Comparison of anti-PR3 capture and anti-PR3 direct ELISA for detection of antineutrophil cytoplasmic antibodies (ANCA) in long-term clinical follow-up of PR3-ANCA-associated vasculitis patients.

A total of 118 sera from 11 patients with anti-neutrophil cytoplasmic antibodies against proteinase-3- (PR3-ANCA) associated vasculitis were retrospectively screened by anti-PR3 capture and anti-PR3 direct ELISA tests. We studied the relationship between capture and direct ELISA scores and the clinical activity of PR3-ANCA-associated vasculitis patients during follow-up. We also studied the ability of the anti-PR3 capture ELISA to detect positive values of PR3-ANCA in clinical vasculitis relapses. Only capture ELISA presented a significant relationship (p < 0.05) with clinical activity of PR3-ANCA-associated vasculitis patients over time. Capture ELISA appears to be a reliable method for detecting clinical relapses in this group of patients. Our results indicate that the new capture ELISA test is more effective than direct ELISA in the follow-up of patients with PR3-ANCA-associated vasculitis and in the detection of relapses.     

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 histology in Chinese patients with anti-myeloperoxidase autoantibody-positive Wegener's granulomatosis.

BACKGROUND: Proteinase-3 antineutrophil cytoplasmic antibody (PR3-ANCA) was the serological marker for Wegener's granulomatosis (WG), while myeloperoxidase (MPO)-ANCA was the serological marker for microscopic polyangiitis (MPA). However, our previous study suggested that patients with MPO-ANCA positive WG were common in Chinese. This study aimed to analyse the renal histology of patients with MPO-ANCA positive WG. METHODS: Patients in our centre with WG were selected according to both the Chapel Hill Consensus Conference (CHCC) definition and American College of Rheumatology classification criteria. Patients with MPA were selected according to the CHCC definition. The renal histology was compared between patients with MPO-ANCA positive WG and with PR3-ANCA positive WG as well as patients with MPO-ANCA positive MPA. RESULTS: Sixty-one patients with WG had complete renal histological data, 39/61 with positive MPO-ANCA and 22/61 with positive PR3-ANCA. Among patients with crescents in glomeruli, those with MPO-ANCA had fewer cellular crescents and more fibrous crescents than those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Interstitial fibrosis and tubular atrophy were more prevalent and severe in patients with MPO-ANCA than in those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Compared with 44 patients with MPO-ANCA positive MPA, patients with MPO-ANCA positive WG had fewer glomeruli with crescents and more normal glomeruli (P < 0.01 and P < 0.01, respectively). CONCLUSION: Patients with MPO-ANCA positive WG are common in Chinese. In renal histology, chronic lesions were more severe and prevalent in patients with MPO-ANCA positive WG than in patients with PR3-ANCA positive WG. Glomerular lesions were less severe and less prevalent in patients with MPO-ANCA positive WG than in those with MPO-ANCA positive MPA.     

Characteristics of Chinese patients with Wegener's granulomatosis with anti-myeloperoxidase autoantibodies

BACKGROUND: Cytoplasmic antineutrophil cytoplasmic autoantibodies (cANCA)/proteinase-3(PR3)-ANCA was considered the serologic diagnostic marker for Wegener's granulomatosis (WG). However, Chinese patients with MPO-ANCA positive WG were frequently diagnosed. We now analyze the characteristics of patients with MPO-ANCA positive WG and investigate the difference between patients with MPO-ANCA and PR3-ANCA. METHODS: Patients with WG were selected according to both Chapel Hill Consensus Conference definition and American College of Rheumatology (ACR) classification criteria in 500 Chinese patients with ANCA-associated systemic vasculitides. The clinical manifestions were compared between patients with MPO-ANCA and with PR3-ANCA. RESULTS: Eight-nine patients fulfilled the diagnostic criteria of WG: 54/89(60.7%) were MPO-ANCA positive, 34/89(38.2%) were PR3-ANCA positive. Patients with MPO-ANCA were predominantly female compared with patients with PR3-ANCA. Patients with MPO-ANCA also had multisystem involvement. However, the prevalences of arthagia, skin rash, ophthalmic and ear involvement were significantly lower in patients with MPO-ANCA than those in patients with PR3-ANCA (46.3% vs. 70.6%, P < 0.05; 20.4% vs. 44.1%, P < 0.05; 27.8% vs. 58.8%, P < 0.01; 40.7% vs. 67.6%, P < 0.05, respectively). The prevalence of elevated initial serum creatinine was significantly higher in patients with MPO-ANCA than that in patients with PR3-ANCA (81.5% vs. 61.8%, chi(2) = 4.20, P < 0.05). CONCLUSION: Patients with MPO-ANCA positive WG were not rare in Chinese.     

