ANCA in haemodialysis patients

The prevalence of positive ANCA as well as the prevalence ofPR-3 and MPO antibodies were examined in a cross-sectional sampleof 1277 haemodialysis patients from 16 German haemodialysiscentres. We found 32 patients positive for c-ANCA (median titre1:40; range 1:20–1:320) and 65 for p-ANCA (1:80; 1:20–1:1280).Twenty-two percent of the c-ANCA-positive and 31% of the p-ANCA-positivepatients had PR-3 and MPO antibodies by ELISA respectively.Clinical evidence of vasculitis was found in 11 of 32 c-ANCA-positiveand 19 of 65 p-ANCA-positive patients. Of the 11 c-ANCA-positive,four had a known diagnosis of Wegener's granulo-matosis (WG);WG was recognized after the test in a further five patientsand two had renal limited RPGN. Of the 19 p-ANCA-positive patients,three had a clinical diagnosis of microscopic polyarteritis(MP), MP was newly diagnosed in a further 12, WG in one andrenal limited RPGN in three. The patients had not received cyclophosphamide(the diagnosis had been non-specified ‘systemic disease’).Thus false-positive ANCA, as defined by absence of vasculitis,was found in 5% of dialysis patients versus 0% in patients withpreterminal renal failure {n=152) or blood donors (n=150). Patientswith vasculitis tended to have higher c-ANCA and p-ANCA titresrespectively, but there was a considerable overlap. Titres werenot higher in patients symptomatic at the time of examination(6 of 11 c-ANCA and 10 of 19 p-ANCA), but PR-3 and MPO ELISAwere positive in all but two. Thus c-ANCA were false positive(i.e. not associated with clinical vasculitis) in 66% and p-ANCAin 71%. In some patients ANCA was positive, but ELISA negative. We conclude that (i) in dialysis patients c-ANCA and p-ANCAare frequently present in the absence of vasculitis; (ii) specificitycan be improved, but sensitivity is lost, by using PR-3 andMPO ELISA respectively; (iii) serology permits the recognitionof WG or MP in cases where the diagnosis has been missed. Itis recommended that all patients with unknown primary renaldisease admitted to renal replacement therapy be examined usingboth ANCA-IF and PR-3/MPO ELISA.     

Does the presence of ANCA in patients with ulcerative colitis necessarily imply renal involvement?

BACKGROUND.: ANCA are thought to play a pathogenic role in renal vasculitis.ANCA may also be detected in patients with diseases not usuallyassociated with renal pathology, such as ulcerative colitis.Our study was conducted to determine if the presence of ANCAin patients with ulcerative colitis is associated with renalpathology. METHODS.: Eight ANCA-positive and five ANCA-negative patients with a histologicaland endoscopic diagnosis of active ulcerative colitis were investigated.Repeated complete urinalyses and determination of microalbuminuriaand creatinine clearance were performed. Serum IgG and IgA ANCAwere evaluated in all patients by indirect immunofluorescenceand ELISA, and when detected the antibodies were further characterizedby alpha granules preparation, myeloperoxidase, lactoferrin,and cathepsin G. RESULTS.: In both ANCA-positive and ANCA-negative patients renal functionwas normal or near normal and urinalyses (including microalbuminuria)failed to disclose any abnormalities. ANCA exhibited a perinuclearpattern in all ANCA-positive patients. Interestingly, none ofthe ANCA-positive patients had antibodies to myeloperoxidaseor to alpha granules which are usually found in the sera ofpatients with ANCA-associated vasculitis, and only one had antibodiesto lactoferrin. The ANCA specificity remained undetermined inthe remaining seven patients. At the end of the 1-year observationperiod, all ANCA-positive patients remained ANCA-positive withoutdeveloping symptoms, signs or laboratory abnormalities consistentwith renal involvement. CONCLUSIONS.: Renal damage was not observed in ANCA-positive patients withulcerative colitis even after 1 year of follow-up, suggestingthat the ANCA found in these patients do not share the antigenictargets with the ANCA commonly found in renal vasculitis. Thereforethe potential of ANCA of inducing renal lesions (if any) isdependent on their own antigenic specificity.     

