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1.
Viruses ; 15(6)2023 05 31.
Article in English | MEDLINE | ID: mdl-37376607

ABSTRACT

Venezuelan equine encephalitis virus (VEEV) is a disease typically confined to South and Central America, whereby human disease is characterised by a transient systemic infection and occasionally severe encephalitis, which is associated with lethality. Using an established mouse model of VEEV infection, the encephalitic aspects of the disease were analysed to identify biomarkers associated with inflammation. Sequential sampling of lethally challenged mice (infected subcutaneously) confirmed a rapid onset systemic infection with subsequent spread to the brain within 24 h of the challenge. Changes in inflammatory biomarkers (TNF-α, CCL-2, and CCL-5) and CD45+ cell counts were found to correlate strongly to pathology (R>0.9) and present previously unproven biomarkers for disease severity in the model, more so than viral titre. The greatest level of pathology was observed within the olfactory bulb and midbrain/thalamus. The virus was distributed throughout the brain/encephalon, often in areas not associated with pathology. The principal component analysis identified five principal factors across two independent experiments, with the first two describing almost half of the data: (1) confirmation of a systemic Th1-biased inflammatory response to VEEV infection, and (2) a clear correlation between specific inflammation of the brain and clinical signs of disease. Targeting strongly associated biomarkers of deleterious inflammation may ameliorate or even eliminate the encephalitic syndrome of this disease.


Subject(s)
Encephalitis Virus, Venezuelan Equine , Encephalomyelitis, Venezuelan Equine , Humans , Horses , Mice , Animals , Tumor Necrosis Factor-alpha , Encephalitis Virus, Venezuelan Equine/physiology , Brain , Inflammation/pathology , Chemokines , Leukocytes
3.
Antibiotics (Basel) ; 11(10)2022 Oct 20.
Article in English | MEDLINE | ID: mdl-36290100

ABSTRACT

Burkholderia pseudomallei is the causative agent of melioidosis, a multifaceted disease. A proportion of the mortality and morbidity reported as a result of infection with this organism may be due to the premature cessation of antibiotic therapy typically lasting for several months. The progression of re-emergent disease was characterised in Balb/c mice following cessation of a 14 day treatment course of co-trimoxazole or finafloxacin, delivered at a human equivalent dose. Mice were culled weekly and the infection characterised in terms of bacterial load in tissues, weight loss, clinical signs of infection, cytokine levels and immunological cell counts. Following cessation of treatment, the infection re-established in some animals. Finafloxacin prevented the re-establishment of the infection for longer than co-trimoxazole, and it is apparent based on the protection offered, the development of clinical signs of disease, bodyweight loss and bacterial load, that finafloxacin was more effective at controlling infection when compared to co-trimoxazole.

4.
ACS Nano ; 15(12): 19284-19297, 2021 12 28.
Article in English | MEDLINE | ID: mdl-34739227

ABSTRACT

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM) encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study, we developed PMs composed of widely available poly(ethylene oxide)-polycaprolactone block copolymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM colocalization with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.


Subject(s)
Burkholderia pseudomallei , Burkholderia , Anti-Bacterial Agents/pharmacology , Macrophages
5.
Transfusion ; 61 Suppl 1: S223-S233, 2021 07.
Article in English | MEDLINE | ID: mdl-34269457

ABSTRACT

BACKGROUND: Parachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer-term RBC survival is undocumented. This study aimed to determine post-drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage. STUDY DESIGN AND METHODS: Twenty-two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6-8 days post-donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4-h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters. RESULTS: Blood units were maintained at 2-6°C and recovered intact after recorded ground impacts of 341-1038 m s-2 . All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB. DISCUSSION: Appropriately packaged aerial delivery by parachute can be successfully used for blood supply.


Subject(s)
Blood Transfusion , Erythrocytes/cytology , Plasma , Transportation , Blood Preservation , Humans , Plasma/chemistry , Quality Indicators, Health Care
6.
Cytometry A ; 95(11): 1167-1177, 2019 11.
Article in English | MEDLINE | ID: mdl-31595661

ABSTRACT

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP-1) lineages. Subsequently, cell populations were fluorescence-activated cell sorted for morphological assessment. Stage-specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP-1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP-1(-)) and mature (RP-1+) granulocytes. Multiple B-cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(-) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.


