Utility of serum complement factors C3 and C4 as biomarkers during therapeutic management of giant cell arteritis.

OBJECTIVE: There is a strong unmet need for biomarkers in giant cell arteritis (GCA), as C-reactive protein (CRP) may be unreliable in patients treated with Tocilizumab (TCZ). We aimed to assess whether C3 and C4 are useful biomarkers in GCA patients, particularly in those treated with TCZ. METHOD: We retrospectively enrolled all patients who underwent C3 and C4 measurement at baseline. All patients were evaluated at 3, 6, 12, and 24 months after diagnosis, as part of routine follow-up. Two assessments after the end of the observational period, in case of further relapses, were also included. RESULTS: At baseline, mean ± sd levels (mg/dL) of C3 (133 ± 28.99) and C4 (25.9 ± 9.04) were within normal ranges. During follow-up, C3 and C4 decreased in patients attaining remission (107.07 ± 19.86, p = 0.0006; 19.86 ± 10.27, p = 0.01, respectively) and sustained remission (95.85 ± 18.04, p = 0.001; 15.61 ± 9.75, p = 0.006). In TCZ-treated patients, even stronger decreases in C3 (83.11 ± 19.66, p = 0.001) and C4 (8.26 ± 3.83, p < 0.0001) were observed, and their values were not correlated with CRP or erythrocyte sedimentation rate. CONCLUSION: C3 and C4 do not seem useful in the diagnosis of GCA, as normal values do not rule out active vasculitis. However, C3 and C4 correlate with disease activity. As the low C4 levels found in TCZ-treated patients are not correlated with CRP, C4 should be evaluated as a potential biomarker of disease activity and treatment response.

[ANCA diagnostics in vasculitis]. / ANCA-Diagnostik bei Vaskulitiden.

The cornerstone of the laboratory diagnostics of small vessel vasculitis is the detection of antineutrophil cytoplasmic antibodies (ANCA). The current international consensus recommendations suggest that proteinase 3 (PR3) and myeloperoxidase (MPO) ANCA immunoassays should be used as a first-line test if there is a justified suspicion of ANCA-associated vasculitis (AAV). A second method is only recommendable when the immunoassay shows a negative or borderline result. The precise identification of all patients with active AAV and avoidance of misdiagnoses due to false positive ANCA measurements is achieved when the ANCA determination is limited to defined clinical situations, which are indicative for AAV. There is increasing evidence that the specificity of ANCA to define homogeneous groups of patients could be better with respect to the prognosis than the clinical subtype.

[Paradigm shift in ANCA diagnostics : New international consensus recommendations]. / Paradigmenwechsel in der ANCA-Diagnostik : Neue internationale Konsensusempfehlungen.

BACKGROUND: Up to now indirect immunofluorescence (IIF) followed by an antigen-specific assay specific for proteinase 3 (PR3) or myeloperoxidase (MPO) has been the standard method for the detection of antineutrophil cytoplasmic antibodies (ANCA). The development of more sensitive and highly specific PR3-ANCA and MPO-ANCA immunoassays for the diagnosis of ANCA-associated vasculitis (AAV) has raised doubts about the two-stage diagnostic strategy currently recommended for ANCA detection. OBJECTIVE: Presentation and discussion of the new international consensus recommendations on ANCA testing in AAV. METHODS: This article presents the new guidelines for ANCA testing that have been developed based on the results of a recent large multicenter study by the European Vasculitis Society (EUVAS). The draft of the author committee was revised by each contributor and subsequently distributed to 12 experts on 4 continents. After further revision the final document was returned for ratification and submitted for publication. RESULTS/CONCLUSION: The current study results confirm the superiority of the diagnostic value of antigen-specific immunoassays compared to IIF. The current consensus recommendations support the primary use of PR3-ANCA and MPO-ANCA immunoassays for diagnostic evaluation of patients with AAV without the categorical need for additional IIF.

Correlation of serum level of high mobility group box 1 with the burden of granulomatous inflammation in granulomatosis with polyangiitis (Wegener's).