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.     

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.     

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.     

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;     

Analysis of prostate specific antigen and alpha1-antichymotrypsin interaction using antipeptide monoclonal antibodies

PURPOSE: The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). MATERIALS AND METHODS: Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. RESULTS: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. CONCLUSIONS: The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.     

DRB1*15 allele is a risk factor for PR3-ANCA disease in African Americans

Anti-neutrophil cytoplasmic autoantibody (ANCA) disease rarely occurs in African Americans and risk factors for the disease in this population are unknown. Here, we genotyped MHC class II alleles and found that, among African Americans, those with proteinase 3-ANCA (PR3-ANCA) had 73.3-fold higher odds of having HLA-DRB1*15 alleles than community-based controls (OR 73.3; 95% CI 9.1 to 591). In addition, a disproportionate number of African American patients carried the DRB1*1501 allelic variant of Caucasian descent rather than the DRB1*1503 allelic variant of African descent. Among Caucasians, those with PR3-ANCA had 2.2-fold higher odds of carrying DRB1*1501 than controls (OR 2.2; 95% CI 1.2 to 4.0). A validation study supported by the Vasculitis Clinical Research Consortium confirmed the strong association between the DRB1*15 allele and PR3-ANCA disease, among African Americans. Furthermore, we found that DRB1*1501 protein binds with high affinity to amino acid sequences of sense-PR3, purportedly an antigenic epitope, and to the amino acid sequence complementary to this epitope in vitro. Peptides of sense-PR3 and complementary-PR3 also bound to TNF-α-induced surface expression of DRB1*1501 on peripheral neutrophils. Taken together, these data suggest HLA-DRB1*15 alleles contribute to the pathogenesis of PR3-ANCA disease.     

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.     

Renal histology in ANCA-associated vasculitis: differences between diagnostic and serologic subgroups.

BACKGROUND: Differences in renal histopathology between microscopic polyangiitis (MPA) and Wegener's granulomatosis (WG), and between anti-neutrophil cytoplasm autoantibody (ANCA) test results in patients with ANCA-associated vasculitis may provide insight into the differences in pathogenesis and raise the opportunity of classifying the vasculitides more accurately. The possible differences in histopathology are investigated in this study. METHODS: We report an analysis of 173 patients with renal disease in microscopic polyangiitis or Wegener's granulomatosis. A total of 173 renal biopsies, performed at diagnosis, were scored by two observers separately, using a previously standardized protocol. Consensus on each biopsy was achieved during a central review. RESULTS: Normal glomeruli were more common in WG than in MPA (P < 0.001). Glomerulosclerosis was more prominent in MPA than in WG (P=0.003). Interstitial fibrosis (P < 0.001), tubular atrophy (P < 0.001), and tubular casts (P=0.005) were more frequently present and more severe in MPA than in WG. Presence of glomerulosclerosis was more extensive in patients with myeloperoxidase (MPO)-ANCA than with proteinase 3 (PR3)-ANCA (P=0.022). Interstitial fibrosis (P=0.008), tubular necrosis (P=0.030), tubular atrophy (P=0.013), and intra-epithelial infiltrates (P=0.006) were more frequently present and more severe in MPO-ANCA than in PR3-ANCA. CONCLUSIONS: Glomerulonephritis in relation to MPA has more characteristics of chronic injury at the time of presentation than glomerulonephritis in relation to WG. This difference may be due to a delayed establishment of diagnosis in patients with MPA compared to patients with WG. Both active and chronic lesions are more abundantly present in MPO-ANCA-positive patients than in patients with PR3-ANCA-positivity, which suggests that the pathogenesis of renal disease in these ANCA subsets could be different. Our results also suggest that ANCA test results may be useful in classifying ANCA-associated vasculitides.     

