Melbourne Dental School - Research Publications

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    Adolescent temperament dimensions as stable prospective risk and protective factors for salivary C-reactive protein
    Nelson, BW ; Byrne, ML ; Simmons, JG ; Whittle, S ; Schwartz, OS ; O'Brien-Simpson, NM ; Walsh, KA ; Reynolds, EC ; Allen, NB (WILEY, 2018-02-01)
    OBJECTIVE: Temperament has associations with later physical health outcomes, yet there is a dearth of research exploring the connection between temperament and mechanisms that have known associations with these health outcomes. Recent research has delineated a connection between personality and inflammation during adulthood, but this association has not yet been studied in adolescent samples. DESIGN: We investigated whether stable adolescent temperament (averaged over two years), specifically effortful control and negative emotionality, provided a more robust prediction of inflammation as measured by salivary C-reactive protein (sCRP), than depressive symptoms. METHODS: Temperament and depressive symptoms were measured in a sample of sixty-three adolescents (37 males) when they were approximately 12 years old (mean age = 12.30, SD = 0.69) and again when they were approximately 14 years old (mean age = 14.84, SD = 0.49). Levels of sCRP were determined approximately 7 months later (mean = 6.77, SD = 2.99) when participants were approximately 15 years old (mean age = 15.49, SD = 0.49). RESULTS: Regression analyses revealed that effortful control (EC) was significantly associated with lower sCRP levels, while higher negative emotionality (NE) was significantly associated with higher sCRP levels. Furthermore, these associations were larger than those for depressive symptoms and were differentially impacted by the addition of covariates. Implications for the role of stable risk and protective factors in inflammatory processes are discussed. CONCLUSIONS: These findings are the first to show associations between adolescent temperament and inflammation. Furthermore, these findings extend previous personality research to temperamental research in a younger sample of adolescents. Statement of contribution What is already known? There is a large extant literature on the association between depressive symptoms and inflammation. There is a smaller extant literature on the association between personality and inflammation. No studies have examined how adolescent temperament traits may relate to inflammation. What does this study add? Longitudinal data collection over the course of 3 years in an adolescent sample. Addresses the question of whether temperament factors relate to inflammation. Temperament provides a more robust predictor of later inflammation than depressive symptoms.
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    Associations Between Observed Parenting Behavior and Adolescent Inflammation Two and a Half Years Later in a Community Sample
    Byrne, ML ; Horne, S ; O'Brien-Simpson, NM ; Walsh, KA ; Reynolds, EC ; Schwartz, OS ; Whittle, S ; Simmons, JG ; Sheeber, L ; Allen, NB (AMER PSYCHOLOGICAL ASSOC, 2017-07-01)
    OBJECTIVE: Family environments have an effect on physical health during adolescence, and a possible underlying mechanism is inflammation. However, little is known about the association between observed parenting behaviors and immune system functioning. The current study examined whether positive and negative emotional parental behaviors observed during family interactions were associated with inflammation in adolescents. METHOD: Sixty-one parent-adolescent dyads (37 male adolescents, 60.6%; 15 male parents, 24.6%) were observed during 2 laboratory-based interaction tasks designed to elicit positive and conflictual emotional behaviors, respectively. Frequency of aggressive and positive parental behavior was coded. Adolescents were followed up approximately 2.5 years later and salivary concentrations of the inflammatory biomarker C-reactive protein (sCRP) were measured. RESULTS: Controlling for BMI and depressive symptoms, lower sCRP was associated both with greater frequency of positive parental behaviors, t = -3.087, p = .003 and less frequency of aggressive parental behavior (t = 2.087, p = .041) in the conflictual task. Trend associations between positive behavior during the positive task and lower sCRP were also found. CONCLUSIONS: This is the first study to show that observed positive parenting is associated with lower levels of inflammation in adolescents. (PsycINFO Database Record
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    Bacterial Fluorescent-dextran Diffusion Assay
    O’Brien-Simpson, N ; Pantarat, N ; Walsh, K ; Reynolds, E ; Sani, M-A ; Separovic, F (Bio-Protocol, LLC, 2014)
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    Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens
    Dashper, S ; O'Brien-Simpson, N ; Liu, SW ; Paolini, R ; Mitchell, H ; Walsh, K ; D'Cruze, T ; Hoffmann, B ; Catmull, D ; Zhu, Y ; Reynolds, E (AMER SOC MICROBIOLOGY, 2014-01-01)
    Bacterial pathogens commonly associated with chronic periodontitis are the spirochete Treponema denticola and the Gram-negative, proteolytic species Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration (IC50) of 2.2 μM and planktonic growth of T. forsythia with a MIC of 295 μM, but it had no effect on the growth of T. denticola. Oxantel treatment caused the downregulation of six P. gingivalis gene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating that P. gingivalis may be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence of T. denticola on the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water of P. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.