The Diagnostic and Prognostic Significance of ANCA

Antineutrophil cytoplasmic antibodies (ANCA) constitute a family of autoantibodies directed against various components of the neutrophil cytoplasm. Their identification and association with vasculitis and rapidly progressive glomerulonephritis has led to considering these diseases as possible autoimmune disorders. In addition, ANCAs constitute a diagnostic tool and a guideline for therapy during follow-up. Originally identified by Davies et al. in 1982 in 8 patients who had necrotizing glomerulonephritis but no immune deposits or systemic vasculitis (1), ANCA are now regarded as a serological marker for active pauci-immune necrotizing and crescentic glomerulonephritis, either in their renal-limited form or associated with systemic vasculitis such as Wegener's granulomatosis (WG), microscopic polyangütis (mPA), and Churg-Strauss syndrome (CSS) (2-9). The usefulness of ANCA detection for the diagnosis of these forms of vasculitis is now established but its usefulness on follow-up remains disputed (10-13). Two major ANCA antigens have already been identified. Proteinase 3 (PR3) in a serine protease of 29 kDa initially identified by Kao and producing a cytoplasmic staining pattern termed cANCA by indirect immunofluorescence (IIF) (14,15). Myeloperoxidase (MPO) is another myeloid lysosomal enzyme producing an artefactual perinuclear staining of ethanol-fixed neutrophils termed pANCA (2,16). Both are localized in the azurophilic granules of neutrophils and monocytes, are translocated to the cell surface during cell activation (17), and are able to interact directly with ANCA. Despite this common location, MPO and PR3 are associated with a broad spectrum of clinical conditions.     

Rapid screening assay for anti-GBM antibody and ANCAs; an important tool for the differential diagnosis of pulmonary renal syndromes

Background. Pulmonary renal syndrome is encountered in several diseases such as Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA) associated systemic vasculitis, systemic lupus erythematosus (SLE) and infection-associated or drug-induced glomerulonephritis. To preserve organ function it is of vital importance to make the correct diagnosis and institute adequate therapy early, in the acute phase. Methods. An enzyme-linked immunosorbent assay (ELISA), specially designed as a rapid screening assay for antiglomerular basement membrane (anti-GBM) antibody proteinase-3 (PR3-) ANCA and myeloperoxidase-(PMPO-) ANCA were evaluated from 1060 serum samples drawn from patients with clinically suspected pulmonary renal syndrome or rapidly progressive glomerulonephritis (RPGN). Results. Of the 1060 serum samples, 142 were positive for anti-GBM antibody (n=19), PR3-ANCA (n=60), or MPO-ANCA (n=73). Of the 142 samples, 10 were double positive. Reanalysis of positive sera with a quantitative ELISA yielded results manifesting good correlation with those of the rapid screening assay. Of 918 sera found to be negative in the rapid screening assay, 105 were also tested with IIF, 11 being found to be positive. However, these 11 sera manifested no specificity for PR3 or MPO, but some were specific for bactericidal/permeability-increasing proteins, lactoferrin or elastase ANCAs. Two of the patients whose sera yielded negative results in the rapid assay had systemic vasculitis. Conclusions. The ELISA thus detects the true antibodies to PR3, MPO, and GBM, whereas IIF detects additional specificities. The findings suggest the rapid assay results to be of high positive predictive value, and the assay to be of high diagnostic specificity and sensitivity and thus useful in the diagnostic workup in suspected cases of RPGN or pulmonary renal syndrome.     

How are antineutrophil cytoplasmic autoantibodies detected?

Antineutrophil cytoplasmic autoantibodies (ANCA) are present in patients with systemic vasculitis with or without renal involvement. These antibodies were first seen using indirect immunofluorescence (IIF). Two types of patterns are seen on ethanol-fixed neutrophils: the cytoplasmic and the perinuclear pattern. The cytoplasmic pattern is called C-ANCA (classical or cytoplasmic ANCA) and the perinuclear, P-ANCA. Antibodies to a serine proteinase, called proteinase 3 or myeloblastin, give rise to the C-ANCA pattern, while antibodies to myeloperoxidase give rise to the P-ANCA pattern. Proteinase 3, as well as myeloperoxidase, is present in the primary granules of neutrophils, and the P-ANCA pattern is thus an artifactual staining pattern. Myeloperoxidase, which is a basic protein, redistributes during ethanol fixation from the primary granules to the negatively charged nucleus. As an alternative to the IF technique, several solid-phase assays have been developed using either 125I or enzyme-labeled secondary antibodies. Depending on the degree of purification of the antigens used, such assays may be used for screening or as a complement to the IF method. Today it is possible to directly screen for both types of ANCA using enzyme-linked immunosorbent assay (ELISA). Simultaneous screening for antiglomerular basement membrane (GBM) antibodies (Goodpasture antibodies) increases the diagnostic yield, especially in patients with renopulmonary syndromes.     