ANCA-associated vasculitis with renal involvement: an outcome analysis.

BACKGROUND: The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a group of heterogeneous diseases. This study was undertaken to investigate the outcome of Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and renal-limited vasculitis (RLV). Furthermore, we analysed the differences in patients with proteinase 3-ANCA (PR3-ANCA) and those with myeloperoxidase-ANCA (MPO-ANCA), which have not been assessed in a homogeneously treated group of patients with renal involvement. METHODS: In this retrospective analysis, 80 patients with a new diagnosis of WG, MPA or RLV with biopsy-proven renal involvement were followed over a median of 46.7 months (range: 0.8-181.9 months). All patients had induction treatment with cyclophosphamide and oral corticosteroids. RESULTS: At the end of follow-up, 23% were dependent on dialysis. Renal survival was significantly worse in patients with WG compared with patients with MPA or RLV (P = 0.04). A higher rate of end-stage renal disease (ESRD) was noticed in PR3-ANCA- vs MPO-ANCA-positive patients. A total of 21 patients (26%) died. Predictors of patient mortality were development of ESRD, older age and the maximum creatinine in the first month. Mortality was found to be higher in patients with WG and was significantly higher in PR3-ANCA-positive cases (P = 0.02). The relative risk of death was 9.32 times higher in PR3-ANCA- vs MPO-ANCA-positive patients. CONCLUSIONS: Our data underscore the pathogenetic potential of ANCA by demonstrating a more aggressive disease state and a poorer outcome in patients with PR3-ANCA.     

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。     

Autoantibodies against myeloid lysosomal enzymes in crescentic glomerulonephritis

To investigate the possible association of crescentic glomerulonephritis (CGN) with autoantibodies to myeloid lysosomal enzymes, we tested sera from 35 consecutive patients with CGN without diagnostic immunohistological findings in their renal biopsy for the presence of antineutrophil cytoplasmic antibodies directed against a 29 kD antigen from azurophilic granules (29 kD-ANCA), against myeloperoxidase (MPO-ANCA) and against elastase (elastase-ANCA), using antigen-catching ELISAs with well-defined monoclonal antibodies. 29 kD-ANCA were present in the sera of all nine patients with CGN as part of biopsy-proven Wegner's granulomatosis (WG), of ten patients with CGN and clinically suspected WG, and of two patients with idiopathic CGN. Sera from the remaining patients with clinically suspected WG (N = 5) or idiopathic CGN (N = 6) were negative for 29 kD-ANCA, but invariably positive for MPO-ANCA. Neither of these antibodies could be detected in sera from patients with CGN of infectious origin (N = 3), different forms of CGN (N = 7), other renal lesions (N = 34), or normal controls (N = 52). None of the sera tested were positive for elastase-ANCA. Our results indicate that both vasculitis-associated CGN and idiopathic CGN are associated with autoantibodies against myeloid lysosomal enzymes. This finding places these disorders within one spectrum of diseases.     

Antigen and epitope specificity of anti-glomerular basement membrane antibodies in patients with goodpasture disease with or without anti-neutrophil cytoplasmic antibodies