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    A therapeutic Porphyromonas gingivalis gingipain vaccine induces neutralising IgG1 antibodies that protect against experimental periodontitis
    O'Brien-Simpson, NM ; Holden, JA ; Lenzo, JC ; Tan, Y ; Brammar, GC ; Walsh, KA ; Singleton, W ; Orth, RKH ; Slakeski, N ; Cross, KJ ; Darby, IB ; Becher, D ; Rowe, T ; Morelli, AB ; Hammet, A ; Nash, A ; Brown, A ; Ma, B ; Vingadassalom, D ; McCluskey, J ; Kleanthous, H ; Reynolds, EC (SPRINGERNATURE, 2016-12-01)
    Porphyromonas gingivalis infected mice with an established P. gingivalis-specific inflammatory immune response were protected from developing alveolar bone resorption by therapeutic vaccination with a chimera (KAS2-A1) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. Protection was characterised by an antigen-specific IgG1 isotype antibody and Th2 cell response. Adoptive transfer of KAS2-A1-specific IgG1 or IgG2 expressing B cells confirmed that IgG1-mediated protection. Furthermore, parenteral or intraoral administration of KAS2-A1-specific polyclonal antibodies protected against the development of P. gingivalis-induced bone resorption. The KAS2-A1-specific antibodies neutralised the gingipains by inhibiting: proteolytic activity, binding to host cells/proteins and co-aggregation with other periodontal bacteria. Combining key gingipain sequences into a chimera vaccine produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis.
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    Streptococcus mutans biofilm disruption by kappa-casein glycopeptide
    Dashper, SG ; Liu, S-W ; Walsh, KA ; Adams, GG ; Stanton, DP ; Ward, BR ; Shen, P ; O'Brien-Simpson, NM ; Reynolds, EC (ELSEVIER SCI LTD, 2013-06-01)
    UNLABELLED: Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities. OBJECTIVES: To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions. METHODS: Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn). RESULTS: CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37μm and a biovolume of 3.88μm(3)/μm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity. CONCLUSIONS: KCG and KCG-Zn may have potential as natural biofilm disruptive agents.
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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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    GM-CSF and uPA are required for Porphyromonas gingivalis-induced alveolar bone loss in a mouse periodontitis model
    Lam, RS ; O'Brien-Simpson, NM ; Hamilton, JA ; Lenzo, JC ; Holden, JA ; Brammar, GC ; Orth, RK ; Tan, Y ; Walsh, KA ; Fleetwood, AJ ; Reynolds, EC (NATURE PUBLISHING GROUP, 2015-09-01)
    Granulocyte-macrophage colony-stimulating factor (GM-CSF) and urokinase-type plasminogen activator (uPA) can contribute to the progression of chronic inflammatory diseases with possible involvement of macrophages. In this study, we investigated the role of both GM-CSF and uPA in Porphyromonas gingivalis-induced experimental periodontitis using GM-CSF-/- and uPA-/- mice. Intra-oral inoculation of wild-type (WT) C57BL/6 mice with P. gingivalis resulted in establishment of the pathogen in plaque and a significant increase in alveolar bone resorption. The infected mice also exhibited a CD11b(+) CD86(+) macrophage infiltrate into the gingival tissue, as well as P. gingivalis-specific pro-inflammatory cytokine and predominantly IgG2b antibody responses. In comparison, intra-oral inoculation of P. gingivalis did not induce bone resorption and there was significantly less P. gingivalis recovered from plaque in GM-CSF-/- and uPA-/- mice. Furthermore, P. gingivalis did not induce a macrophage gingival infiltrate or activate isolated peritoneal macrophages from the gene-deficient mice. Pro-inflammatory P. gingivalis-specific T-cell cytokine responses and serum interferon-gamma (IFN-γ) and IgG2b concentrations were significantly lower in GM-CSF-/- mice. In uPA-/- mice, T-cell responses were lower but serum IFN-γ and IgG2b levels were comparable with WT mice levels. These results suggest that GM-CSF and uPA are both involved in the progression of experimental periodontitis, possibly via a macrophage-dependent mechanism(s).
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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, 2013-03-01)
    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.
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    Acute phase protein and cytokine levels in serum and saliva: A comparison of detectable levels and correlations in a depressed and healthy adolescent sample
    Byrne, ML ; O'Brien-Simpson, NM ; Reynolds, EC ; Walsh, KA ; Laughton, K ; Waloszek, JM ; Woods, MJ ; Trinder, J ; Allen, NB (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013-11-01)
    Recent research has examined associations between inflammation and mental health, and has increasingly focused on utilising younger samples to characterise the temporal relationship between inflammatory responses and the emergence of other symptoms. These studies have typically used blood to measure inflammation, although rates of detection for many inflammatory markers appear to be low. Saliva is a safe and low-cost alternative, and adult research has shown that levels of some salivary markers correlate well with those in serum. However, no research has examined this association in young people. This study examined 16 inflammatory markers in serum and saliva in 17 depressed adolescents and 18 healthy controls, aged 13-18 years. In general, detection rates were higher in saliva compared to in serum. When non-detectable levels were excluded, serum levels of C-reactive protein (CRP) correlated with salivary CRP (r=0.424, p=0.015), and this correlation appeared to only exist for those individuals with high levels of serum CRP (r=0.599, p=0.014). However, when non-detectable levels were included as zero, salivary levels of CRP, interleukin (IL)-2, IL-12p70, and interferon (IFN)-γ correlated with their serum counterparts. No significant clinical group differences in any acute phase proteins or cytokines were present. This study suggests that saliva can be used to measure inflammation in studies with adolescent participants, especially CRP, as it appears to correlate with systemic inflammation for those individuals who are expected to have high levels of inflammation. Implications for future directions in research on salivary inflammatory markers are discussed.