OBJECTIVES: To investigate the correlation of serum levels of high mobility group box 1 (HMGB1) with the extent of granulomatous inflammation in granulomatosis with polyangiitis (GPA). METHODS: From 169 patients with GPA, 17 patients with granulomatous inflammation, without evidence of vasculitis were identified and 36 patients without measurable 'granuloma' formation. HMGB1 serum levels were determined and compared between the two groups, using a Mann-Whitney U test. Serum levels of 26 healthy individuals served as controls. In a further 21 patients with GPA with a pulmonary granulomatous manifestation from the study population, CT volumetry of 'granuloma' was performed. Volumes were compared with serum levels of HMGB1 (Spearman rank order test). RESULTS: Serum levels of HMGB1 were significantly higher in patients with predominant granulomatous disease than in patients without measurable 'granuloma' manifestations (6.44 ± 4.53 ng/ml vs 3.85 ± 2.88 ng/ml; p=0.0107). In both groups, levels of HMGB1 were significantly higher than in controls (2.34 ± 2.01 ng/ml; p<0.01). A positive correlation of HMGB1 serum levels with volumes of pulmonary 'granuloma' (r=0.761, p<0.0017) was seen. CONCLUSIONS: HMGB1 serum levels are significantly higher in GPA with predominant granulomatous manifestations and correlate with volumes of pulmonary 'granuloma'. HMGB1 may be used as a marker of the burden of granulomatous inflammation in GPA.

Neutrophil activation by anti-proteinase 3 antibodies in Wegener's granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation.

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

Wegener's granulomatosis: anti-proteinase 3 antibodies are potent inductors of human endothelial cell signaling and leakage response.

Anti-neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor alpha induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng-2.5 microg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen-antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis-related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3-induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.

Eotaxin-3 is involved in Churg-Strauss syndrome--a serum marker closely correlating with disease activity.

OBJECTIVE: Churg-Strauss Syndrome (CSS) is characterized by excessive eosinophil accumulation in peripheral blood and affected tissues with development of granulomatous vasculitic organ damage. The contribution of eosinophil-chemotactic cytokines (eotaxin family) to eosinophilia and disease activity in CSS is unknown. Thus, we compared serum levels of the eotaxin family members in CSS patients with healthy and disease controls. METHODS: Forty patients with CSS diagnosed according to ACR 1990 criteria, 30 healthy controls (HC) and 57 disease controls (28 asthma, 20 small vessel vasculitis, 9 hypereosinophilic syndrome) were studied. Clinical data were collected and serum levels of eotaxin-1, -2 and -3 were determined by ELISA. Further, immunohistochemistry was applied to identify eotaxin-3 expression in tissue biopsies from patients with CSS. RESULTS: In contrast to eotaxin-1 and -2, eotaxin-3 was highly elevated in serum samples of active CSS patients and correlated highly significantly with eosinophil counts, total immunoglobulin E (IgE) levels and acute-phase parameters. Moreover, eotaxin-3 was not elevated in other eosinophilic and vasculitic diseases. Immunohistochemical analysis revealed strong expression of eotaxin-3 in endothelial and inflammatory cells in affected tissues of active CSS patients. CONCLUSIONS: This study reveals the specific association of elevated eotaxin-3 expression with high disease activity and eosinophilia in CSS patients. Eotaxin-3 might thus be a pathogenic player, biomarker and potential therapeutic target in CSS.

Proteinase 3, protease-activated receptor-2 and interleukin-32: linking innate and autoimmunity in Wegener's granulomatosis.

Proteinase 3 (PR3) is a multifunctional neutrophil-derived serine protease influencing cell cycle, differentiation, and cell death. This molecule is the main target antigen of autoantibodies in Wegener's granulomatosis (WG) known as antineutrophil cytoplasmic antibodies (PR3-ANCA). WG usually starts as granulomatous inflammation of the upper respiratory tract (localized phase) and progress to systemic disease with PR3-ANCA-associated vasculitis (generalized phase). PR3-ANCA is thought to play a critical role in the pathogenesis of vascular damage in WG. In contrast, it is not clear how the granulomatous inflammation, the hallmark of WG, is driven, and what is the relationship between granuloma and autoimmunity. Recent findings provide evidence that PR3 might function as endogenous "danger/alarm" signal that communicates the presence of tissue injury to dendritic cells (DC) via protease-activated receptor-2 (PAR-2), triggers their maturation and instructs DC to induce Th1-type cell responses in WG. Furthermore, PR3 has the capacity to bind and activate IL-32, a recently discovered proinflammatory cytokine that has emerged as an important player in innate and adaptive immune response.Collectively, these results delineate new pathogenic pathways at the molecular level and provide insights into the mechanisms by which PR3 may contribute to the early pathogenesis of WG supporting the pivotal role of the interaction of Wegener's autoantigen with the "gateway" receptor PAR-2 in mediating both innate and adaptive immune response in WG.