The role of flow cytometric ANCA detection in screening for acute pauci-immune crescentic glomerulonephritis.

BACKGROUND: Most cases of pauci-immune crescentic glomerulonephritis (PICGN) are associated with serum anti-neutrophil cytoplasmic antibodies (ANCA). This article studied the sensitivity and specificity of serum ANCA, determined by flow cytometry and indirect immunofluorescence (IIF), to identify patients with acute PICGN. METHODS: 577 adults presenting for first biopsy of their native kidneys with serum taken for ANCA (flow cytometry and IIF) determination were studied. A positive ANCA was defined using a flow cytometric ANCA assay as a screening test, followed by a slide-based indirect IIF technique. Pathological confirmation of acute PICGN was used to assess the sensitivity and specificity of this combined approach and its positive predictive value (PPV) and negative predictive value (NPV) in patients presenting for renal biopsy due to abnormal urinary sediment. RESULTS: Forty-nine patients were found to have acute PICGN on renal biopsy. Of these 47 were ANCA positive (sensitivity 95.9%). Overall 93 of the renal biopsy patients were ANCA positive, (specificity 91.3%). A further seven patients (two ANCA positive) had advanced sclerosing disease consistent with PICGN but without evidence of current disease activity. The PPV and NPV of ANCA, assessed by flow cytometry and slide IIF, in predicting that patients presenting with undifferentiated renal disease would have acute PICGN was 50.5 and 99.8%, respectively. CONCLUSIONS: Flow cytometric screening of serum for ANCA in patients undergoing renal biopsy has a high NPV for determining those with acute PICGN. It may provide a rapid, simple screening test for this lesion in laboratories using diagnostic flow cytometry and may complement IIF/ELISA in evaluating ANCA positive patients.     

Anti-neutrophil cytoplasmic antibodies and anti-endothelial cell antibodies

Anti-netrophil cytosolic antibodies (ANCA) and anti-endothelial cell antibodies (AECA) have been identified in a wide variety of disorders, but their pathophysiological role remains unclear. ANCA appear to be particularly associated with various forms of vasculitis including Wegener's granulomatosis, Kawasaki disease and microscopic polyartiritis. Cytoplasmic staining (cANCA) on indirect immunofluorescence is associated with extrarenal disease and a perinuclear pattern (pANCA) with renal limited disease. The cANCA antigen appears to be proteinase 3 and that for pANCA myeloperoxidase. AECA have been detected in systemic lupus erythematosus, scleroderma and dermatomyositis but are also found in systemic vasculiitis. Kawasaki disease, haemolytic uraemic syndrome, thrombotic thrombocytopenic purpura and renal allograft recipients at the time of rejection. Their presence appears to be correlated with disease activity and they may be directed against epitopes on as yet unidentified infective agents that precipitate some of the diseases in which they are found that cross-react with antigenic sites exposed on endothelial cells. Measurement of these antibodies has a diagnostic role, facilitates monitoring of disease activity and may prove valuable in understanding the pathogenesis of the diseases in which they are found.     

ANCA titres, even of IgG subclasses, and soluble CD14 fail to predict relapses in patients with ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic autoantibodies (ANCA) are presumed to reflect disease-activity and to be useful for guidance of immunosuppressive therapy of ANCA-associated systemic vasculitis (AASV), but with respect to conventional ANCA assays this is controversial. ANCA titres, measured in the IgG3 subclass and modern capture ELISAs, have been said to be superior predictors of relapses of AASV. METHODS: In this retrospective study serial measurements of ANCA parameters and soluble CD14 (sCD14) were performed in 169 consecutive sera over a median of 21 months in 18 patients with AASV and related to disease activity, assessed by Birmingham Vasculitis Activity Score (BVAS) for new or deteriorated (BVAS1), and for chronic disease activity (BVAS2). Fourteen patients had Wegener's granulomatosis (WG) and were C-ANCA positive with Pr 3-antibodies and four patients had microscopic polyangiitis (MPA) with P-ANCA and MPO-antibodies. In WG patients ANCA by IIF, Pr 3-ELISA for IgG, IgG1, IgG3, IgG4 and sCD14 were measured, as well as capture ELISA for Pr 3, and in MPA patients ANCA by IIF, MPO-ELISA for IgG and IgG1, IgG3, IgG4, and sCD14 respectively. In eight patients, data collection started at diagnosis, in 10 patients at remission. RESULTS: The parameters predicted neither the nine major relapses (increase of immunosuppression necessary), nor the 26 minor relapses (increase of BVAS1>2) with sufficient sensitivity (>80%) or specificity (> 90%90%), and they also failed to predict relapses within the following 2 months. ANCA-IgG3 and capture ELISA for Pr 3 were not advantageous for prediction of relapses (sensitivity 0.45 and 0.19 respectively), and sCD14 remained elevated in all samples irrespective of disease activity. CONCLUSIONS: There is no rationale for serial measurements of ANCA in AASV. For changes of therapy, the ANCA parameters should only be used in conjunction with clinical information.     

