• Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation.

      Ni Ainle, Fionnuala; Preston, Roger J S; Jenkins, P Vincent; Nel, Hendrik J; Johnson, Jennifer A; Smith, Owen P; White, Barry; Fallon, Padraic G; O'Donnell, James S; Haemostasis Research Group, St James's Hospital, Dublin, Republic of Ireland. (2009-08-20)
      Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.
    • A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins.

      Ryan, Anthony; Lynch, Mark; Smith, Sinead M; Amu, Sylvie; Nel, Hendrik J; McCoy, Claire E; Dowling, Jennifer K; Draper, Eve; O'Reilly, Vincent; McCarthy, Ciara; et al. (2011-06)
      Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4(-/-) and Myd88(-/-), but not TRIF(-/-) mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.
    • Skin microbiome before development of atopic dermatitis: Early colonization with commensal staphylococci at 2 months is associated with a lower risk of atopic dermatitis at 1 year.

      Kennedy, Elizabeth A; Connolly, Jennifer; Hourihane, Jonathan O'B; Fallon, Padraic G; McLean, W H Irwin; Murray, Deirdre; Jo, Jay-Hyun; Segre, Julia A; Kong, Heidi H; Irvine, Alan D (Journal of Allergy and Clinical Immunology, 2017-01)
      Disease flares of established atopic dermatitis (AD) are generally associated with a low-diversity skin microbiota and Staphylococcus aureus dominance. The temporal transition of the skin microbiome between early infancy and the dysbiosis of established AD is unknown.