Evaluation of the FIDIS vasculitis multiplex immunoassay for diagnosis and follow-up of ANCA-associated vasculitis and Goodpasture's disease.

We have evaluated a new-multiplex immunoassay (FIDIS Vasculitis) for simultaneous detection and quantification of anti-MPO, -PR3, and -glomerular basement membrane (GBM) antibodies in diagnosis and follow-up of ANCA-associated vasculitides (AAV) and Goodpasture's disease. ANCA were determined in sera of (a) 87 consecutive patients with biopsy-proven pauci-immune NCGN and 72 controls; (b) 9 patients with Goodpasture's disease; and (c) 60 WG patients and 60 controls, previously used in a multicenter comparison of direct and capture ELISA for PR3-ANCA. Finally, for prediction of relapses, PR3-ANCA was measured in samples preceding relapse in 23 PR3-AAV patients and in 23 matched PR3-AAV patients without relapse. The relative sensitivity of the FIDIS Vasculitis assay was 97.4% for MPO-ANCA and 92.3% for PR3-ANCA; specificity was 100% and 97.2%, respectively. Evaluation of the anti-GBM antibody detection revealed a sensitivity of 100% and a specificity of 99.6%. The sensitivity for WG of the PR3-ANCA detection (71.6%) approached the performance of capture ELISA (74%), although at the cost of specificity (96.7% versus 100%). For prediction of relapses a rise of 50% in ANCA level by FIDIS Vasculitis appeared optimal (ROC curve) for prediction of relapses. However, as compared to capture ELISA, both positive (63% versus 76%) and negative (68% versus 72%) predictive values were reduced. In conclusion, simultaneous detection of anti-MPO, -PR3, and -GBM antibodies in the multiplex FIDIS Vasculitis assay has excellent performance in terms of diagnosis of patients with AAV or Goodpasture's disease. However, detection of rises in PR3-ANCA for prediction of relapses gives less optimal results when compared to capture ELISA.

A novel high sensitivity ELISA for detection of antineutrophil cytoplasm antibodies against proteinase-3.

OBJECTIVE: Conventional direct enzyme-linked immunosorbent assays (ELISA) for the detection of anti-neutrophil cytoplasm antibodies (ANCA) often lack sensitivity because epitopes of the target antigen are hidden by binding to the ELISA plate. This study was designed to evaluate a novel ELISA method for detection of ANCA against proteinase-3 (PR3) for the diagnosis of Wegener's granulomatosis (WG) using PR3 presented in its native form. METHODS: Sera from four subgroups of patients with a diagnosis of WG (n=86), 80 healthy controls and 450 disease controls were tested for the presence of C-ANCA/PR3-ANCA by anchor ELISA, direct ELISA, capture ELISA, indirect immunofluorescence (IFT) and immunoblotting. RESULTS: In prospectively analysed consecutive patients, anchor ELISA showed the highest sensitivity for a diagnosis of WG of 96.0% (95% CI: 79.6-99.3), followed by IFT 92.0% (73.9-98.8), capture ELISA 72.0 (50.6-87.9) and direct ELISA 60.0 (38.7-78.8). Specificity was high for all methods and ranged from 98.5 (97.0-99.4) to 95.5% (97.9-99.8). Receiver operating characteristics curve analysis revealed that the overall diagnostic performance of the anchor ELISA was significantly superior compared to the direct ELISA and the capture ELISA in patients with generalized WG, and also compared to IFT and immunoblotting in patients with localised WG. CONCLUSION: Anchor ELISA is a novel highly sensitive and specific method for the detection of PR3-ANCA in patients with WG, which may replace the need for a combined analysis with IFT and ELISA in the future.

Proinflammatory cytokines and autoimmunity in Churg-Strauss syndrome.

