Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA)

In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.     

Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).     

Detection of anti-neutrophil cytoplasmic antibodies in MRL/Mp-lpr/lpr mice and analysis of their target antigens

Anti-neutrophil cytoplasmic antibodies (ANCA) have been widely studied and recognized to be clinically very important for some human diseases including systemic rheumatic diseases. We analyzed ANCA response and their target antigens in MRL/Mp-lpr/lpr (MRL-lpr) mice, an animal model of systemic rheumatic disease. P-ANCA was detected in 57% of the mice. Antibodies to the known P-ANCA target antigens at the same age were examined. Among these, antibodies to high mobility group (HMG) proteins HMG1 and HMG2 were detected in 57% of the mice, 75% of which were also positive for P-ANCA. These anti-HMG1/HMG2 activities were absorbed by preincubation with a mixture of HMG1 and HMG2. In contrast, antibodies to myeloperoxidase and cathepsin G were detected in 14% and 7%, respectively, but these activities were not inhibited by preincubation with corresponding antigens. In addition, the titers of P-ANCA and anti-HMG1/HMG2 antibodies in MRL-lpr mice were significantly correlated with each other. Thus, HMG1 and HMG2 were considered to be significant target antigens of P-ANCA in MRL-lpr mice.     

Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody.

Features of tissue damage in invasive amoebiasis, in particular polymorphonuclear neutrophil (PMN) degranulation and vasculitis, bear resemblance to that seen in Wegener's granulomatosis, the latter being associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We therefore tested sera from patients with confirmed amoebic liver abscess (ALA) for the presence of ANCA by means of an indirect fluorescent antibody test using pure neutrophils as substrate. ANCA was detected in 97.4% of amoebic sera; the pattern of staining was cytoplasmic, homogeneous, without central accentuation (C-ANCA). A proteinase 3 (PR3) ELISA demonstrated PR3 specificity in 75% of C-ANCA-positive ALA sera. Possible explanations are (i) a cross-reacting antibody to a component of Entamoeba histolytica, or (ii) an antibody to PMN components released, and possibly modified, by the action of E. histolytica on PMN. It is possible that this antibody contributes to the pathogenesis of invasive amoebiasis.     

Uveitis and anti-neutrophil cytoplasmic antibodies.

Serum samples of 485 uveitis patients were screened for the presence of anti-neutrophil cytoplasmic antibodies using a standardized immunofluorescence test (IIF) on neutrophil granulocytes. Seventeen of these sera contained cytoplasmic (C)-ANCA antibodies, while two of the sera contained perinuclear (P)-ANCA antibodies (both antinuclear antibody (ANA)-positive, one anti-myeloperoxidase (MPO)-positive). None of the C-ANCA-positive sera reacted with proteinase-3 in ELISA using a highly purified proteinase-3 preparation. Four C-ANCA and one P-ANCA-positive serum reacted with MPO. The majority of the sera did react with azurophilic granules in ELISA. The implication of these results is that in patients with uveitis a positive C-ANCA test is not diagnostic for Wegener's granulomatosis, but is most probably caused by the presence of autoantibodies against as yet unknown constituents of azurophilic granules.     

Agreement of anti-neutrophil cytoplasmic antibody measurements obtained from serum and plasma

Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrollment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluorescence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using kappa-coefficients. These were > 0.8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were > or = 0.95 (P < 0.0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.     

Identification of target antigens of anti-endothelial cell antibodies in patients with anti-neutrophil cytoplasmic antibody-associated vasculitides: A proteomic approach

Anti-endothelial cell antibodies (AECAs) have been reported to cause endothelial cell dysfunction, but their specific targets have never been identified in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs). Proteins from human umbilical vein endothelial cells (HUVECs) were separated by 2-dimensional electrophoresis (2-DE). 2-D immunoblots were used to compare serum IgG reactivities from 30 patients with AAV and 12 healthy controls (HCs). Proteins identified as target antigens by MALDI- TOF-TOF mass spectrometry included lamin A/C, vimentin, α-enolase, far upstream binding protein 2 (FUBP2) and protein disulfide-isomerase A3 precursor (PDIA3). Antibodies targeting lamin A, vimentin, α-enolase, FUBP2 and PDIA3 were identified in 57.1%, 64.3%, 35.7%, 50% and 0% of patients with microscopic polyangiitis and 8%, 3.3%, 7.2%, 0% and 6.7% of HCs respectively. IgG from patients with microscopic polyangiitis had stronger reactivity against HUVEC than other groups and HCs and induced stronger Erk phosphorylation in HUVECs than IgG from HCs.     

