Detection rate and antigenic specificities of antineutrophil cytoplasmic antibodies in chinese patients with clinically suspected vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.     

狼疮性肾炎中抗中性粒细胞胞浆抗体及其靶抗原的分析

目的通过对于抗中性粒细胞胞浆抗体(ANCA)在活动期狼疮性肾炎(LN)患者中的发生率及其靶抗原的分析,探讨ANCA在LN发病机制中所起的作用。方法通过间接免疫荧光法(IIF)及酶联免疫吸附分析法(ELISA)检测33例处于活动期的LN患者和20例正常对照血清ANCA,并分析ANCA与LN临床表现及其他实验室检查结果之间的关系。结果用IIF方法检测时,ANCA在LN的阳性率为45.4%,全部是核周型ANCA(p-ANCA)。用ELISA方法检测靶抗原,乳铁蛋白(LF)为主要的靶抗原,占80.0%,且其荧光模型全为甲醛敏感型。其余靶抗原为髓过氧化物酶(MPO),占20.0%,其荧光模型为甲醛抵抗型。而正常对照血清ANCA及其靶抗原全部阴性。ANCA阳性组中LN患者平均年龄为24.3岁±4.5岁,而ANCA阴性组中LN患者平均年龄为31.9岁±9.3岁,ANCA阳性组合并皮肤血管炎、病情活动及抗ds-DNA抗体的阳性率均显著高于ANCA阴性组,具有统计学差异。但2组间病理分型未见显著差异。结论 ANCA可能参与了LN的发病过程,且与LN的进展有关。     

Detection Rate and Antigenic Specificities of Antineutrophil Cytoplasmic Antibodies in Chinese Patients with Clinically Suspected Vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.     

Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

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.     

Antigen specificities and clinical distribution of ANCA in kidney diseases

Summary The antigenic specificity and clinical distribution of the antineutrophil cytoplasmic antibodies (ANCA) in kidney diseases have recently been extensively studied. In patients with systemic vasculitis, the great predominance of two major ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO), is now established. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation, and thus are able to interact with autoantibodies after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, it has been shown that PR3 is identical to myeloblastin, which has been described independently and is involved in the control of growth and differentiation of leukemic cells. Aside from the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been identified, including human leukocyte elastase, lactoferrin, CAP57, and cathepsin G. These rare ANCA specificities occur in a limited number of patients. The variety of ANCA antigen specificities contrasts, however, with the fact that the vast majority of ANCA-positive sera are monospecific for one single ANCA antigen.With regard to clinical distribution, ANCA have major diagnostic significance in the four conditions in which they are frequently detected: Wegener's granulomatosis (WG), Churg and Strauss Syndrome (CSS), microscopic periarteritis (MPA), and necrotic and crescentic glomerulonephritis (NCGN). However, the initial dichotomy between MPO-associated vasculitis (NCGN, MPA) and that associated with anti-PR3 antibodies (WG) appears far from absolute.Abbreviations ANCA antineutrophil cytoplasm antibodies - PR3 proteinase 3 - MPO myeloperoxidase - CAP57 cationic antimicrobial protein 57 kDa - WG Wegener's granulomatosis - CSS Churg and Strauss syndrome - MPA microscopic periarteritis - NCGN necrotic and crescentic glomerulonephritis - IIF indirect immunofluorescence - HLE human leukocyte elastase - GBM glomerular basement membrane - IgAN IgA nephropathy - HSP Henoch-Schönlein purpura Preprint of a lecture to be read at the 22nd Congress of the Gesellschaft für Nephrologie, Heidelberg, September 15–18, 1991 (Editor: Prof. Dr. E. Ritz, Heidelberg)     

Anti-neutrophil cytoplasmic antibodies in echinococcus granulosus hydatid disease

The authors studied the presence of ANCA, evaluated by indirect immunofluorescence (IIF) and ELISA for anti-lactoferrin (LF), and anti-myeloperoxidase antibodies (anti-MPO), in sera of 69 patients with cystic echinococcosis (CE). According to Caremani's classification, 27 patients were considered to have active cysts and 42 patients were considered to have inactive cysts. ANCA were detected in 9 out of 27 patients (33.3%) with active cysts and in 3 out of 42 patients (7.1%) with inactive cysts. Differences between the two groups were statistically significant (P < 0.05). Anti-LF antibodies were found in seven patients (10.14%) and anti-MPO antibodies in ten patients (14.5%).     

Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA)

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjögren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P= 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P= 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P< 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.     

Antibodies to proteinase 3 prime human oral, lung, and kidney epithelial cells to secrete proinflammatory cytokines upon stimulation with agonists to various Toll-like receptors, NOD1, and NOD2

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies, the detection of which in serum can be used in the diagnosis of Wegener's granulomatosis (WG). Proteinase 3 (PR3) is a major target antigen of ANCA in WG patients, and the interaction of PR3 ANCA with leukocytes causes a debilitating autoimmune disease. The first signs and symptoms in WG patients are observed in the oral cavity, lungs, and kidneys. Human epithelial cells generally do not secrete proinflammatory cytokines upon stimulation with pathogen-associated molecular patterns (PAMPs). In this study, anti-PR3 antibodies (Abs) and PR3 ANCA-containing sera from WG patients endowed human oral, lung, and kidney epithelial cells with responsiveness to PAMPs in terms of the production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor alpha. Protease-activated receptor-2 (PAR-2) agonist peptides mimicked the priming effects of PR3 ANCA against PAMPs. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeting PAR-2 and NF-kappaB. This is the first report of priming effects of anti-PR3 Abs (PR3 ANCA) on epithelial cells. The results suggest that anti-PR3 Abs (PR3 ANCA) prime human epithelial cells to produce cytokines upon stimulation with various PAMPs, and these mechanisms may be involved in severe chronic inflammation in WG.     

Alpha 1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.     

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.     

Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA)

BACKGROUND: The "International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)" requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time. AIMS: To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA). METHODS: Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO). RESULTS: IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears. CONCLUSIONS: Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.     

The value of anti-neutrophil cytoplasmic antibodies (ANCA) testing for the diagnosis of ANCA-associated vasculitis,a systematic review and meta-analysis

The testing of anti-neutrophil cytoplasmic antibodies (ANCA) takes an important place in the diagnostic workup to ANCA-associated vasculitis (AAV). Nowadays, it is recommended to screen for the presence of PR3 and MPO specific antibodies first using immunoassay, without the need for ANCA measurement by indirect immunofluorescence (IIF). A literature search was performed to assess the diagnostic test value of ANCA IIF and PR3- and MPO-antibody immunoassay to diagnose AAV. This meta-analysis shows that the c-ANCA testing by IIF has a pooled sensitivity of 75.2% and a pooled specificity of 98.4%. For PR3-antibody immunoassay, the pooled sensitivity depended on the immunoassay method used, and ranged from 79.8% to 86.6%, whereas the pooled specificity ranged from 96.8% to 98.3%. For both p-ANCA IIF and MPO-antibody immunoassay (all methods) sensitivity varied considerably showing pooled values of respectively 46.3% and 58.1%, whereas respective pooled specificity was 91.4% and 95.6%. These findings support the 2017 international consensus that primary anti-PR3 and anti-MPO screening by immunoassay, based on superior immunoassay sensitivity without the need for IIF ANCA testing, improves the diagnostic workup of AAV.     

Audit of the clinical usefulness of a rapid qualitative ELISA screen for antimyeloperoxidase and antiproteinase 3 antibodies in the assessment of patients with suspected vasculitis

BACKGROUND: The need for urgent antineutrophil cytoplasmic antibody (ANCA) results when assessing patients with acute renal failure, pulmonary renal syndrome, or mononeuritis multiplex has led to the development of a rapid qualitative ELISA screening assay for antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3). AIMS: To report the use of a rapid qualitative ELISA screen for PR3-ANCA and MPO-ANCA in a regional immunology laboratory and its correlation with standard indirect immunofluorescence (IIF) and quantitative ELISA for PR3-ANCA and MPO-ANCA. METHODS: Over 12 months, 103 samples requiring urgent ANCA testing were screened by a rapid qualitative ELISA and the results compared with IIF and quantitative ELISA assays for PR3-ANCA and MPO-ANCA. RESULTS: There was an excellent correlation between the rapid qualitative ELISA and standard ANCA IIF and a routine ELISA for MPO/PR3-ANCA, with sensitivities ranging from 82% to 100%. There were two false negatives, which gave weak to moderately positive values as determined by routine ELISA. However, the clinical relevance of these two cases is doubtful. CONCLUSIONS: The rapid ELISA for anti-MPO and anti-PR3 correlates well with quantitative ELISA and IIF ANCA, and urgent management decisions in patients with suspected small vessel vasculitis can be based with confidence on this test.     

