Marjan C. Slot

Evaluation of a new fluorescent-enzyme immuno-assay for diagnosis and follow-up of ANCA-associated vasculitis

In this study we have evaluated a new, fully automated fluorescent-enzyme immuno-assay (FEIA) for detection and quantification of anti-PR3 and anti-MPO ANCA in diagnosis and follow-up of ANCA-associated small vessel vasculitis (AAV). PR3- and MPO-ANCA were determined by FEIA technology in (1) sera of 87 consecutive patients with biopsy-proven, pauci-immune necrotizing crescentic glomerulonephritis (NCGN) and 72 controls; (2) 120 sera (60 patients with Wegener’s granulomatosis and 60 controls) that were previously used in a multicentre comparison of direct and capture ELISAs for PR3-ANCA; (3) in samples preceding relapse in 23 PR3-AAV patients with and 23 matched PR3-AAV patients without relapse for prediction of relapses. PR3- and/or MPO-ANCA detection in pauci-immune NCGN by FEIA revealed an overall sensitivity of 82.8%. The FEIA specificity was 96% and 100% for PR3- and MPO-ANCA, respectively. The overall sensitivity of MPO- and PR3-ANCA could be increased to 88.5% by lowering the cut-off values without affecting the specificity (ROC-curve analysis), which is similar to a multistep ANCA procedure that combines indirect immunofluorescence with direct and capture ELISAs. The sensitivity for Wegener’s granulomatosis (WG) of the PR3-ANCA FEIA (60%) was more comparable to direct ELISAs (64%) than to capture ELISAs (74%). A rise of 100% in ANCA level as measured by FEIA appeared optimal (ROC-curve) for prediction of relapses and such a rise was observed in 26 patients. In 18 of these 26 patients the rise was followed by a relapse (PPV 69%), whereas in 15 of the 20 patients without a rise no relapse was observed (NPV 75%). In conclusion, detection of PR3- and MPO-ANCA by FEIA has excellent performance in terms of diagnosis of AAV patients. Furthermore, detection of rises in PR3-ANCA by FEIA for prediction of relapses gives results comparable to other techniques.

Evaluation of antibodies against human HSP60 in patients with MPO-ANCA associated glomerulonephritis: a cohort study

BackgroundHuman Heat Shock Protein 60 (hHSP60) has been implicated in autoimmunity through molecular mimicry, based on the high degree of homology with HSP65 of micro-organisms leading to autoimmune recognition of the human protein. Additionally, sequence homology between hHSP60 and myeloperoxidase (MPO) has been described. MPO is a major autoantigen in vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA). We hypothesized that infections may trigger the ANCA response against MPO through hHSP60.MethodsIn 86 consecutive patients with ANCA-associated vasculitis (AAV), anti-hHSP60 and anti-mycobacterial HSP65 were measured by ELISA. Patients were compared with 69 healthy controls (HC). Continuous data between groups were compared using Wilcoxon signed rank test and Kruskal-Wallis test with Dunns post-test when appropriate. Correlations between data were derived using Spearman correlation. Odds ratios and 95% confidence intervals were obtained using Fishers exact test.ResultsAt diagnosis, median anti-mHSP65 level was higher in AAV (median [range]: 42.5 [0–500]), and subsequently in MPO-ANCA (44 [7–500]), compared to HC (22 [0–430]). Anti-hHSP60 levels in AAV were not higher compared to HC (18 [0–319] and 18.5 [0–98], respectively). However, in MPO-ANCA anti-hHSP60 levels were increased (32.5 [0–319]) compared to PR3-ANCA (13 [0–79]) and HC. We could not detect cross-reactivity between hHSP60 and MPO-ANCA. There was a correlation between anti-mHSP65 and anti-hHSP60 levels (r = 0.32, P = 0.003) but not between anti-hHSP60 and MPO-ANCA (r = -0.064, P = 0.69).ConclusionAntibodies against mHSP65 are higher in AAV compared to HC, and anti-hHSP60 antibodies are higher in patients with MPO-ANCA than in patients with PR3-ANCA and HC. Although this finding may be indicative for cross-reactivity between MPO-ANCA and hHSP60, additional assays did not support this hypothesis.