Pathological classification of anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis

What drives human beings to classify? It seems as if it is within our nature to do so. Clinical classification systems for the systemic vasculitides were composed a long time ago, and they are constantly being revised and altered. The histopathological features of many diseases are so diverse that classification is called for. The histopathological classification for anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis was the culmination of results produced from a number of clinicopathological studies conducted within the European Vasculitis Study Group (EUVAS). The classification scheme has four general categories, named focal, crescentic, sclerotic and mixed. The first three categories are based on the predominance of normal glomeruli, glomeruli with cellular crescents and globally sclerotic glomeruli. The mixed category represents a heterogeneous phenotype of biopsies in which none of the aforementioned features is dominant. Results from a validation study incorporating 100 patients with at least 1-year follow-up showed that the phenotypical order of the four classes corresponded to the severity of renal function impairment. The new histopathological classification for ANCA-associated glomerulonephritis provides a logical structure for the categorization of patients into four subgroups defined according to glomerular features. This classification will be of use for future studies, such as clinical trials.     

Immunogenetic risk factors for anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

Wegener's granulomatosis (WG) and microscopic polyangiitis are systemic autoimmune diseases characterized by the presence of ANCA in the sera of patients. Little is known about the aetiologic factors and genetic predisposition as well as the pathogenesis of these disease entities. A slightly decreased representation of HLA-DRB1*13 and HLA-DQB1*0603 individuals was observed in our cohort of ANCA-associated systemic vasculitis (AASV) patients compared with controls. In addition, HLA-DRB1*04 individuals were over-represented in a subgroup of patients with WG in end-stage renal disease as a result of renal vasculitis. In order to identify other genes relevant for these diseases, we investigated highly polymorphic markers in the vicinity of several immunorelevant genes, i.e. tumour necrosis factor (TNF)alpha, IL-2, IL-5 receptor alpha (IL-5RA), in a group of 102 patients with AASV and compared the representation with controls. Furthermore, functional polymorphisms were directly analysed in the promotor region of TNFalpha as well as in the coding region of the FcgammaIIRA genes. Polymorphisms of the TNFalpha promotor (TNF-308) as well as in the FcgammaIIRA gene were excluded as risk factors for the disease in our cohort. No major phenotype distribution differences were observed between patients and controls for the IL-2 and IL-5RA microsatellites. Most importantly, several haplotypes on chromosome 6p appeared strongly associated with proteinase 3 (PR3)-ANCA+ AASV. Thus, as in other autoimmune diseases, different predisposing factors play differential aetiopathogenic roles in various groups of AASV patients.     

Diverse target antigens recognized by circulating antibodies in anti-neutrophil cytoplasm antibody-associated renal vasculitides.