Goodpasture disease (GP) is defined by the presence of anti-glomerular basement membrane (anti-GBM) antibodies and rapidly progressive glomerulonephritis. Besides anti-GBM, many patients with GP produce anti-neutrophil cytoplasmic antibodies (ANCA). For elucidation of the pathophysiologic significance of ANCA in this setting, epitope and antigen specificity of the anti-GBM antibodies and antigen specificity of ANCA were studied. Bovine testis alpha(IV)NC1 (tNC1); recombinant human alpha1, alpha3, alpha4, and alpha5(IV)NC1 (ralpha1 through ralpha5); and three chimeric proteins that contain previously defined epitope regions designated E(A), E(B), and S2 were used to examine the anti-GBM antibodies by ELISA in 205 Chinese patients with GP with or without ANCA. In the 205 anti-GBM antibody-positive sera, 63 (30.7%) were also ANCA positive (61 myeloperoxidase-ANCA and six proteinase 3-ANCA, four being triple positive). All 205 sera recognized tNC1 and ralpha3(IV)NC1. In the double-positive group, 54.0, 66.7, 71.4% of the sera could recognize ralpha1, ralpha4, and ralpha5, respectively, compared with 49.3, 60.6, and 55.6% for patients with anti-GBM antibodies alone. The levels of the antibodies to ralpha3, tNC1, and the alpha3/alpha1 ratio were lower in the double-positive group than that in patients with anti-GBM antibody alone (P < 0.05). Most of the sera could recognize the epitope regions E(A), E(B), and S2, but the absorbance values to E(A), E(B), and S2 were lower in double-positive group (P < 0.05). Double-positive patients had a broader spectrum of anti-GBM antibodies and lower levels of antibodies against alpha3(IV)NC1 compared with that of patients with anti-GBM antibodies alone.     

Antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with anti-GBM crescentic glomerulonephritis

OBJECTIVE: To study the prevalence of ANCA and their target antigen in Chinese patients with anti-GBM crescentic glomerulonephritis (CGN), and to evaluate the possible role of ANCA in Chinese anti-GBM CGN patients with coexisting serum ANCA by studying clinicopathologic features of this disease. MATERIAL AND METHODS: Twenty-three sera were collected from 23 renal biopsy-proven anti-GBM CGN patients. According to the standardized procedures, all of the sera were determined by both, indirect immunofluorescence (IIF) ANCA, and enzyme-linked immunosorbent assay (ELISA) MPO-ANCA, PR3-ANCA and BPI-ANCA. The patients were divided into two groups according to serum ANCA positivity (Group A) or negativity (Group B). Thirty-three ANCA-associated pauci-immune CGN patients were regarded as control group (Group C). Their clinicopathologic features were compared to reveal whether ANCA correlated with disease activity. RESULTS: There were 11 (47.8%) cases with positive serum ANCA in 23 anti-GBM glomerulonephritis patients. There were 4/11 MPO-ANCA (one with positive PR3-ANCA and C-ANCA, three with negative IIF-ANCA), 1/11 PR3-ANCA (with positive MPO-ANCA and C-ANCA), 3/11 P-ANCA (with negative ELISA-ANCA) and 5/11 C-ANCA (one with positive PR3-ANCA and MPO-ANCA, and the other four with negative ELISA-ANCA). No BPI-ANCA was detected. No different clinicopathologic features were found between Groups A and B. Both were different from Group C in age, sex ratio, frequence of anuria and ESRD, variety of crescents, glomerular sclerosis, vessel lesion and prognosis. CONCLUSION: Our data demonstrate that ANCA in Chinese patients with anti-GBM CGN is not rare. The major target antigen of ANCA is MPO. ANCA seems not to be correlated with disease activity.     

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.     

ANCA patients have T cells responsive to complementary PR-3 antigen

Some patients with proteinase 3 specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) also have antibodies that react to complementary-PR3 (cPR3), a protein encoded by the antisense RNA of the PR3 gene. To study whether patients with anti-cPR3 antibodies have cPR3-responsive memory T cells we selected conditions that allowed cultivation of memory cells but not na?ve cells. About half of the patients were found to have CD4+TH1 memory cells responsive to the cPR3(138-169)-peptide; while only a third of the patients had HI-PR3 protein responsive T cells. A significant number of T cells from patients responded to cPR3(138-169) peptide and to HI-PR3 protein by proliferation and/or secretion of IFN-gamma, compared to healthy controls while there was no response to scrambled peptide. Cells responsive to cPR3(138-169)-peptide were not detected in MPO-ANCA patients suggesting that this response is specific. The HLADRB1(*) 15 allele was significantly overrepresented in our patient group and is predicted to bind cPR3(138-169) peptide with high affinity. Regression analysis showed a significant likelihood that anti-cPR3 antibodies and cPR3-specific T cells coexist in individuals, consistent with an immunological history of encounter with a PR3-complementary protein. We suggest that the presence of cells reacting to potential complementary protein pairs might provide an alternative mechanism for auto-immune diseases.