Anti‐oxidized low‐density lipoprotein antibodies in myeloperoxidase–positive vasculitis patients preferentially recognize hypochlorite‐modified low density lipoproteins

Many patients surviving vasculitis are prone to accelerated atherosclerosis and often have enhanced levels of antibodies to oxidized low‐density lipoprotein (oxLDL). To measure anti‐oxLDL antibodies, oxidation of LDL is achieved with copper (Cu) or malondialdehyde (MDA). Because, in vivo, LDL may be oxidized with myeloperoxidase (MPO) or its product hypochlorite, we measured anti‐hypochlorite LDL antibodies in patients with vasculitis, haemodialysis patients and healthy controls. A newly developed enzyme‐linked immunosorbent assay (ELISA) was used to detect antibodies to oxLDL as modified by hypochlorite. Results are compared with data obtained by standard LDL oxidation using MDA–LDL or Cu–LDL as substrate. Results were compared between anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis (AAV) patients (n = 93), haemodialysis (HD) patients (n = 59) and healthy controls (HC; n = 43). Furthermore, patients with MPO–ANCA‐associated vasculitis (n = 47) were compared with patients with proteinase 3 (PR3)–ANCA associated vasculitis (n = 46). Optimal cut‐off points were determined by receiver operator characteristic (ROC) curve analysis. Anti‐oxLDL antibodies are enhanced in AAV patients (MDA–LDL and hypochlorite–LDL) and in HD patients (hypochlorite–LDL), when compared to HC. Furthermore, patients with MPO–ANCA‐associated vasculitis had higher levels of antibodies to hypochlorite–LDL than patients with PR3–ANCA‐associated vasculitis. Our newly developed assay, in which hypochlorite–LDL is used as substrate, seems a more sensitive assay than traditional assays to measure oxLDL antibodies. Furthermore, our results suggest that enhanced MPO‐mediated LDL oxidation occurs in patients with MPO–ANCA.

Wegener's Granulomatosis

Wegener’s granulomatosis (WG) is a disease of presumed autoimmune origin characterized by necrotizing granulomatous inflammation of the upper and lower airways and necrotizing vasculitis that is especially likely to involve the kidneys. Untreated, WG results in death within weeks to months. Since the introduction of cyclophosphamide and prednisolone as standard treatment, survival has improved dramatically from ,20% at 1 year reported in 1958 to at least 74% 5-years survival reported in the past 10 years. – 6] WG and other small-vessel vasculitides, such as microscopic polyangiitis (MPA) and the Churg–Strauss syndrome are strongly associated with antineutrophil cytoplasmic antibodies (ANCA) which are either directed to myeloperoxidase (MPO) or proteinase 3 (PR3). – 9] There is evidence that ANCA play a pathophysiologic role in the induction of these vasculitides. First, rises in ANCA level predict disease reactivation. Second, it has been demonstrated in vitro that ANCA induce neutrophil activation, i.e. degranulation and production of a respiratory burst. In addition, ANCA activate other cells such as monocytes and endothelial cells in vitro. Finally, in vivo, experimental data also suggest an important role for ANCA in the pathophysiology of vasculitis. The mechanism by which ANCA are induced are yet unclear. One favored hypothesis is that infections may trigger an ANCA response. Certain drugs have been linked to the induction of ANCA and the onset of ANCAassociated vasculitis (AAV). Recently, it has been described that an autoimmune response may be induced by the presence of a peptide that is antisense or complementary to the autoantigen, for instance, PR3. This immune response may induce anti-idiotypic antibodies (autoantibodies) that cross-react with the autoantigen. AUTOANTIBODIES AS DIAGNOSTIC MARKERS

Phosphorylcholine antibodies are diminished in ANCA-associated vasculitis.

ANCA‐associated (AAV) vasculitis is an autoimmune small‐vessel vasculitis and may be associated with accelerated atherosclerosis as suggested by current literature. Antibodies against oxidized lipoproteins (OxLDL) and phosphorylcholine (Pc) protect from atherosclerosis. This study characterizes these antibodies in AAV.