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.

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.

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.

[Autoantibody detection by indirect immunofluorescence on HEp-2 cells]. / Autoantikörpernachweis mittels indirekter Immunfluoreszenz an HEp-2-Zellen.

Systemic autoimmune diseases are characterized by the presence of antinuclear autoantibodies (ANA). Diluted patient sera are typically used to screen for the presence of ANA by immunfluorescence microscopy with fixed HEp-2 cells. Despite high-quality test kits, reports of different laboratories frequently present controversial results. This article recommends unified processing and interpretation of HEp-2 based screening for autoantibodies. Suggestions are made for the selection of high-quality test kits, optimized processing and diagnostic procedures. In addition to a relevant clinical diagnosis and an experienced laboratory specialist, the following procedure is highly recommended to achieve good laboratory practice: Initial HEp-2 based screening by indirect immunofluorescence, starting with a 1:80 serum dilution, and evaluation in a bright fluorescence microscope, pathological values from a titer of 1:160 upwards, internal quality checks and unified interpretation. We aim to improve diagnosis and care of patients with autoimmune diseases as a central focus of the European Autoimmunity Standardization Initiative (EASI).

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.

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.

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.

Variations in performance characteristics of commercial enzyme immunoassay kits for detection of antineutrophil cytoplasmic antibodies: what is the optimal cut off?

BACKGROUND: Previous studies have shown considerable variation in diagnostic performance of enzyme linked immunosorbent assays (ELISAs) for measuring antineutrophil cytoplasmic antibodies (ANCA) specific for proteinase 3 (PR3) and myeloperoxidase (MPO). OBJECTIVE: To analyse the performance characteristics of different commercially available direct ANCA ELISA kits. METHODS: ELISA kits for detecting PR3-ANCA and MPO-ANCA from 11 manufacturers were evaluated. Serum samples were taken from patients with Wegener's granulomatosis (15), microscopic polyangiitis (15), other vasculitides (10), and controls (40). RESULTS: were compared with data obtained by indirect immunofluorescence (IFT). The diagnostic performance of the tests was analysed and compared by receiver operating characteristic (ROC) curve analysis.Results: Applying the manufacturers' cut off resulted in great variation in sensitivity of the commercial PR3-ANCA kits for diagnosing Wegener's granulomatosis (ranging from 13.3% to 66.7%), and of the MPO-ANCA kits for diagnosing microscopic polyangiitis (ranging from 26.7% to 66.7%). Specificities were relatively constant (from 96.0% to 100%). IFT was superior to all ELISAs (C-ANCA for Wegener's granulomatosis: sensitivity 73.3%, specificity 98%; P-ANCA for microscopic polyangiitis: sensitivity 86.7%, specificity 98%). The sensitivities of PR3-ANCA and MPO-ANCA ELISA kits were increased by lowering the cut off values. This reduced specificity but increased overall diagnostic performance. CONCLUSIONS: The low sensitivity of some commercial kits reflects the high cut off levels recommended rather than methodological problems with the assays. Comparative analyses using sera from well characterised patients may help identify optimum cut off levels of commercial ANCA ELISA tests, resulting in better comparability of results among assays from different manufacturers.

Association study of Wegener granulomatosis and the functionally relevant A645G polymorphism in the bactericidal/permeability increasing protein (BPI) gene.

In antineutrophil cytoplasmatic antibody (ANCA)-associated vasculitides (AAV), bactericidal/permeability increasing protein (BPI) ANCAs are detected. Recent observations suggest that BPI-ANCAs can potentially contribute to a proinflammatory setting in the absence of proteinase 3 (PRTN3) ANCAs during the development of a pulmonary relapse by impeding the elimination of Gram-negative bacteria (GNB). However, it is as yet not clear whether the genetic background contributes to the generation of BPI-ANCAs in Wegener granulomatosis (WG) or if BPI polymorphisms are associated with WG. In this study we genotyped the functionally relevant single nucleotide polymorphism (SNP) A645 (Glu216Lys) of the BPI gene in 201 WG patients and 608 healthy controls. To investigate whether further SNPs might be associated with WG, we also examined an intragenic microsatellite marker. No significant differences were found between patients and controls. Thus BPI polymorphisms do not appear to contribute to genetic predisposition to WG. Moreover, our data do not suggest a genetic background for the generation of BPI-ANCAs in WG.

