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Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 58 - 58
1 Oct 2022
Cecotto L van Kessel K Wolfert M Vogely H van der Wal B Weinans H van Strijp J Yavari SA
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Aim

In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants.

Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018.

Method

S. aureus, strain SH1000, was incubated with different concentrations of each HDP and bacterial growth was monitored overnight. Primary human monocytes were isolated from buffy coats using Ficoll-Paque density and CD14 microbeads, and differentiated for 7 days to macrophages. After 24h incubation in presence of LPS and HDPs, macrophages cytokines production was measured by ELISA. Macrophages cultured for 24h in presence of HDPs were infected with serum-opsonized S. aureus. 30 min and 24h after infection, bacterial phagocytosis and intracellular killing by macrophages were measured by flow cytometry and colony forming units (CFU) count respectively.


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_14 | Pages 25 - 25
1 Dec 2019
de Vor L Van Kessel K De Haas C Aerts P Viveen M Boel E Fluit A van Dijk B Vogely C van der Wal B van Strijp J Weinans H Rooijakkers S
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Aim

“Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”

Method

“We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”