Development of a prototype wound dressing technology which can detect and report colonization by pathogenic bacteria

A new methodology for detecting the microbiological state of a wound dressing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we report how stabilized lipid vesicles containing self-quenched carboxyfluorescein dye are sensitive to lysis only by toxins/virulence factors from P. aeruginosa and S. aureus but not by a non-toxic Escherichia coli species. The development of the stabilized vesicles is discussed and their response to detergent (triton), bacterial toxin (α-hemolysin) and lipases (phospholipase A₂). Finally, fabrics with stabilized vesicles attached via plasma deposited maleic anhydride coupling are shown visibly responding to S. aureus (MSSA 476) and P. aeruginosa (PAO1) but not E. coli DH5α in a prototype dressing.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine) coordination complexes as cell death imaging agents

The aim of this study was the development of (??m)Tc labeled bis(zinc(II)-dipicolylamine) (Zn2?-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 was labeled with (??m)Tc via the (??m)Tc-tricarbonyl core ([(??m)Tc(CO)?-1]3?) or via HYNIC ((??m)Tc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for [(??m)Tc(CO)?-1]3?. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding to apoptotic cells. (??m)Tc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-reperfusion injury was used to further explore the ability of the (??m)Tc-labeled Zn2?-DPA coordination complexes to target cell death. Selective accumulation could be detected for both tracers in the area at risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were 3.82 for [(??m)Tc(CO)?-1]3? and 5.45 for (??m)Tc-HYNIC-1.     

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids

The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO.     

Effect of 1918 PB1-F2 expression on influenza A virus infection kinetics

Relatively little is known about the viral factors contributing to the lethality of the 1918 pandemic, although its unparalleled virulence was likely due in part to the newly discovered PB1-F2 protein. This protein, while unnecessary for replication, increases apoptosis in monocytes, alters viral polymerase activity in vitro, enhances inflammation and increases secondary pneumonia in vivo. However, the effects the PB1-F2 protein have in vivo remain unclear. To address the mechanisms involved, we intranasally infected groups of mice with either influenza A virus PR8 or a genetically engineered virus that expresses the 1918 PB1-F2 protein on a PR8 background, PR8-PB1-F2(1918). Mice inoculated with PR8 had viral concentrations peaking at 72 hours, while those infected with PR8-PB1-F2(1918) reached peak concentrations earlier, 48 hours. Mice given PR8-PB1-F2(1918) also showed a faster decline in viral loads. We fit a mathematical model to these data to estimate parameter values. The model supports a higher viral production rate per cell and a higher infected cell death rate with the PR8-PB1-F2(1918) virus. We discuss the implications these mechanisms have during an infection with a virus expressing a virulent PB1-F2 on the possibility of a pandemic and on the importance of antiviral treatments.