Small vessel vasculitis and relapsing panniculitis in tumour necrosis factor receptor associated periodic syndrome (TRAPS).

CASE REPORTS: A 66 year old female patient had relapsing fever and non-suppurative panniculitis suggestive of enigmatic "Weber-Christian disease" (WCD). Antineutrophil cytoplasmic antibodies with specificity for human leucocyte elastase (HLE-ANCA) were detected. A biopsy showed small vessel vasculitis and panniculitis. A 53 year old man had recurrent episodes of abdominal pain, erythematous rash, and myalgia. Fever attacks had stopped a few years ago. A biopsy showed panniculitis and fasciitis. In both patients mutations (R92Q, T50M) of the tumour necrosis factor receptor super family (TNFRSF) 1A gene were disclosed. Mutations of the TNFRSF 1A gene are the cause of tumour necrosis factor receptor associated periodic syndrome (TRAPS). Both patients responded favourably to treatment with the human soluble p75 TNF alpha receptor fusion protein etanercept (2 x 25 mg subcutaneously/week). DISCUSSION: Small vessel vasculitis and panniculitis have not been reported in TRAPS so far. The cases underline the importance of TNF alpha regulation in inflammatory processes including vasculitis. Genetically determined causes of fever may account for some cases of WCD.

Update on the pathogenesis of Churg-Strauss syndrome.

Churg-Strauss syndrome (CSS) is a rare form of systemic vasculitis occurring in patients with asthma. The cause of CSS is unknown, and yet little data are available regarding its pathogenesis. The presence of a marked tissue- and blood-eosinophilia, as well as secretory products of eosinophils in blood and tissues, implicates a pathogenetic role of eosinophil granulocytes. Prolonged survival of eosinophils due to inhibition of CD95-mediated apoptosis by soluble CD95 seems to contribute to eosinophilia in CSS. Although the mechanisms involved in eosinophil-activation in CSS have not been elucidated, recent data suggest a possible role of T lymphocytes secreting eosinophil-activating cytokines. This review describes the current insights into the pathogenesis of CSS in the light of its putative nature as a type 2 granulomatous disease. Recent clinical, experimental and epidemiologic data regarding the possible role of inflammatory cells and their secretory products, anti neutrophil cytoplasm antibodies (ANCA), epidemiologic factors and anti-asthma treatments are summarized.

Prevalence of ANCA in mixed cryoglobulinemia and chronic hepatitis C virus infection.

OBJECTIVE: To determine the prevalence, target antigens and clinical associations of antineutrophil cytoplasmic antibodies (ANCA) in chronic hepatitis C without extrahepatic manifestations and in chronic hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) in two European centers. METHODS: 50 sera from patients with chronic hepatitis C and 116 sera from HCV-associated MC were tested for cytoplasmic or perinuclear pattern (C-ANCA/P-ANCA) by indirect immunofluorescence test (IFT). ANCA target antigens were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Clinical characteristics of the patients were not different between the two centers. Cryoglobulinemic vasculitis (CV) was biopsy-proven in about 90% of the MC patients. Two patients with HCV-associated MC and 1 patient with chronic hepatitis C had a P-ANCA. A C-ANCA was detected in 1 patient with HCV-associated MC. Eight patients with a HCV-associated MC and 5 patients with chronic hepatitis C had an ANCA either directed against bactericidal/permeability increasing protein (BPI) or cathepsin G (CG). BPI- or CG-ANCA positivity was not associated with a more severe disease course. The C-ANCA titer followed disease activity in one C-ANCA positive HCV-associated MC patient. The subspecificity of the C-ANCA was not determinable in that patient. CONCLUSION: Two new target antigens of ANCA have been identified in HCV-associated MC and chronic hepatitis C in this study. BPI-ANCA and GC-ANCA were present in about 10% of patients with HCV-associated MC or chronic hepatitis C. ELISA proved to be more sensitive in the detection of ANCA than IFT. The present study on chronic HCV infection adds to various reports on the induction of CG- and BPI-ANCA in chronic infections.