Antibodies that are directed against cytoplasmic constituents of neutrophils and monocytes (anti-neutrophil cytoplasm antibodies, ANCA) have been described in Wegener's granulomatosis, microscopic polyarteritis (MPA) and some cases of segmental necrotizing glomerulonephritis (SNGN). Other antibodies occasionally described in Wegener's granulomatosis and MPA include anti-nuclear antibodies (ANA) and anti-glomerular basement membrane (GBM) antibodies. We have studied the diversity of the corresponding antigens in ANCA-associated renal diseases. Sera from 46 patients with active histologically proven Wegener's granulomatosis, MPA and SNGN were tested for ANCA by indirect immunofluorescent examination of normal peripheral blood neutrophils. Thirty-four sera (74%) were positive; 16 were associated with diffuse cytoplasmic staining (cANCA) and 18 with perinuclear staining (pANCA). In addition, five demonstrated antineutrophil-specific nuclear staining (ANNA). On Western blotting of the neutrophil extract, five sera recognized a 29-kD molecule recently identified as neutrophil proteinase 3. Two sera with typical cANCA bound to molecules of 36, 38 and 116 kD and another to a molecule of 22 kD. The final serum associated with pANCA bound to a molecule of about 12 kD. Thirteen sera out of 46 (28%) tested in an ELISA contained anti-myeloperoxidase antibodies; 10 of these were associated with pANCA and two others with ANNA. Three sera of 17 (18%) tested contained anti-elastase antibodies; these also contained anti-myeloperoxidase antibodies and were associated with pANCA. However, eight sera with pANCA were negative for anti-myeloperoxidase antibodies and three of these were also negative for anti-elastase antibodies, suggesting further unidentified target antigen or antigens associated with the pANCA. Fifteen of the 34 sera positive for ANCA also demonstrated anti-nuclear staining on Hep-2 cells (53%) in a speckled, homogeneous, or nucleolar pattern. ANA were significantly associated with the presence of pANCA (P less than 0.01), and levels of ANA and ANCA fell in parallel after treatment. One serum with a pANCA was also positive for anti-GBM antibodies. Inhibition studies using ELISAs for anti-GBM antibodies indicated that there was no cross-reactivity between target molecules recognized by these antibodies. The diversity of target molecules recognized by ANCA suggests that cross-reactivity with bacterial structures is less likely as the primary aetiological event in the development of these antibodies than tissue destruction; and that cross-reactivity with vascular endothelium is also unlikely as the pathogenetic basis of vessel disease.     

Vasculitis and its renal manifestation with special reference to anti-neutrophil cytoplasmic antibody]

The nomenclature and classification of vasculitis have been problematic, with each researcher proposing their own system. Since the discovery of anti-neutrophil cytoplasmic antibody and the consensus conference on the nomenclature of vasculitis in Chapel Hill, order has been brought to the chaos to some extent. Measurement of P-ANCA and C-ANCA in patients' sera has exerted an immeasurable influence on the clinical aspects of vasculitis, since they are shown to be valuable for the diagnosis and assessment of the vasculitic activity in most cases. Pathogenetic significance is also given to ANCA. ANCA has been reported to release myeloperoxidase(MPO) and proteinase-3 antigens from neutrophils activated by inflammatory cytokines. When both MPO antigen in serum as well as ANCA were measured serially in the same patients, however, clinical condition was better reflected by the amount of serum MPO than the titer of ANCA in some patients. Thus, the amount of serum MPO increased without concomitant rise in ANCA titer in exacerbation of vasculitis in one patient. In two more, high serum titers of ANCA and low MPO antigen in the active phase of vasculitis were reversed when pulmonary hemorrhage occurred, and fatal outcome ensued. It could be speculated from these observations that some ANCA might be directed against the active sites of MPO and even suppress the vasculitic activity through blocking MPO. Since definitive evidence has not been obtained so far on the pathogenetic role of ANCA, the clinical significance of ANCA should be addressed at the same level as anti-dsDNA antibody in systemic lupus erythematosus: aid for diagnosis and disease activity.     

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

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

Steroid-1-dehydrogenases in nocardioform bacteria studied by electrophoresis and immuno blotting techniques

Fifteen noncardioform bacteria strains, capable of transforming steroid compounds were investigated with regard to their range of inducible steroid-1-dehydrogenase (St1DH)1 activities. The St1DHs of these bacteria were compared due to their immuno reactivity in Western blot experiments with a rabbit antiserum raised against the purified St1DH of Rhodococcus rhodochrous 7030. Four strains exhibited a strong immuno reactivity, irrespective of differences in the electrophoretic mobility of the enzymes. Five strains revealed significantly diminished reactivities, and in five strains with a very low St1DH content, no reactivity was found. One strain, designated as Nocardiaspec. 7151, exhibited a high, inducible St1DH activity, but no immunoreaction was found. The absence of immuno reactivity is discussed in connection with the considerably diminished electrophoretic mobility of this enzyme.     

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。     

No association between neutrophil FcgammaRIIa allelic polymorphism and anti-neutrophil cytoplasmic antibody (ANCA)-positive systemic vasculitis.