Churg-Strauss syndrome (CSS) belongs to the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides and is further characterized by severe eosinophilia and, often, granulomatous inflammation. The therapeutic efficacy of recombinant interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) blockade point toward a central role of cytokines in the pathogenesis of CSS. Recent data show that, in contrast to other primary systemic vasculitides, peripheral blood mononuclear cells (PBMCs) secrete not only large amounts of T helper type 1 (Th1) cytokines, particularly IFN-gamma, but also release T helper type 2 (Th2) cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13). Interleukin-5 is the most potent stimulator of eosinophil production and functional activation of mature eosinophils, the key effector cells in CSS. Data are presented showing that PBMCs from patients with CSS cultured with T cell-specific stimuli secrete significantly increased amounts of IL-5 compared with healthy controls, suggesting that IL-5 contributes substantially to the development of eosinophilia in CSS. As recombinant IFN-alpha downregulates IL-5 production of CD4(+) T cells in vitro, the increased secretion of IL-5 in patients with CSS may provide the clue for the therapeutic efficacy of recombinant IFN-alpha in the disease. Variations in the balance between Th1 and Th2 cytokines at different disease stages could contribute to the distinct clinical courses seen in patients with CSS, which can range from prominent Th1-mediated generalized vasculitis and granulomatous inflammation on one end of the spectrum to Th2-mediated systemic hypereosinophilia on the other. Although the association of ANCAs with CSS point toward an autoimmune origin of the disease, there is no direct evidence as yet for a direct pathogenic role of ANCAs in CSS.

Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis.

Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 microg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5-7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis.

Diagnostic Value of Procalcitonin in ANCA-Associated Vasculitis (AAV) to Differentiate Between Disease Activity, Infection and Drug Hypersensitivity.

OBJECTIVE: Procalcitonin (PCT) is considered to be a specific marker for severe bacterial infections and sepsis. Elevated PCT levels have been reported in active autoimmune diseases without infection. The aim of this study was to assess the diagnostic value of PCT serum levels in ANCA-associated vasculitis (AAV) patients with respect to infection, disease activity and drug fever using a high sensitive PCT detection method. METHODS: In 53 AAV patients with elevated C-reactive protein (CRP) PCT was determined by the Thermo Scientific BRAHMS PCT sensitive KRYPTOR assay. Patients underwent standardized diagnostic procedures for evaluation of disease activity and infection. RESULTS: 53 patients with AAV and elevated CRP (7.7±6.9 mg/dl, PCT 0.34±1.02 ng/ml) were assessed, 10 had infection with elevated CRP levels of 11.2±10.2 mg/dl and PCT levels of 1.06±2.07 ng/dl. 43 patients had no evidence of infection, 36 of them were presented with AAV with normal or only slightly positive PCT levels in active disease (n=36) (PCT 0.06±0.06 ng/ml). 7 patients had increased PCT levels due to azathioprine hypersensitivity (0.76±1.01 ng/ml). For discrimination between infection and vasculitis activity PCT was more useful than CRP with the best cut-off at 0.1 ng/ml (sensitivity 60%, specificity 92%). CONCLUSION: In contrast to previous studies using semiquantitative PCT assays, the KRYPTOR performs better with respect to discrimination of infection from active AAV. In all patients assessed with active AAV (and without infection) PCT levels remained below the PCT reference limit (0.5 ng/ml) for infections. Drug hypersensitivity seems to be an important differential diagnosis in the setting of elevated CRP and PCT in patients who receive azathioprine.

Comparison of eight commercial kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA).

Antineutrophil cytoplasmic antibodies (ANCA) are used as diagnostic markers for systemic vasculitis. However, the specificity and sensitivity of ANCA detection differs from centre to centre due in large part to variations in methodology. We compared 8 commercial ELISA kits and an in-house method (HM) for their specificity and sensitivity in detecting ANCA against proteinase 3 (PR3-ANCA, 7 kits) and meyloperoxidse (MPO-ANCA, 8 kits). Sera from 5 patients with systemic lupus erythematosus (SLE), 28 with Wegener's granulomatosis (WG), 22 with microscopic polyangiitis (MPA), 5 with idiopathic rapidly progressive glomerulonephritis (RPGN), and 5 healthy controls were examined by both the indirect immunofluorescence technique (IFT) and the ELISA kits. Sera from healthy controls and patients with SLE or cANCA-negative WG were shown to be PR3-ANCA negative by all 7 PR3-ANCA kits. In 25 cANCA-positive sera from WG patients, PR3-ANCA positivity ranged from 44% to 84%. An absolute concordance among the 7 kits was noted in 56% of the cANCA-positive samples. The PR3-ANCA levels in 5 of the 7 kits correlated with the cANCA titers in IFT. Sera from the healthy controls and 4 out of the 5 SLE and pANCA-negative patients were found to be MPO-ANCA negative in all 8 MPO-ANCA kits. In 20 pANCA-positive sera, MPO-ANCA positivity ranged from 25% to 75%. Thirty-five percent of MPO-ANCA-positive sera were confirmed by capture ELISA, immunoblot and inhibition assay. The concordance rate was only 30% among pANCA-positive sera in the 8 MPO-ANCA kits. No significant correlation was observed between pANCA titers and MPO-ANCA levels. The HM showed that 65% of cANCA-positive sera were PR3-ANCA positive, and 45% of pANCA-positive sera were MPO-ANCA positive. Our results indicate that the sensitivities and specificities for ANCA detection differ significantly among the commercial kits tested and underline the necessity of establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of data.