Subject(s)
Bone Marrow Cells/cytology , Femoral Fractures/immunology , Hematopoietic Stem Cells/cytology , Shock, Hemorrhagic/immunology , Wounds and Injuries/immunology , Animals , Antimicrobial Cationic Peptides/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , CD11b Antigen/metabolism , Cell Lineage/immunology , Flow Cytometry , Granulocytes/cytology , Granulocytes/metabolism , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Immunophenotyping , Inflammation/immunology , Inflammation/metabolism , Leukocyte Common Antigens/metabolism , Lymphopoiesis/immunology , Male , Multiple Organ Failure/immunology , Multiple Organ Failure/pathology , Myeloid Cells/cytology , Myeloid Cells/metabolism , Rats , Rats, Wistar , Shock, Hemorrhagic/metabolism , Thy-1 Antigens/metabolism , Wounds and Injuries/metabolism
8.
J Immunol Res ; 2018: 8087916, 2018.
Article in English | MEDLINE | ID: mdl-30622981

ABSTRACT

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.


Subject(s)
Bacterial Vaccines/immunology , Francisella tularensis/physiology , Glycoconjugates/immunology , Hexosyltransferases/immunology , Tularemia/immunology , Animals , Cells, Cultured , Disease Models, Animal , Female , Humans , Inhalation , Mice , Mice, Inbred BALB C , Protein Binding , Pseudomonas aeruginosa/metabolism , Rats , Rats, Inbred F344 , Vaccination
9.
Methods ; 134-135: 41-49, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29122725

ABSTRACT

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model.


Subject(s)
Endocytosis/genetics , Flow Cytometry/methods , Microscopy, Fluorescence/methods , Ricin/metabolism , A549 Cells , Biological Transport/genetics , Cytosol/metabolism , Epithelial Cells/metabolism , Humans , Protein Transport/genetics
10.
Toxins (Basel) ; 9(10)2017 10 18.
Article in English | MEDLINE | ID: mdl-29057798

ABSTRACT

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')2 antitoxin as a treatment for ricin intoxication.


Subject(s)
Antitoxins/immunology , Ricin/immunology , Animals , Antibodies, Neutralizing/analysis , Chlorocebus aethiops , Female , Mice, Inbred BALB C , Ricin/pharmacokinetics , Ricin/toxicity , Sheep , Vero Cells
11.
Nanomedicine (Lond) ; 12(8): 845-863, 2017 04.
Article in English | MEDLINE | ID: mdl-28351228

ABSTRACT

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.


Subject(s)
Cell Differentiation/drug effects , Liposomes/pharmacology , Osteogenesis/drug effects , Wnt3A Protein/pharmacology , Bone Marrow Cells/drug effects , Dimyristoylphosphatidylcholine/chemistry , Dimyristoylphosphatidylcholine/pharmacology , Humans , Liposomes/chemistry , Phosphatidylcholines/chemistry , Phosphatidylcholines/pharmacology , Polyethylene Glycols/chemistry , Stem Cells/drug effects , Wnt3A Protein/chemistry
12.
PLoS One ; 11(7): e0158575, 2016.
Article in English | MEDLINE | ID: mdl-27391310

ABSTRACT

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.


Subject(s)
Autophagy/physiology , Bacillus anthracis/metabolism , Bacterial Proteins/metabolism , Microtubules/metabolism , Animals , Anthrax/microbiology , Autophagy/genetics , Bacillus anthracis/genetics , Bacterial Proteins/genetics , Cell Line , Cell Survival/physiology , Female , HEK293 Cells , Humans , Mice , Microscopy, Confocal , Phylogeny , Protein Binding , Signal Transduction , Virulence/genetics , Virulence/physiology
13.
Cytometry A ; 89(4): 328-37, 2016 04.
Article in English | MEDLINE | ID: mdl-26841315

ABSTRACT

The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC.


Subject(s)
Burkholderia Infections/diagnosis , Flow Cytometry , Image Cytometry , Animals , Bacterial Load/methods , Burkholderia Infections/microbiology , Flow Cytometry/methods , Image Cytometry/methods , Intracellular Space , Mice , Microscopy, Fluorescence/methods
14.
Microb Pathog ; 92: 50-53, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26724738

ABSTRACT

Plague is a highly pathogenic disease caused by the bacterium Yersinia pestis. There is currently no vaccine available for prophylaxis and antibiotic resistant strains have been isolated, thus there is a need for the development of new countermeasures to treat this disease. Survival protein A (SurA) is a chaperone that has been linked to virulence in several species of bacteria, including the close relative Yersinia pseudotuberculosis. In this study, we aimed to evaluate the role of SurA in virulence of the highly pathogenic Y. pestis by creating an unmarked surA deletion mutant. The Y. pestis ΔsurA mutant was found to be more susceptible to membrane perturbing agents and was completely avirulent in a mouse infection model when delivered up to 2.1 × 10(5) CFU by the subcutaneous route. This provides strong evidence that SurA would make a promising antimicrobial target.


