Private specificities can dominate the humoral response to self-antigens in patients with cryptogenic fibrosing alveolitis

BACKGROUND: The pathogenetic mechanisms that underlie the interstitial lung disease cryptogenic fibrosing alveolitis (CFA) may involve an immunological reaction to unidentified antigens in the lung, resulting in tissue damage. METHOD: In order to identify the range of target autoantigens, we used expression cloning, employing serum from an index patient as the probe against an expressed cDNA library that was derived from a tumour cell line. We screened over 5 × 10(5) recombinants and obtained sequence information on three antigens that had provoked strong responses with immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. RESULTS: All of the antigens were identifiable by comparison with sequence data from the US National Center for Biotechnology Information. Alanyl tRNA synthetase (ATS) was picked on six occasions; five of these incidences reflected independent recombination events, indicating that the library was not biased. Antibodies to ATS (anti-PL-12) represent the most common reactivity that defines the antisynthetase syndrome, which is typically expressed as polymyositis, dermatomyositis and interstitial lung disease (ILD). The index patient never showed symptoms other than those associated with alveolitis, even though sera obtained from him over a period of 2 years contained antibodies with the same specificity. Autoantibodies to ATS were never detected in serial bleeds from 11 other patients with CFA, and neither did we detect antibodies to the other two antigens identified from the serum of the index patient. CONCLUSION: The humoral response in patients with CFA can be dominated by autoantibodies with private specificities. This suggests that the antibodies are epiphenomenal and are a secondary feature of tissue damage induced by some other mechanism

Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling

Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context