Citations

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA‐containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in‐person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

SUMMARYANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ-rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With δ-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.