Subject(s)
Bacterial Proteins/genetics , Plague/microbiology , Yersinia pestis/physiology , Yersinia pestis/pathogenicity , Animals , Bacterial Proteins/metabolism , Disease Models, Animal , Female , Gene Deletion , Genetic Complementation Test , Mice , Plague/mortality , Virulence/genetics , Virulence Factors
15.
Stem Cells ; 34(2): 418-30, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26573091

ABSTRACT

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.


Subject(s)
Bone Marrow Cells/metabolism , Leukocytes, Mononuclear/metabolism , Osteogenesis , Stem Cells/metabolism , Wnt Signaling Pathway , Wnt3A Protein/metabolism , Aged , Aged, 80 and over , Arthroplasty, Replacement, Hip , Bone Marrow Cells/cytology , Female , Flow Cytometry , Humans , Leukocytes, Mononuclear/cytology , Male , Middle Aged , Stem Cells/cytology , Stromal Cells/cytology , Stromal Cells/metabolism
16.
Nanoscale ; 6(14): 8376-86, 2014 Jul 21.
Article in English | MEDLINE | ID: mdl-24941427

ABSTRACT

Aqueous bifunctional semiconductor polymer nanoparticles (SPNs), approximately 30 nm in diameter (as measured from electron microscopy), were synthesised using hydrophobic conjugated polymers, amphiphilic phospholipids and a gadolinium-containing lipid. Their fluorescence quantum yields and extinction coefficients were determined, and their MRI T1-weighted relaxation times in water were measured. The bimodal nanoparticles were readily taken up by HeLa and murine macrophage-like J774 cells as demonstrated by confocal laser scanning microscopy, and were found to be MRI-active, generating a linear relationship between T1-weighted relaxation rates and gadolinium concentrations The synthesis is relatively simple, and can easily result in milligrams of materials, although we fully expect scale-up to the gram level to be easily realised.


Subject(s)
Contrast Media/chemistry , Gadolinium/chemistry , Magnetic Resonance Imaging , Nanoparticles/chemistry , Polymers/chemistry , Animals , Cell Line , Contrast Media/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Nanoparticles/metabolism , Particle Size , Radiography , Spleen/diagnostic imaging
17.
Antimicrob Agents Chemother ; 58(3): 1458-67, 2014.
Article in English | MEDLINE | ID: mdl-24366729

ABSTRACT

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.


Subject(s)
Anti-Infective Agents/pharmacology , Bacterial Proteins/drug effects , Burkholderia pseudomallei/drug effects , Drug Discovery/methods , Immunophilins/drug effects , Anti-Infective Agents/therapeutic use , Bacterial Proteins/ultrastructure , Binding Sites , Crystallography, X-Ray , Immunophilins/ultrastructure , Nuclear Magnetic Resonance, Biomolecular , Virulence Factors
18.
Peptides ; 43: 96-101, 2013 May.
Article in English | MEDLINE | ID: mdl-23500517

ABSTRACT

Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.


Subject(s)
Antimicrobial Cationic Peptides/therapeutic use , Tularemia/drug therapy , Administration, Intranasal , Animals , Antimicrobial Cationic Peptides/administration & dosage , Antimicrobial Cationic Peptides/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Francisella tularensis/drug effects , Francisella tularensis/immunology , Immunomodulation , Macrophages/drug effects , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Structure-Activity Relationship , Tularemia/immunology , Cathelicidins
19.
Microbiology (Reading) ; 158(Pt 6): 1593-1606, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22403187

ABSTRACT

The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1ß- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.


Subject(s)
Bacterial Proteins/metabolism , Interleukin-1/metabolism , Plague/metabolism , Plague/microbiology , Toll-Like Receptors/metabolism , Yersinia pestis/metabolism , Yersinia pestis/pathogenicity , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Plague/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Signal Transduction , Toll-Like Receptors/genetics , Virulence , Yersinia pestis/chemistry , Yersinia pestis/genetics
20.
FEMS Immunol Med Microbiol ; 62(3): 348-61, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21569124

ABSTRACT

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.


Subject(s)
Francisella tularensis/immunology , Macrophages/immunology , Macrophages/microbiology , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/microbiology , Tularemia/immunology , Animals , Anti-Bacterial Agents/pharmacology , Cytokines/metabolism , Female , Flow Cytometry , Francisella tularensis/drug effects , Francisella tularensis/pathogenicity , Lung/drug effects , Lung/immunology , Lung/microbiology , Mice , Mice, Inbred BALB C , Phagocytosis/drug effects , Phagocytosis/immunology , Statistics, Nonparametric , Tularemia/drug therapy , Tularemia/microbiology
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