ANCA, implicated as having a pathogenic role in systemic vasculitis, can activate tumour necrosis factor-alpha (TNF-alpha)-primed neutrophils by cross-linking surface-expressed ANCA antigens with neutrophil FcgammaRIIa receptors to release reactive oxygen species. The FcgammaRIIa receptor exists as polymorphic variants, R131 and H131, which differ in their ability to ligate human IgG2 and IgG3. Neutrophils homozygous for the FcgammaRIIa-H131 allotype bind more efficiently to IgG3 than the FcgammaRIIa-R131 allotype and are the only human FcgammaR which bind IgG2. Our aim was to determine whether the homozygous FcgammaRIIa-H131 individuals are more susceptible to developing ANCA-associated systemic vasculitis and nephritis due to differential IgG binding and activation. FcgammaRIIa allotype was determined by both allele-specific polymerase chain reaction (PCR) and Southern blotting with allele-specific oligonucleotide probes end-labelled with 32P-gammaATP, after PCR amplification of genomic FcgammaRIIa DNA in 107 Caucasian patients with ANCA+ vasculitis (of whom 89 had renal disease) and 100 ethnically matched controls. Phenotyping of neutrophil FcgammaRIIa alleles was confirmed in some patients by quantitative flow cytometry using murine MoAbs 41H16 and IV.3. Of the patients with ANCA+ systemic vasculitis, 75 had ANCA with specificity for proteinase 3 and 32 with specificity for myeloperoxidase. Overall, no skewing in FcgammaRIIa allotypes was seen in patients compared with controls. No significant increase of the FcgammaRIIa-H131 allotype was found amongst patients irrespective of ANCA specificity, and no association between the FcgammaRIIa allotype and nephritis was found. Our data suggest that the FcgammaRIIa receptor allotype is not a major factor predisposing to the development of ANCA+ systemic vasculitis, or to nephritis.     

Protein analysis of newly isolated variants of echovirus type 18 by electrophoresis and western blotting

The protein patterns of two newly isolated antigenic variants of echovirus type 18 were compared with those of the prototype strain. Electrophoretic mobilities of VP1 and VP2 of the variants differed from those of the prototype strain. The antiserum against the prototype strain reacted to VP0 and VP2 in the Western blotting experiment. In addition, the variants differed from the prototype strain in growth behavior in the cultured cells.     

Comparison of methods for the analysis of outer membrane antigens of Neisseria meningitidis by western blotting.

The method of extraction of outer membrane proteins (OMPs), the conditions of electrophoretic transfer, and the conditions of antibody binding, were compared in Western blotting studies of Neisseria meningitidis outer membrane antigens. The OMP profiles obtained by SDS-PAGE of outer membrane vesicles extracted with lithium chloride/sodium acetate were compared with profiles obtained by Sarkosyl extraction; these profiles were further compared with the patterns obtained by 125I-labelling of surface-exposed proteins. Sarkosyl extracts gave profiles most closely resembling those of 125I-labelled whole-cells and gave the best resolution of the major proteins. After transfer in Tris-glycine-methanol buffer some proteins, including the major proteins, were not completely transferred and remained in the gel, with the class 2/3 and 5 proteins not effectively detected on nitrocellulose by amido black staining. There was weak antibody recognition of the class 1 and 4 proteins but good recognition of lipooligosaccharide (LOS) and H8 antigen. Empigen BB had no effect on renaturation of the class 1 protein. When 0.1% SDS was incorporated in the same buffer all of the proteins were removed from the gel, and although the major proteins bound to nitrocellulose other proteins did not. There was weak antibody recognition of the class 1 and 4 proteins, stronger reaction to the class 5 protein, but no recognition of the class 2 protein, LOS or H8 antigen, Empigen BB slightly enhanced antibody recognition of the class 1 protein. After transfer in Tris-glycine buffer, all the major proteins were transferred and bound to nitrocellulose and, other than the class 2 protein, were recognised by antibody, both in the presence or absence of Empigen BB, as were LOS and the H8 antigen. Differences existed in the patterns of antibody recognition between the lithium and the Sarkosyl extracts; additional proteins were recognised in the lithium extracts. The surface-labelling studies indicated, however, that some of these proteins were not surface-exposed. Some minor proteins appeared to be more highly immunogenic than the major proteins.     