C-antineutrophil cytoplasmic antibody positivity in vasculitis patients is associated with the Z allele of alpha-1-antitrypsin, and P-antineutrophil cytoplasmic antibody positivity with the S allele

BACKGROUND.: Antineutrophil cytoplasmic antibodies (ANCA) in vasculitis haveeither cANCA or pANCA patterns as defined by immunofluorescence.The target autoantigen of cANCA is usually proteinase 3 (PR3),whereas that of pANCA is usually myeloperoxidase (MPO). Alpha-1-antitrypsin(1AT) is the major physiological inhibitor of PR3, while MPOis an inhibitor of 1AT. METHODS.: To determine whether there was an association between ANCA positivevasculitis, ANCA pattern, and 1AT deficiency alleles, we studied1AT phenotypes of 99 cANCA and 99 pANCA positive vasculitispatients by isoelectric focusing and immunoblotting, and comparedthem with 2310 controls from the same geographical area. RESULTS.: C-ANCA patients showed an increased frequency of the Z allele(0.055 versus 0.018 in controls), conferring a relative riskof 3. They showed no increase in frequency of the S allele.P-ANCA patients showed an increased frequency of the S allele(0.091 versus 0.046 in controls) conferring a relative riskof 2. The frequency of the Z allele also appeared to be increased(0.030 versus 0.018 in controls), but this was not statisticallysignificant. CONCLUSIONS.: These findings demonstrate an association between ANCA-positivevasculitis and deficiency phenotypes of 1AT, and suggest a rolefor 1AT in the development of systemic vasculitis.     

Antineutrophil-cytoplasmic antibodies and antiglomerular basement membrane antibodies in Goodpasture's syndrome and in Wegener's granulomatosis.

Antiglomerular basement membrane (anti-GBM) diseases-including Goodpasture's (GP) syndrome-and Wegener's granulomatosis (WG) are systemic diseases, which may be diagnosed by means of circulating autoantibodies. Possible overlap syndromes may exist; however, they remain imperfectly defined. We analyzed sera from 31 patients with WG and from 23 patients with anti-GBM disease. All underwent biopsy. Anti-cytoplasmic antibodies (ANCA) were demonstrated by indirect immunofluorescence (IIF); a perinuclear (P-ANCA) or diffuse-cytoplasmic (C-ANCA) staining was discerned. In addition, myeloperoxidase (MPO) antibodies (P-ANCA) and protein 3 (SP3) antibodies (C-ANCA) were analyzed by specific ELISA systems. Anti-GBM antibodies (anti-NC1 antibodies) were detected by ELISA and immunoblotting; the globular domain NC1 of collagen IV was employed as antigen. All 31 WG patients, as defined by clinical and histological criteria, showed ANCA by IIF. Twenty-nine of 31 showed a C-ANCA pattern; all were also positive for SP3 antibodies by ELISA. Three of 31 WG patients were P-ANCA positive by IIF and also had anti-MPO antibodies by ELISA. In one of these patients, SP3 antibodies were additionally found by IIF and by ELISA (double positive). No patient with WG had anti-NC1 antibodies. All 23 serum samples from patients with GP syndrome (N = 19) or anti-GBM glomerulonephritis (N = 4) had anti-NC1 antibodies. In seven of these patients, low titers of anti-MPO antibodies were detected by ELISA; however, the IIF for ANCA was negative. None of these seven patients had extraglomerular vasculities. In addition, the clinical prognosis of these patients was similar to that of those patients who lacked these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antineutrophil cytoplasmic autoantibody-associated diseases: a rheumatologist's perspective