Use of native and recombinant bactericidal/permeability-increasing proteins (BPI) as antigens for detection of BPI-ANCA.

Anti-neutrophil cytoplasmic antibodies (ANCA) against native bactericidal/permeability-increasing protein (nBPI) have gained increasing diagnostic significance in inflammatory bowel disease and cystic fibrosis. However, routine detection of BPI-ANCA requires pure antigen in large quantities. As nBPI is difficult to isolate and is very susceptible to proteolytic cleavage with subsequent epitope loss, it was the aim of this study to determine whether recombinant BPI (rBPI) can be used as an alternative to nBPI as target antigen for ANCA in diagnostic procedures. Therefore, 93 BPI-ELISA-positive sera and controls were compared in different ELISAs using nBPI, rBPI, unglycosylated rBPI and a 21-kDa amino-terminal fragment of rBPI. ELISA results were confirmed by immunoblotting and all sera were tested in indirect immunofluorescence (IFT). There was an 88% (82/93) agreement in recognition of nBPI and rBPI by ANCA in both ELISA systems, yet the quantitation of BPI-ANCA in relative units showed a less optimal result and correlated only by 45% (p < 0.01). Most sera recognized nBPI, rBPI and unglycosylated rBPI equally suggesting that glycosylation has no influence on antigen recognition. Only two sera were positive for the 21-kDa nBPI indicating that the binding sites for ANCA are either conformational epitopes and/or are located mainly on the carboxy-terminal part of the BPI molecule. Most BPI-ELISA-positive sera were negative in IFT (43%), but a perinuclear (pANCA, 30%), a cytoplasmic (cANCA,10%) or an atypical ANCA (aANCA, 2%) staining pattern, as well as a cytoplasmic pattern only on formaldehyde-fixed granulocytes (13%) were also observed. Overall, no characteristic pattern was seen for BPI-ELISA-positive sera in IFT. Taken together, these data suggest that rBPI offers an excellent alternative to nBPI for broad-based BPI-ANCA ELISA and will be of great value in further investigations of BPI-ANCA interactions.

A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis.

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegener's granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in WG by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line (SK-RC11) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA+ sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a SK-RC11 extract and a high-titer C-ANCA+ serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with MHC II. In addition, 21 WG patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non-leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins.

Development and standardization of solid phase assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). A report on the second phase of an international cooperative study on the standardization of ANCA assays.

Anti-neutrophil cytoplasmic antibodies (ANCA) are diagnostic markers for systemic vasculitis. They are classically detected by an indirect immunofluorescence test using normal donor neutrophils as substrate. This assay lacks antigenic specificity and is not quantitative. The 'EC/BCR Project for ANCA Assay Standardization' is an international collaboration study with the aim to develop and standardize solid phase assays for ANCA detection. In this part of the study the isolation and characterization of proteinase-3 and myeloperoxidase, the two main target molecules for ANCA, and the development and standardization of ELISAs with these antigens are described. Six laboratories successfully isolated purified proteinase-3 preparations that could be used. Three of these preparations, together with one myeloperoxidase preparation, were subsequently used for ANCA testing by ELISA. The ELISA technique was standardized in two rounds of testing in the 14 participating laboratories. The coefficient of variation of these new assays decreased from values of approx. 50% in the first round to approx. 20% in the second round. We conclude that purified proteinase-3 and myeloperoxidase can be used in standardized ELISAs for ANCA detection. Whether such procedures offer advantages over the IIF test will be determined in a prospective clinical study.