Denosumab

Eligible patients were randomly assigned, in a 1:1 ratio, to receive in a blinded fashion denosumab (Amgen) or placebo during the placebo-controlled double-blind phase of the study, by use of a randomization scheme with a fixed block size of four. The randomization list was generated by a coworker independent of the study and not involved in any procedure during the study. The study medication was provided by the pharmacy department. The medication and placebo syringes were identical in terms of color and shape and labeled with an unique sample number and study patient identification number. Patients and investigators retained unaware of the initial allocation during the entire trial, including the open-label extension.

Fab fragments of pooled human IgG were purchased from Calbiochem, pooled human Fc fragments from Athens Research, human IgG (IVIG; Kiovig) from Baxalta. Immunoglobulins were purchased from Sigma (IgA, IgM, IgG3, and IgG4), Amgen (denosumab), Athens Research and Technology (IgG1, IgG2), Abcam (IgD), Thermo Fisher (IgE), and goat control IgG from Molecular Innovations. denosumab (Amgen) is a monoclonal hu-IgG2 antibody that recognizes human RANKL and is used to treat osteoporosis, but does not recognize S. pyogenes (31 (link)). For flow cytometric analysis, the following Abs were used: mouse anti–hu-C4BP MK104 (33 (link)) coupled to biotin, goat anti human IgG (H+L)−647 (Invitrogen). Streptavidin PE (eBioscience) was used to detect MK104-biotin. Human C4BP was purified from human plasma; Protein H was expressed and purified from E. coli, all according to previously described protocols (11 (link), 34 (link)). The AHP peptide was a gift from Prof. Lars Björck (35 (link)). α1-antitrypsin [α1AT; a gift of the late Prof. Bertil Laurell (36 (link))] was used as a negative control for binding experiments. Plasma purified C4BP preparations of 2 mg/ml contained between 2 and 10 μg/ml human IgG, as determined by a sandwich ELISA for human IgG.