelial cells play a role in ANCA-GN. De novo glomerular expression of VCAM-1 is associated more with ANCA directed against PR3 than with ANCA directed against MPO. Upregulated glomerular expression of VCAM-1 may reflect a higher histological activity in patients with PR3-ANCA, and supports the existence of specific immune activation mechanisms in the different serologic subgroups in ANCA-GN. The de novo tubular expression of ICAM-1 and VCAM-1 suggests that the epithelial cells may participate in adhesive interactions in ANCA-GN.     

Clinical characteristics and prognosis of ANCA-associated vasculitis in patients with renal injury

Objective To investigate the characteristics and outcome of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in patients with renal injury. Methods AAV patients with renal injury diagnosed in the Department of Nephrology, Renmin Hospital of Wuhan University, from January 2012 to January 2017 were included into this study. Patients were divided into MPO-ANCA positive and PR3-ANCA positive groups for further study. The clinical characteristics, pathological and laboratory indexes, treatment and prognosis were retrospectively analyzed. Results A total of 68 cases were enrolled, among which 52 cases (76.5%) were MPO-ANCA positive and 16 cases (23.5%) were PR3-ANCA positive, and 41 patients (60.3%) were over 65 years old. The incidences of interstitial lung disease, digestive and nervous system damage in PR3-ANCA positive group were significantly higher than those MPO-ANCA positive group (P＜0.05). There were significant differences of hemoglobin, complement C3, complement C1q, IgE, 24 h urinary protein, erythrocyte sedimentation rate, procalcitonin, BVAS score and eGFR in two groups (P＜0.05). 19 cases had done renal biopsy,among them 14 cases were MPO-ANCA positive and 5 cases were PR3-ANCA positive. Incidence of crescentic necrotizing glomerulonephritis in PR3-ANCA positive group was significantly higher than that in MPO-ANCA positive group, and incidence of diffuse global glomerulosclerosis in MPO-ANCA positive group was significantly higher than that in PR3-ANCA positive group (all P＜0.05). At the median follow-up time of 32 months, the relapse rate at 6 month of MPO-ANCA-positive and PR3-ANCA-positive patients were 46.2% and 75.0%, respectively (P＜0.05). Multivariate logistic regression analysis showed that PR3-ANCA positive, age≥65 years old, baseline eGFR＜30 ml?min-1?(1.73 m2)-1, and combined with pulmonary interstitial lesions were all independent risk factors for relapse. And the incidence of ESRD were 42.3% and 75.0% during the follow-up period and 10 patients (14.7%) died. COX regression analysis showed that patients older than 65 years old, BVAS score≥18 points, eGFR＜30 ml?min-1?(1.73 m2)-1 and complicated with pulmonary interstitial disorders at the onset were independent risk factors causing ESRD or death. Conclusion The PR3-ANCA-positive patients had more severe renal injury than those with MPO-ANCA-positive patients, and the injury of extrarenal organs was more serious, recurrence rate was higher, and the prognosis was worse.     

PR3-ANCA-positive crescentic necrotizing glomerulonephritis accompanied by isolated pulmonic valve infective endocarditis, with reference to previous reports of renal pathology

Patients with infective endocarditis (IE) often have renal complications which may include infarcts, abscesses and glomerulonephritis (GN). Furthermore, it is generally accepted that there is an association between IE and anti-neutrophil cytoplasmic antibody (ANCA). Here, we report the case of a 24-year-old man who developed rapidly progressive GN in the course of IE due to infection with alpha-streptococcus. The initial clinical manifestation of the condition was severe sacroiliitis without fever. Sandwich ELISA showed that the patient was positive for PR3-ANCA at low titer, and the classical complement pathway was also activated. Renal biopsy demonstrated several lesions: focal embolic GN, GN with immune deposits and focal and segmental crescentic necrotizing GN. Treatment with antibiotics and steroids led to eradication of the infection, and resolution of the renal disease was accompanied by immediate disappearance of PR3-ANCA and hypocomplementemia. During a 4-year follow-up period, no recurrence was observed. There have only been 7 case reports of GN associated with IE and PR3-ANCA in which the renal pathology has been described, and the current report is the first to document renal pathology in a patient with isolated pulmonic valve IE and PR3-ANCA. Moreover, this report is the first to show a change in renal biopsy findings in response to treatment. A review of the 7 literature cases and that of our patient showed that none involved pauci-immune GN. Hence, further studies are needed to clarify the prevalence of pauci-immune GN in ANCA-positive IE patients.     

Antibodies with dual reactivity to plasminogen and complementary PR3 in PR3-ANCA vasculitis

Patients with inflammatory vascular disease caused by anti-neutrophil cytoplasmic autoantibodies (ANCA) can harbor antibodies not only to the autoantigen proteinase 3 (PR3) but also to complementary PR3 (cPR3(105-201)), a recombinant protein translated from the antisense strand of PR3 cDNA. The purpose of this study was to identify potential endogenous targets of anti-cPR3(105-201) antibodies. Patients' plasmapheresis material was tested for the presence of antigens reactive with affinity-purified rabbit and chicken anti-cPR3(105-201) polyclonal antibodies. Antigen-containing fractions were tested with patients' anti-cPR3(105-201) affinity-purified IgG, and putative protein targets were sequenced by mass spectrometry. Unexpectedly, plasminogen was identified as a target of anti-cPR3(105-201). Reactivity of affinity-purified antibodies from two patients was lost when plasminogen was converted to plasmin, indicating restricted specificity. Antiplasminogen antibodies from five patients bound plasminogen at a surface-exposed loop structure within the protease domain. This loop contains an amino acid motif that is also found in a portion of recombinant cPR3(105-201); site-directed mutagenesis of this sequence decreased antibody reactivity by 30%. Functionally, antiplasminogen antibodies delayed the conversion of plasminogen to plasmin and increased the dissolution time of fibrin clots. Serologically, antiplasminogen antibody levels were higher in PR3-ANCA patients (n = 72) than healthy control subjects (n = 63), myeloperoxidase-ANCA patients (n = 34), and patients with idiopathic thrombosis (n = 57; P = 0.001). Of the patients with PR3-ANCA, nine had documented deep venous thrombosis events, five of whom were positive for antiplasminogen antibodies. In summary, capitalizing on interactions with complementary proteins, specifically complementary PR3, this study identified plasminogen as a previously undescribed autoantigen in PR3-ANCA vasculitis.