Analysis of the UNC group emphasises the differences in patient selection and assays between it and the Kain studies. The 103 patients were selected from patients attending the UNC vasculitis service and disease activity was scored according to an established clinical index (BVAS) . The patients were segregated into two groups: 58 in remission with BVAS scores of 0; and 45 (including those newly presenting) with BVAS scores greater than 0 and defined as having active disease. This is a very different level of activity from the patients studied by Kain et al.  in which those at presentation or in clinical relapse generally had BVAS scores between 10 and 15.
Only 15 of the UNC sera were newly presenting patients, and thus suitable for testing the Vienna group's central proposition; some of these were already on treatment. Sera from seven of these (47%) were positive for antibodies to hLAMP-2 in an ELISA that had been validated using positive and negative control sera supplied by the Vienna group. Thus, the incidence is lower than in the cohorts assayed in Vienna but still much higher than healthy controls [2 of 52 (4%), P = 0.0002, Fisher's Exact Test]. By contrast, all 15 sera were negative when tested by Western blotting and IIF, as were all the other sera that were positive in the UNC LAMP-2 ELISA. Importantly, the Vienna positive control sera were also negative in both assays, which demonstrates that neither detects antibodies to hLAMP-2 in serum. The four Vienna positive controls were also negative in the UNC peptide ELISA. This highlights the lack of concordance between the UNC assays and the difficulty in interpreting results from them, as discussed by others [61▪▪,62▪▪].
The critical question about newly discovered autoantibodies is whether they are pathogenic or simply an epiphenomenon. Rodent models have become the standard strategy for addressing this issue in AAV [15–17]. The two approaches to induce injury are injection of antibodies specific for the proposed target antigen and active immunisation with the antigen or a closely related molecule: both have been used to test the potential pathogenicity of antibodies to LAMP-2. Kain et al. passively immunised Wystar Kyoto rats (WKY) – a rat strain commonly used in vasculitis research – with high titre rabbit IgG to recombinant hLAMP-2 that bound purified rLAMP-2 in ELISA and Western blot and rLAMP-2 in rat liver, kidney and neutrophils by IIF. WKY rats injected intravenously with this IgG had circulating anti-hLAMP-2 antibodies but the concentrations decreased rapidly over 24 h. Rabbit IgG was detected bound to glomerular endothelium 2 h after injection but not at later time points. The injected rats developed glomerulonephritis as evidenced by dipstix positive haematuria, severe proteinuria and development of a piFNGN with crescents in around 25% of glomeruli . None of these effects was seen in rats injected with normal rabbit globulin.
The cross-reactivity between FimH and hLAMP-2 provided another opportunity to test the pathogenicity of anti-LAMP-2 antibodies because the common hLAMP-2/FimH epitope recognised by patients’ autoantibodies is partially conserved in rLAMP-2. WKY rats immunised with recombinant FimH developed antibodies to FimH and eight of the 10 studied developed antibodies that reacted with rat and human LAMP-2. The sera bound to a synthetic peptide P41–49 by dot blot and affinity purified IgG to P41–49 from these sera bound to human glomerular endothelium by IIF. Immunoelectron microscopy confirmed the binding and showed the antibodies to P41–49 bound the same structures within cells as a monoclonal antibody to hLAMP-2. The immunised rats had positive ANCA assays using rat neutrophils and developed piFNGN. This supports the results of the passive immunisation experiments and confirms by a different strategy that anti-LAMP-2 antibodies can be pathogenic and cause piFNGN in rats.
These studies demonstrate that immunization with FimH induces antibodies to rat and human LAMP-2 accompanied by the development pauci-immune FNGN. This proves the molecular mimicry between the two molecules – at least under these experimental conditions – and raises the question whether natural infection with fimbriated bacteria could induce AAV in the same way. Two sets of clinical data are consistent with this: Kain et al.  reported that nine of 13 consecutive patients presenting with AAV had had a microbiologically proven infection with a fimbriated organism within the preceding 3 months; and Roth et al. [24▪▪] reported that 12% of a sample of 105 patients with UTIs had positive assays for LAMP-2 in their ELISA. The large prospective multicentre study should determine whether infections with type 1 fimbriated bacteria induce antibodies to hLAMP-2 in man and correlate with the development of AAV (http://http://www.intricate.eu/).
All four published studies show that the frequency of autoantibodies to hLAMP-2 is greatly increased in new onset patients with AAV, and that the autoantibodies are no longer detectable once remission has been achieved. Current controversies concern their absolute frequency, and how closely their presence correlates with disease activity. These controversies are largely attributable to the inadequacies of the current assays for the autoantibodies and will be easily resolved once robust ‘clinical grade’ assays have been developed. However, it is already clear that anti-hLAMP-2 antibodies become undetectable after treatment more quickly than antibodies to MPO and PR3 and so assays for them are unlikely to replace standard ANCA testing for diagnosis (except for ANCA-negative patients). It remains to be seen whether anti-hLAMP-2 antibodies more faithfully reflect disease activity than current assays. If so, their measurement would greatly improve tailoring immunosuppression in the individual patient. The answer will come from large-scale clinical studies.
Papers of particular interest, published within the annual period of review, have been highlighted as:
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 146–147).
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