Autoantibodies against neutrophil cytoplasmic antigens (ANCA) produce two major immunofluorescence (IF) patterns on ethanol-fixed granulocytes: the "classical" (centrally accentuated) C-ANCA, associated with Wegener's granulomatosis (WG), and P-ANCA (perinuclear), which mainly occur in renal vasculitis. Rheumatic manifestations are an important clinical finding in systemic vasculitis, often preceding a fulminant course and sometimes imitating various rheumatic disorders. We analyzed the incidence of ANCA in rheumatic patients and looked for the frequency of rheumatic symptoms in systemic vasculitis. In WG (n = 186), we found rheumatic symptoms in 55% (myalgia, 45%; arthritis, 21%); in 90%, rheumatic complaints were associated with active vasculitis. In 730 patients with various rheumatic conditions (eg, 268 rheumatoid arthritis, 130 systemic lupus erythematosis [SLE], 32 sharp-S, 50 ankylosing spondylitis, 43 systemic sclerosis) no C-ANCA were found. On the contrary, the P-ANCA pattern was seen in seven of 62 giant cell arteritis, five of 27 Felty's/Still's syndrome, and four of 130 SLE patients in addition to renal vasculitis (21/74). We demonstrated that 95% of C-ANCA-positive sera react with proteinase 3 (PR3 or myeloblastin). Using monoclonal antibodies, we showed that PR3 is expressed on the plasma membrane of neutrophil granulocytes and monocytes; thus, PR3 autoantigens are accessible for circulating antibodies. The detection of ANCA in sera from vasculitis and other rheumatic diseases is of immunodiagnostic value and provides new insight in the pathogenesis of systemic vasculitides.     

Anti-neutrophil cytoplasmic antibody-associated vasculitis, large vessel vasculitis and Kawasaki disease in Japan

Based on studies comparing the prevalence of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) between Japan and Europe, we have learned that the difference may be due to genetic background and environmental factors, but not to diagnosis or ELISA system for myeloperoxidase and proteinase-3 ANCA. In Japan, microscopic polyangiitis is the most common among AAV, but Wegener's granulomatosis was present in less than 2 per million patients. Also, one study from Hokkaido reported only 16 patients in a 27-year time frame. A recent retrospective study of renal vasculitis between 2000 and 2004 from Miyazaki prefecture in Japan reported an incidence of microscopic polyangiitis of 14.8 per million, but no patients with Wegener's granulomatosis or Churg-Strauss syndrome. In the present review, we focus on ANCA-related vasculitis in Japan: (1) AAV and large vessel vasculitis - Takayasu's arteritis and giant cell arteritis; (2) primary renal vasculitis; (3) epitopes of myeloperoxidase-ANCA in vasculitis in the Japanese population and comparison of ANCA-ELISA systems in Japan and Europe, and finally (4) children with vasculitis in Japan involving Kawasaki disease - a systemic vasculitis.     

Analysis of anti-neutrophil cytoplasmic antibodies (ANCA): frequency and specificity in a sample of 191 homozygous (PiZZ) alpha1-antitrypsin-deficient subjects.

BACKGROUND: ANCA are autoantibodies directed against polymorphonuclear cell antigens, mainly proteinase 3 (PR3) and myeloperoxidase (MPO), which are implicated in the pathogenesis of small-vessel necrotizing vasculitis. Alpha1-antitrypsin is the main inhibitor of neutral serine proteinase [i.e. human leukocyte elastase (HLE) and PR3] present in PMN alpha-granules (alphaGr). An association first reported by us between PR3 ANCA and the deficient PiZZ phenotype in ANCA-positive systemic vasculitis, now widely confirmed by others, led us to study the incidence and specificity of ANCA among PiZZ subjects. METHODS: We tested a population of 191 PiZZ (273 sera) for ANCA activity versus 272 PiMM matched control subjects using alphaGr or antigen-specific ELISA [PR3, HLE, MPO, lactoferin (LF) and bactericidal/ permeability increasing protein (BPI)]. RESULTS: The incidence of antibodies directed against alphaGr and HLE but not PR3, MPO, LF or BPI was increased in the PiZZ as compared to the PiMM group (Fisher probability respectively P < 0.0001 and P < 0.05). CONCLUSIONS: ANCA not directed against classical antigens (MPO and PR3) may be found in PiZZ patients. However, these patients do not develop systemic vasculitis features. Therefore, alpha1-antitrypsin deficiency is not sufficient to induce ANCA positive vasculitides, and may only act as a second hit amplifying factor.     

