Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.     

The legacy of nuclear risk and the founder effect in biotechnology organizations

In the wake of the Chernobyl nuclear accident and a decline in the public trust of science, the founders of modern biotechnology recognized the strategic importance of risk assessment and regulatory affairs. In an effort to avoid the demonization that was attached to the nuclear industry, the pioneers of modern biotechnology delegated authority for regulatory negotiation and risk management to senior positions in the firm. At the same time, the Biotechnology Industry Organization was handed great latitude and trust with making public pronouncements on issues of bioethics and public policy. The way in which founders and leaders embed norms for negotiating regulation and responding to public perceptions has proved important in the maturation and acceptance of a biotechnology sector.     

Window of opportunity: an episode of recruitment in a Banksia hybrid zone demonstrates continuing hybridization and phenotypic plasticity

Background and Aims

In perennial plants (especially post-fire resprouters), extant populations may reflect recruitment events in the distant past. This is true of hybrid zones formed by two Banksia species of swamps and woodlands in south-eastern Australia, Banksia robur and B. oblongifolia. Both resprout after fire but recruitment is dependent on periodic fires. Although plants of intermediate morphology have also been identified as hybrids using allozyme markers, the extent of ongoing hybridization is unknown. This study investigates whether both microsatellite markers and morphological measurements can be used to distinguish between the two species and their hybrids. A recent recruitment event and microsatellite markers allow the frequency of ongoing hybridization to be estimated, and also the effects of environmental variation on the morphology of plants and seedlings to be tested.

Methods

Variation at seven microsatellite loci was scored and seven leaf characteristics within putatively pure stands and mixed stands of both species were measured, revealing that the two species were genetically and morphologically distinct and that mixed stands also contained genetically and sometimes morphologically distinct hybrids. An opportunity created by wildfires was used to analyse the genetics and morphometrics of adults and seedlings from two hybrid zones.

Key Results

Approximately 9 % of adults and 21 % of seedlings were identified as genetic hybrids in both hybrid zones. Within these sites, the genotype of mature plants correlated well with morphology, except for some hybrid plants that had parental morphology. However, seedling morphology was highly variable and insufficient to describe the composition of the hybrid zone in this cohort. Greater phenotypic plasticity was evident among seedlings growing within the hybrid zones than seedlings growing in pots.

Conclusions

The hybrid zones are complex and the range of genotypes detected in seedlings reveals both continuing hybridization and introgression.     

Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice

Introduction

Rheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.

Methods

DTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.

Results

We demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4⁺ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.

Conclusions

Our findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4⁺ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.     

Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA

Mutations in the Drosophila retinal degeneration A (rdgA) gene, which encodes diacylglycerol kinase (DGK), result in early onset retinal degeneration and blindness. Whole-cell recordings revealed that light-sensitive Ca2+ channels encoded by the trp gene were constitutively active in rdgA photoreceptors. Early degeneration was rescued in rdgA;trp double mutants, lacking TRP channels; however, the less Ca2+-permeable light-sensitive channels (TRPL) were constitutively active instead. No constitutive activity was seen in rdgA;trpI;trp mutants lacking both classes of channel, although, like rdgA;trp, these still showed a residual slow degeneration. Responses to light were restored in rdgA;trp but deactivated abnormally slowly, indicating that DGK is required for response termination. The findings suggest that early degeneration in rdgA is caused by uncontrolled Ca2+ influx and support the proposal that diacylglycerol or its metabolites are messengers of excitation in Drosophila photoreceptors.     

Decisions reduce sensitivity to subsequent information

Behavioural studies over half a century indicate that making categorical choices alters beliefs about the state of the world. People seem biased to confirm previous choices, and to suppress contradicting information. These choice-dependent biases imply a fundamental bound of human rationality. However, it remains unclear whether these effects extend to lower level decisions, and only little is known about the computational mechanisms underlying them. Building on the framework of sequential-sampling models of decision-making, we developed novel psychophysical protocols that enable us to dissect quantitatively how choices affect the way decision-makers accumulate additional noisy evidence. We find robust choice-induced biases in the accumulation of abstract numerical (experiment 1) and low-level perceptual (experiment 2) evidence. These biases deteriorate estimations of the mean value of the numerical sequence (experiment 1) and reduce the likelihood to revise decisions (experiment 2). Computational modelling reveals that choices trigger a reduction of sensitivity to subsequent evidence via multiplicative gain modulation, rather than shifting the decision variable towards the chosen alternative in an additive fashion. Our results thus show that categorical choices alter the evidence accumulation mechanism itself, rather than just its outcome, rendering the decision-maker less sensitive to new information.     

Behaviour of tritium-labelled isopenicillin N and 6-aminopenicillanic acid as potential penicillin precursors in an extract of Penicillum chrysogenum.

1. 3H was incorporated into solvent-soluble penicillin from isopenicillin N and 6-aminopenicillanic acid 3H-labelled in the 2beta-methyl group when the labelled compounds were incubated with a crude extract of Penicillum chrysogenum. 2. With a soluble protein fraction of the extract incorporation from isopenicillin N occurred on addition of phenyl-acetyl-CoA. 3. Labelled benzylpenicillin was isolated after incubation of the crude extract with phenylacetyl-CoA and isopenicillin and the addition of unlabelled benzylpenicillin as a carrier. 4. No incorporation of 3H into solvent-soluble penicillin was detected on incubation of these extracts with penicillin N.     

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a

The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr-/-), and apolipoprotein E-deficient (Apoe-/-) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr-/- mice and greatly accelerated in Apoe-/- mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr-/- mice was similar to that in wild-type mice and was only slightly accelerated in Apoe-/- mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance. In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.     

Biodiversity in agricultural landscapes: the ground beetle communities of woody uncultivated habitats

Agricultural landscapes can be defined as mosaics of landscape elements which are affected by farming practices. Woodland habitats, even though they are managed, are amongst the most stable elements of agricultural landscapes and can play a key role in the maintenance of biodiversity. This study of the ground beetle (carabid) communities of woodlands and woody linear features in a Scottish agricultural landscape shows that these habitats contribute significantly to the overall landscape diversity of these beetles. Communities in woods and hedgerows display the same species diversity and are both characterized by the presence of forest species. The main factors constraining carabid communities in both environments are the grazing intensity and, to a lesser extent, the type of soil. Heavily grazed locations are characterized by the occurrence of grassland species while forest species are restricted to ungrazed locations. At the landscape scale, the distribution of the forest species is limited by spatial isolation, indicating that there are insufficient functional links between woodland habitats in the study area. Isolation could be compensated for either by a better control of grazing so that linear features can be used as dispersal corridors for forest carabids or by planting more linear features and woods in the area.