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ABSTRACT: The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.
PLoS ONE 01/2011; 6(10):e25333. · 4.09 Impact Factor
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ABSTRACT: Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.
American Journal of Respiratory Cell and Molecular Biology 12/2010; 43(6):692-702. · 5.13 Impact Factor
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ABSTRACT: To develop a more effective vaccination method against H5N1 virus, we investigated the immunogenicity and protective efficacy after skin vaccination using microneedles coated with influenza virus-like particles containing hemagglutinin derived from A/Vietnam/1203/04 H5N1 virus (H5 VLPs). A single microneedle vaccination of mice with H5 VLPs induced increased levels of antibodies and provided complete protection against lethal challenge without apparent disease symptoms. In contrast, intramuscular injection with the same vaccine dose showed low levels of antibodies and provided only partial protection accompanied by severe body weight loss. Post-challenge analysis suggested that improved protection was associated with lower lung viral titers and enhanced generation of recall antibody secreting cells by microneedle vaccination. Thus, this study provides evidence that skin delivery of H5 VLP vaccines using microneedles designed for self-administration induces improved protection compared to conventional intramuscular immunization.
Antiviral research 11/2010; 88(2):244-7. · 3.61 Impact Factor
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ABSTRACT: In 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model.
We generated two adenovirus(Ad5)-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA) from the recently emerged swine influenza isolate A/California/04/2009 (H1N1)pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFNgamma Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus.
A single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunization.
PLoS ONE 01/2010; 5(5):e10492. · 4.09 Impact Factor
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Sarah Fox,
Tom S Wilkinson,
Paul S Wheatley,
Bo Xiao,
Russell E Morris,
Alistair Sutherland,
A John Simpson, Peter G Barlow,
Anthony R Butler,
Ian L Megson,
Adriano G Rossi
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ABSTRACT: Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn(2+)-exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the anti-bacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties.
Acta biomaterialia 10/2009; 6(4):1515-21. · 3.98 Impact Factor
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Hsin-Ni Li, Peter G Barlow,
Johan Bylund,
Annie Mackellar,
Ase Björstad,
James Conlon,
Pieter S Hiemstra,
Chris Haslett,
Mohini Gray,
A John Simpson,
Adriano G Rossi,
Donald J Davidson
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ABSTRACT: Cathelicidins are CHDP with essential roles in innate host defense but also more recently associated with the pathogenesis of certain chronic diseases. These peptides have microbicidal potential and the capacity to modulate innate immunity and inflammatory processes. PMN are key innate immune effector cells with pivotal roles in defense against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and dysregulation is implicated in disease pathogenesis. The efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. We demonstrate that the human cathelicidin LL-37 induced rapid secondary necrosis of apoptotic human PMN and identify an essential minimal region of LL-37 required for this activity. Using these LL-37-induced secondary necrotic PMN, we characterize the consequence for macrophage inflammatory responses. LL-37-induced secondary necrosis did not inhibit PMN ingestion by monocyte-derived macrophages and in contrast to expectation, was not proinflammatory. Furthermore, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated after LL-37-induced secondary necrosis. However, this process of secondary necrosis did induce the release of potentially harmful PMN granule contents. Thus, we suggest that LL-37 can be a potent inducer of PMN secondary necrosis during inflammation without promoting macrophage inflammation but may mediate host damage through PMN granule content release under chronic or dysregulated conditions.
Journal of leukocyte biology 08/2009; 86(4):891-902. · 4.99 Impact Factor
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ABSTRACT: Increased levels of particulate air pollution (PM10) have been implicated as a causal agent in pulmonary disease exacerbation and increased deaths from respiratory and cardiovascular disorders. The exact mechanism by which PM10 drives toxicity in the lung is still unknown, but studies have focused on inhibition of macrophage function and impaired alveolar clearance mechanisms. To assess the effects of PM10 on pulmonary macrophage clearance mechanisms ex vivo, Wistar rats were instilled with 125 or 250 microg of PM10 collected from the North Kensington, London. Control rats were instilled with sterile saline. The rats were sacrificed after 18 h and a bronchoalveolar lavage (BAL) was performed. Macrophages isolated from the BAL fluid were assessed for ability to migrate towards a positive chemoattractant (ZAS) ex vivo and to perform phagocytosis. Macrophages isolated from the PM10-exposed rats showed inhibition of potential to migrate. Macrophage phagocytic ability ex vivo was also significantly reduced by the presence of PM10 inside the cells. This study indicates that acute PM10 exposure diminishes macrophage motility and phagocytosis in a manner that could prove deleterious to particle clearance from the alveolar region of the lung. Decreased particle clearance promotes inflammation, and hence, warrants further investigation in relation to the effects of chronic PM10 exposure on macrophage clearance mechanisms and establishing the mechanisms behind decreased macrophage migration.