ANCA相关性血管炎肾损害临床特征及早期诊治探讨

目的 分析抗中性粒细胞胞浆抗体(ANCA)相关性血管炎的临床表现和肾脏病理特征,探讨早期诊断和治疗对预后的影响.方法选取本院2000年1月至2009年8月明确诊断的ANCA相关性血管炎共21例,18例行肾活检.总结患者的临床病理资科.分析不同治疗时机对肾功能转归的影响.结果本组21例ANCA相关性血管炎平均年龄(52.5±11.5)岁,显微镜下多血管炎(MPA)16例,韦格纳肉芽肿(WG)3例,变应性肉芽肿性血管炎(CSS)2例.肾外表现主要为发烧17例(80.1%)、下呼吸道症状18例(85.7%)、肺影像学改变21例(100%)、贫血16例(76.2%)、眼耳鼻受累8例(38.1%);肾脏表现血尿21例(100%),蛋白尿19例(90.1%),血肌酐正常6例(28.5%),升高15例(71.4%),8例需透析替代.ANCA检测pANCA和MPO-ANCA阳性16例,cANCA和PR3-ANCA阳性3例.pANCA/MPO-ANCA和cANCA/PR3-ANCA均阳性1例,全阴性1例.肾活检可见节段性小血管壁纤维素样坏死,新月体多见.免疫荧光无或微量免疫复合物沉积.治疗采用糖皮质激素联合环磷酰胺,重症加用血浆置换.7例血肌酐异常但不需透析者5例治疗后血肌酐恢复正常;8例需透析者2例治疗后血肌酐恢复正常,2例脱离透析但血肌酐异常,4例未能脱离透析.结论ANCA相关性小血管炎临床表现多样,肺、肾是最常见的受累器官.ANCA检测和肾活检有助于早期诊断,尽早积极治疗有助于肾功能的恢复.     

抗中性粒细胞胞浆抗体相关性小血管炎30例临床诊治分析

目的：探讨抗中性粒细胞胞浆抗体（ANCA）相关性小血管炎的临床特点、诊断和治疗。方法：回顾性分析2002年6月~2009年6月检测并明确诊断的30例ANCA相关性小血管炎患者的临床病理资料。结果：30例患者中胞浆型ANCA（c-ANCA）阳性4例,3例识别蛋白酶3（PR3）,1例识别髓过氧化物酶（MPO）;核周型ANCA（p-ANCA）阳性26例,均识别MPO。临床表现呈多器官受累,以肾、肺受累为主。多数患者有贫血、血沉增快和C反应蛋白增高。糖皮质激素联合免疫抑制剂治疗,诱导缓解的缓解率为83.3%。结论：ANCA相关性小血管炎临床表现为多器官受累,缺乏特异性,其诊断要结合临床表现、ANCA检测和病理活检综合考虑,糖皮质激素联合免疫抑制剂治疗有较好疗效,吗替麦考酚酯和硫唑嘌呤等免疫抑制剂较环磷酰胺毒副作用更小。     

Clinical implications of antineutrophil cytoplasmic antibody test in lupus nephritis

To elucidate the prevalence and clinical implications of antineutrophil cytoplasmic antibody (ANCA) in lupus nephritis (LN), we examined ANCA by indirect immunofluorescence and by ELISA against antilactoferrin (anti-LF) and antimyeloperoxidase (anti-MPO) antibody. To discriminate perinuclear ANCA (pANCA) with antinuclear antibody (ANA), all the ANCA-positive sera were tested again after incubating patients' sera with single-stranded (SS) and double-stranded (ds) DNA. These results were compared with clinicopathologic manifestations and clinical courses of LN. ANCA was positive in 19 (37.3%) of 51 LN patients. Among these LN patients, 3 had cytoplasmic ANCA (cANCA) and 16 had pANCA. ANCA was not found in 8 SLE patients without nephritis and 30 normal controls. The presence of ANCA, particularly pANCA, was associated with the presence of nephritis (18/51 cases vs. 0/8 cases, p < 0.05), especially with diffuse proliferative lupus nephritis, WHO class IV (17/18 cases vs. 21/31 cases, p < 0.05) as well as the presence of anti-dsDNA antibody (17/19 cases vs. 18/30 cases, p < 0.05). Patients with ANCA frequently had deterioration of renal function (3/16 vs. 0/26 cases). Anti-LF antibody was positive in 13 patients. Among those, 12 patients had nephritis. Five patients with anti-LF antibody did not have ANCA, but 7 had pANCA, and 1 had cANCA. Patients with anti-LF antibody had lower initial creatinine levels than those without it [serum creatinine (mg/dl): 0.78 (0.6-1.0) vs. 1.43 (0.5-5.0), p < 0.05]. Anti-MPO antibody was positive in only 1 patient, suggesting that MPO is a rare antigen for ANCA in LN.     