Identification of antigens diagnostic for European isolates ofBabesia equi by two-dimensional electrophoresis and Western blotting

A lysate ofBabesia equi-infected erythrocytes (USDA strain) was separated by two-dimensional electrophoresis and anlysed by Western blotting. Nine major antigens or antigen groups with mol. wts. ranging from 43 to 19 kDa were recognized by sera from horses experimentally infected with the USDA strain. Four antigens or antigen groups were also recognized by some or all sera from horses infected withB. caballi or not infected withBabesia spp. Of the remaining five antigens, four were recognized by all sera from field-infected horses from Europe. Thus four antigens with mol. wts. of 33, 31, 19 and 20 kDa were identified as diagnostic antigens for European isolates ofB. equi. None of the antigens diagnostic for European isolates was recognized by sera from field-infected horses from Brazil.     

Immunization with anti-neutrophil cytoplasmic antibody (ANCA) induces the production of mouse ANCA and perivascular lymphocyte infiltration.

Wegener's granulomatosis (WG) is a granulomatous necrotizing vasculitis associated with the presence of ANCA, predominantly directed against proteinase 3 (PR3). The titres of ANCA correlate with disease activity and titre increases may precede disease exacerbations. Previously, we have shown that it is possible to induce autoimmune disease (systemic lupus erythematosus (SLE) and anti-phospholipid syndrome) in naive mice following active immunization with human autoantibodies, namely anti-DNA and anti-cardiolipin, respectively. The mice developed first anti-autoantibodies and, after about 4 months anti-anti-autoantibodies (Ab3), simulating auto-antibodies (Ab1) in their binding activities, and their presence was associated with the development of disease manifestations, characteristic of the human disease. So far, there is no good animal model for WG. In the current study we have immunized mice with human ANCA with the aim of inducing experimental WG. In two separate studies 30 mice were immunized in their footpads with autoantigen-purified IgG fraction (ANCA) from the sera of two patients with untreated WG, emulsified in Freund's complete adjuvant, followed 3 weeks later by ANCA injection in PBS. In the first experiment mice immunized with ANCA developed sterile microabscesses in the lungs after 8 months, and died after 8-15 months. In the second experiment, mice immunized with ANCA developed after 4 months mouse ANCA, with specificity both to PR3 and to myeloperoxidase, as well as anti-endothelial autoantibodies (AECA), as shown by radioimmunoprecipitation. Pathologically, the immunized mice developed proteinuria but not haematuria, and histological sections of the lungs demonstrated mononuclear perivascular infiltration, while diffuse granular deposition of immunoglobulins was noted in the kidneys. Our results point to a pathogenic role of ANCA in WG, and confirm the importance of the idiotypic network in the etiopathogenesis of autoimmune conditions.     

抗中性粒细胞胞浆抗体检测在类风湿性关节炎并发新月体性肾小球肾炎中的临床意义

目的:探讨类风湿性关节炎(rheumatoid arthritis,RA)并发新月体性肾小球肾炎(crescenticglomerulonephritis,CrGN)抗中性粒细胞胞浆抗体(antineutrophil cytoplasmic antibody,ANCA)检测的临床意义。方法:回顾性分析2000年1月至2010年12月萍乡市人民医院诊治的5例RA并发CrGN患者(RA并发CrGN组)、86例RA(RA组)和16例ANCA相关性小血管炎患者(ANCA相关性小血管炎组)的ANCA检测结果及临床病理特征。结果:RA并发CrGN组中,5例都为中年妇女,且具有7~14年的RA病龄,最初的临床症状都为血尿,从血尿发展到具有明显组织病变平均时间为17个月,ANCA检测均为核周型ANCA(pANCA)阳性,靶抗原为髓过氧化物酶(myeloperoxidase,MPO),被检肾小球平均有31%形成新月体,同时也存在肾小球硬化和间质性纤维化等症状;RA组ANCA检测均为阴性,ANCA相关性小血管炎组ANCA检测阳性,阳性率为87.5%(14/16),且均为pANCA;与ANCA相关性小血管炎组相比,RA并发CrGN组确诊年龄明显小于前者(P<0.05)。结论:RA并发CrGN发病年龄趋于年轻化,疾病的发展过程趋于漫长并且伴随轻微肾功能损伤,ANCA检测对RA并发CrGN患者早期发现、治疗具有重要的意义。