Cell Biology and Toxicology 07/2008; 24(3):243-52. · 2.51 Impact Factor
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ABSTRACT: Particulate air pollution (PM(10)) consists of a mixture of components, including nanoparticles and metals. Studies from our laboratory have demonstrated that transition metals can potentiate the ability of nanoparticles to induce lung inflammation and that the zinc content of PM(10) was largely responsible for their potential to induce inflammation. These results are also relevant to zinc-containing engineered nanoparticles.
To investigate the potential of ZnCl(2) and FeCl(3) to interact with nanoparticle carbon black in cell-free and biological systems to generate ROS, express pro-inflammatory mediators and cytotoxic ability.
ROS production was examined using DCFH-DA. J774 cells were treated for 4 h with 14 nm CB and/or ZnCl(2) before measuring TNF-alpha by ELISA. Cytoskeletal changes were investigated using confocal microscopy. Flow cytometry was used to examine apoptotic/necrotic cells and phagocytic ability.
In a cell-free system the particles generated significant ROS, whereas ZnCl(2) did not. Treatment of cells with 100 microM ZnCl(2), but not FeCl(3), increased TNF-alpha. Treatment with 14 nm CB alone induced TNF-alpha, which was synergistically enhanced by ZnCl(2). No interactions were observed in cells treated with 14 nm CB and FeCl(3). Cytoskeletal changes were observed with increasing concentrations of ZnCl(2). These results were confirmed by flow cytometry indicating that ZnCl(2) induced markers of apoptosis and necrosis. The phagocytic ability of cells was also significantly decreased. Nanoparticle carbon black alone did not induce changes in apoptosis/necrosis or the phagocytosis activity of the cells.
Despite an inability to induce ROS production, ZnCl(2) stimulated TNF-alpha production which was synergistically enhanced by 14 nm carbon black. The ability of zinc to induce morphological changes and cell death was not altered by nanoparticle treatment.
Toxicology and Applied Pharmacology 12/2007; 225(1):80-9. · 4.45 Impact Factor
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ABSTRACT: The human cathelicidin LL-37 is a cationic host defense peptide (antimicrobial peptide) expressed primarily by neutrophils and epithelial cells. This peptide, up-regulated under conditions of inflammation, has immunomodulatory and antimicrobial functions. We demonstrate that LL-37 is a potent inhibitor of human neutrophil apoptosis, signaling through P2X(7) receptors and G-protein-coupled receptors other than the formyl peptide receptor-like-1 molecule. This process involved modulation of Mcl-1 expression, inhibition of BID and procaspase-3 cleavage, and the activation of phosphatidylinositol-3 kinase but not the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. In contrast to the inhibition of neutrophil apoptosis, LL-37 induced apoptosis in primary airway epithelial cells, demonstrating alternate consequences of LL-37-mediated modulation of apoptotic pathways in different human primary cells. We propose that these novel immunomodulatory properties of LL-37 contribute to peptide-mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic-resistant infectious diseases.
Journal of Leukocyte Biology 10/2006; 80(3):509-20. · 4.99 Impact Factor
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ABSTRACT: Alveolar macrophages are a key cell in dealing with particles deposited in the lungs and in determining the subsequent response to that particle exposure. Nanoparticles are considered a potential threat to the lungs and the mechanism of pulmonary response to nanoparticles is currently under intense scrutiny. The type II alveolar epithelial cell has previously been shown to release chemoattractants which can recruit alveolar macrophages to sites of particle deposition. The aim of this study was to assess the responses of a type II epithelial cell line (L-2) to both fine and nanoparticle exposure in terms of secretion of chemotactic substances capable of inducing macrophage migration.
Exposure of type II cells to carbon black nanoparticles resulted in significant release of macrophage chemoattractant compared to the negative control and to other dusts tested (fine carbon black and TiO2 and nanoparticle TiO2) as measured by macrophage migration towards type II cell conditioned medium. SDS-PAGE analysis of the conditioned medium from particle treated type II cells revealed that a higher number of protein bands were present in the conditioned medium obtained from type II cells treated with nanoparticle carbon black compared to other dusts tested. Size-fractionation of the chemotaxin-rich supernatant determined that the chemoattractants released from the epithelial cells were between 5 and 30 kDa in size.
The highly toxic nature and reactive surface chemistry of the carbon black nanoparticles has very likely induced the type II cell line to release pro-inflammatory mediators that can potentially induce migration of macrophages. This could aid in the rapid recruitment of inflammatory cells to sites of particle deposition and the subsequent removal of the particles by phagocytic cells such as macrophages and neutrophils. Future studies in this area could focus on the exact identity of the substance(s) released by the type II cells in response to particle exposure.
Particle and Fibre Toxicology 02/2005; 2:11. · 7.25 Impact Factor
