Melbourne Dental School - Research Publications

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Author: O'Brien-Simpson, Neil × Author: Walsh, Katrina × Author: Attard, Troy × Start date: 2013 × End date: 2018 ×

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    A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity
    O'Brien-Simpson, NM ; Pantarat, N ; Attard, TJ ; Walsh, KA ; Reynolds, EC ; Castanho, MARB (PUBLIC LIBRARY SCIENCE, 2016-03-17)
    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.
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    Polymerisation of a T Cell Epitope with an Immunostimulatory C3d Peptide Sequence Enhances Antigen Specific T Cell Responses
    O'Brien-Simpson, NM ; Attard, TJ ; Zheng, B ; Walsh, KA ; Reynolds, EC (SPRINGER, 2013-03)
    The complement protein C3d and C3d derived peptides that bind CD21 are known to enhance immunity to co-immunised antigens. In this study we have synthesised the minimal CD21 binding sequence of C3d (1227LYNVEA 1232) as mono, di and tri tandem repeats and derivatised the N-terminus with an acryloyl moiety. These acryloyl-(C3d)n peptides were co-polymerised with a acryloyl-T cell epitope (PAS1K) from the Porphyromonas gingivalis antigen the RgpA–Kgp proteinase–adhesin complex. The ability of C3d containing polymers to enhance T cell immunity in vitro and in vivo was evaluated. When used to stimulate in vitro PAS1K-primed or RgpA–Kgp complex-primed T cells the C3d containing PAS1K polymers induced a mixed and significantly (p\0.05) higher IL-4 and IFNc T cell response compared to that induced by the PAS1K peptide or polymer. PAS1K polymers containing tandem repeats of C3d induced a significantly (p\0.05) stronger maximal proliferative response, at the same antigenic dose, compared to that induced by the PAS1K peptide or polymer. When used as immunogens to prime T cells all of the C3d containing PAS1K polymers induced a dominant IFNc T cell response and reduced the antigen dose required for maximal proliferation 150-fold compared to that required for the PAS1K-peptide or polymer primed T cells. In conclusion, the 6 residue sequence LYNVEA from C3d is sufficient to enhance immunity to an antigen and that the effect is more pronounced when C3d is part of the immunising antigen rather than an in vitro stimulating antigen.