Antineutrophil cytoplasmic autoantibodies and associated diseases: a review

Antineutrophil cytoplasmic autoantibodies (ANCA) are specific for constituents of neutrophil primary granules and monocyte lysosomes. There are different types of ANCA with different specificities. By indirect immunofluorescence microscopy using alcohol-fixed neutrophils as substrate, two major categories of ANCA can be recognized, one with cytoplasmic staining (C-ANCA) and the other with artifactual perinuclear staining (P-ANCA). Some C-ANCA have specificity for proteinase 3 (PR3-ANCA) and some P-ANCA have specificity for myeloperoxidase (MPO-ANCA), but there are additional C-ANCA and P-ANCA specificities. ANCA are found in the blood of patients with necrotizing systemic vasculitis, such as Wegener's granulomatosis and polyarteritis nodosa, and patients with idiopathic crescentic glomerulonephritis. The glomerular lesion in patients with systemic and renal-limited ANCA-associated diseases is the same, ie, a pauci-immune necrotizing and crescentic glomerulonephritis. No matter where the vascular lesions of ANCA-associated disease are (eg, kidney, lung, gut, muscle, skin), they are characterized by necrotizing inflammation and a paucity of immune deposits. The distribution of disease correlates to a degree with the ANCA specificity, although there is substantial overlap. For example, patients with Wegener's granulomatosis most often have C-ANCA and patients with renal-limited disease most often have P-ANCA. In patients with P-ANCA-associated glomerulonephritis, approximately 90% of the P-ANCA have specificity for MPO. The clinical manifestations of ANCA-associated diseases often begin following a flu-like illness. The onset is most often in the winter and least often in the summer. The renal disease usually presents as rapidly progressive renal failure with nephritis. One of the most life-threatening components of the systemic involvement is pulmonary hemorrhage caused by a necrotizing alveolar capillaritis. Intravenous cyclophosphamide plus steroids is as effective as oral cyclophosphamide plus steroids for controlling ANCA-associated diseases. Using life-table analysis, the 2-year patient and renal survival rate in both patients with renal-limited and systemic disease is greater than 70%. There is evidence that in addition to being a useful serologic marker, ANCA are directly involved in the pathogenesis of the vascular injury in patients with ANCA-associated diseases. Although ANCA antigens are normally in the cytoplasm of neutrophils and monocytes, priming of these cells, as occurs following exposure to certain cytokines, results in the release of small amounts of ANCA antigens at the cell surface. In vitro, ANCA-IgG causes cytokine-primed neutrophils to undergo a respiratory burst and degranulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Urinary enzymes in acute renal failure

