Recombinant Human b-Defensin-3 in an Animal Model of Infection: A Feasibility Pilot Study

Human b-defensin-3 (HBD-3) is an antimicrobial peptide with bactericidal effects on many gram-positive and gram-negative bacteria and some yeast species and, if radiolabeled, might be used to distinguish bacterial infection from sterile inflammation. The goals of the present study were to develop methods for radiolabeling HBD-3 with 99mTc and to perform preliminary investigations on 99m Tc-labeled HBD-3 as a means to evaluate induced infection in an animal model. To this purpose, Staphylococcus aureus‐induced infection was used to evaluate the capability of 99m Tc-HBD-3 to distinguish infection from aseptic inflammation in rats. Methods: Twenty to 40 mg of recombinant HBD-3 were labeled with 99m Tc 1 hexa-coordinated with 3 molecules of CO and H2O and separated by a column from free 99m Tc. 99m Tc-HBD-3 was added to cultures of a bacterial suspension of S. aureus and Escherichia coli to evaluate in vitro antibacterial activity. A bacterial suspension of S. aureus and a carrageenansolutionwereusedtoinduceinfectionandsterileinflammation, respectively, in opposite thighs of 9 adult rats. Three separate experiments were performed on groups of 3 rats each. The animals received different doses of 99m Tc-HBD-3 injected through a cannula into the jugular vein. After sacrifice of the animals, tissue samples were obtained from sites of infection, inflammation, and control muscle (left foreleg) at 1, 3, and 5 h after 99m Tc-HBD-3 administration. Tissue samples were weighed and then counted in a well-counter. Simultaneously, 1 mL of a standard solution of 99m Tc-HBD-3 corresponding to each administered dose was counted. Results: 99m Tc-HBD-3 retained antibacterial activity. Radioactivity in tissue samples fromtheinfectedsiteswassignificantlyhigherthanthatinsamples of either induced inflammation or normal control muscle (ratio, ;3:1) at 3 and 5 h after injection, whereas similar radioactivity countswere observed fortissuesamplesfrom asepticinflammation sites and normal control muscle. Conclusion: In this investigation, 99m Tc-HBD-3 retained antibacterial activity and successfully distinguished infection from aseptic inflammation in adult rats.