Anti-idiotypes against anti-neutrophil cytoplasmic antigen autoantibodies in normal human polyspecific IgG for therapeutic use and in the remission sera of patients with systemic vasculitis.

Anti-neutrophil cytoplasmic antigen (ANCA) activity was inhibited in 15 out of 21 sera from patients with acute systemic vasculitis following incubation with normal polyspecific IgG for therapeutic use (IVIg). ANCA antibodies reacted with IVIg through idiotypic-anti-idiotypic interactions, as shown in competitive binding assays using F(ab')2 fragments from IVIg and affinity chromatography of ANCA IgG on Sepharose-bound F(ab')2 fragments from IVIg. Co-incubation of sera from patients with acute systemic vasculitis with paired autologous remission stage sera also resulted in inhibition of ANCA activity in acute sera. Remission sera contain IgM and IgG capable of interacting with beta and or alpha idiotypes of ANCA IgG from acute sera. Anti-idiotypic IgM may account for the lack of expression of ANCA activity in whole serum from patients in remission from systemic vasculitis, which were found to contain high titres of ANCA IgG. These observations suggest that remission of systemic vasculitis is associated with the generation of anti-idiotypes against autoantibodies rather than the suppression of production of ANCA autoantibodies. IVIg may modulate the activity of systemic vasculitis in vivo.     

肾脏疾病患者血清中ANCA的荧光模式及其意义

目的：进一步探讨肾脏疾病患者血清中抗中性粒细胞细胞质抗体(ANCA)的荧光模式及其意义。方法：应用间接免疫荧光(IIF)和ELISA方法检测了251例肾脏疾病患者血清中ANCA及其靶抗原。结果：在251例肾脏疾病患者中，ANCA阳性51例占20．3％，其中10例为cANCA阳性(10／251)；30例为cANCA阳性(30／251);11例为aANCA阳性(11／251)。结论：同时用IIF法和ELISA法检测ANCA，可以提高ANCA检出的阳性率；不同ANCA荧光模式与疾病的种类病情及预后密切相关；提高对不典型ANCA和pANCA伴ANA荧光模式的鉴别，有助于临床对血管炎和其它自身免疫性疾病的诊断。     

Antineutrophil cytoplasmic antibodies induce decreased CD62L expression and enhanced metabolic activity in monocytes

Monocyte in vitro activation by antimyeloperoxidase (anti-MPO)- and antiproteinase-3 (anti-PR3)-positive sera, corresponding immunoglobulin G (IgG) fractions and monoclonal antibodies against MPO and PR3 was evaluated. The expression of adhesion molecules, l-selectin (CD62L) and CR3 (CD11b), involved in leucocyte endothelial adhesion, and metabolic activity, measured as the production of hydrogen peroxide, were analysed. Decreased expression of CD62L was demonstrated in monocytes after incubation with antineutrophil cytoplasmic antibody (ANCA)-positive sera. This finding was not accompanied by changes in CD11b expression. Metabolic activity was increased in monocytes after incubation with ANCA-positive IgG fractions as well as after incubation with monoclonal anti-MPO and anti-PR3. These findings support the concept that the pathophysiological effect of ANCA is partly mediated through the action on crucial events in monocyte activation, such as CD62L downregulation and oxygen radical production.     

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA)

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.     

Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55.6%), 276/516 (53.3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15.8%, anti-ssDNA in 15.6%, anti-dsDNA in 8.5%, aCL in 5%, anti-SS-B/La in 4.1%, anti-SS-A/Ro (60 kD) in 3.9%, anti-E2 in 3.3% and anti-SSA/Ro (52 kD) in 1.2%, anti-MPO in 4.8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and alpha-Fetoprotein (alpha-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated alpha-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-alpha). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.