Intestinal-type alkaline phosphatase (IAP) has been localizedto the S3 segment of the renal tubule in previous studies, asite believed to be particularly vulnerable to toxic and ischaemicdamage. During a 17-month period a pilot study of the valueof urinary enzyme measurements (IAP and tissue non-specificalkaline phosphatase—TNAP, using monoclonal antibody-basedimmunoassays, and N-acetyl-beta-glucosaminidase—NAG, usingcolorometric assay) in 50 prospectively followed cases of acuterenal failure (ARF) was performed. Urinary enzymes were measuredat initial evaluation (‘start’), and then each dayfor 14 days, with the highest enzyme value (‘peak’)also used for analysis. Patients were divided into prerenal(n=16), renal (n=28), postrenal (n=6) categories according tostandard criteria. Of the renal ARF patients 23 of 28 had acutetubular necrosis (ATN), 3 of 28 acute interstitial nephritis(AIN), and 2 of 28 acute glomerulonephritis (AGN); 18 of 50had a fatal outcome and 1 of 50 was dialysis-dependent at discharge(‘poor’ prognosis group), while 31 of 50 survivedhospital without becoming dialysis-dependent (‘good’prognosis group). Median enzyme concentration were increased in ‘poor’compared to ‘good’ prognosis patients: start IAP3.2 versus 2.2 U/g creat (NS), start NAG 48.6 versus 13.7 (P<0.01),start TNAP 3.5 versus 0.9 (P<0.02). When renal ARF patientsalone were analysed, only IAP (3.2 versus 1.3 U/g creat at start)and NAG (57.9 versus 7.8 U/g creat at start) were significantlyincreased in the poor compared to the good prognosis group.Peak values showed similar trends. Of all patients, five witha start IAP>12 U/g creat died, and all survivors had a startIAP<12, but 14 of 19 poor prognosis patients also had a startIAP<12. All urinary enzymes were less in the postrenal group,but only the IAP significantly so. None of the enzymes weresignificantly different between prerenal and renal ARF groups. Urinary enzymes IAP, NAG, TNAP appear to be unhelpful in determiningthe site of renal injury in ARF, except for postrenal cases,where IAP was significantly lower. There were too few patientswith AGN or AIN to test the hypothesis that the enzymes wouldbe less in glomerular compared to tubular pathologies. Despitea low sensitivity, the start IAP may be a marker of outcomein ARF if the high positive predictive value for death is confirmedin larger studies.     

SCG/Kinjoh mice: a model of ANCA-associated crescentic glomerulonephritis with immune deposits

BACKGROUND: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice spontaneously develop crescentic glomerulonephritis (CGN), systemic vasculitis, and perinuclear ANCA (pANCA), and have been suggested as an animal model for human antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AASV). Since no systematic serologic, immunohistologic, or structural evaluation had been performed thus far, we reinvestigated the development of ANCA and CGN in these mice. METHODS: SCG/Kj mice were subjected to serologic and urinary analysis, as well as histologic evaluation of the kidneys by standard light, immunofluorescence, and electron microscopy at regular intervals during the course of the disease. RESULTS: Perinuclear ANCA developed as early as the 6th week of life, increasing both in frequency and titer in up to 100% of animals at week 20. Crescent formation began at week 10 and peaked at week 16, maximally affecting 57% of glomeruli. Crescent formation was initiated by "activated" podocytes that formed cell bridges between tuft and Bowman's capsule. The typical picture of a diffuse immune complex nephritis was found in all animals as early as 8 weeks. Fluorescence intensity increased with age and became strongly positive for immunoglobulin (Ig)A, IgM, IgG, and C3 in the mesangium and along the peripheral capillary loops. CONCLUSION: Although ANCAs were found in the majority of animals, the massive presence of glomerular immune deposits differed from the pauci-immune pattern found in human AASV, making this model not completely representative for human ANCA-associated CGN. However, the spontaneous and concomitant development of pANCA, small vessel vasculitis, and CGN raises the opportunity to analyze pathogenetic links between these disease manifestations in vivo.     

Classic and perinuclear anti-neutrophil cytoplasm antibodies and antimyeloperoxidase antibodies in rapidly progressive glomerulonephritis.

Classic anti-neutrophil cytoplasm antibodies (cANCA), perinuclear ANCA (pANCA) and antibodies directed against myeloperoxidase (MPO-Ab) were evaluated in 25 patients with either idiopathic or secondary rapidly progressive glomerulonephritis (RPGN). While cANCA were found almost exclusively in Wegener's granulomatosis, pANCA were detectable in several disorders, including microscopic polyarteritis (mPA), but also idiopathic RPGN. MPO-Ab were frequently found in sera from patients with all types of idiopathic but not of secondary RPGN. These results support the hypothesis that some cases of RPGN are early or limited forms of systematic vasculitis. We then looked for the presence of IgA-ANCA in Henoch-Schoenlein purpura (HSP): we found IgA-ANCA with immunoenzymatic assay but not with immunofluorescence in HSP, in primary IgA-GN and in membranous GN as well, thus suggesting the poor specificity of this type of ANCA. The possible pathologic implications of ANCA were examined in vitro. Serum samples from several patients with ANCA were assessed for their capacity to enhance chemiluminescence generation from resting or PMA-stimulated macrophages. Sera from RPGN and mPA patients displaying anti-MPO activity induced granulocytes to enhance the production of oxygen free radicals, thus suggesting a phlogistic effect of MPO-Ab positive sera.