Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.     

Effect of the initial dose of foot-and-mouth disease virus on the early viral dynamics within pigs

This paper investigates the early viral dynamics of foot-and-mouth disease (FMD) within infected pigs. Using an existing within-host model, we investigate whether individual variation can be explained by the effect of the initial dose of FMD virus. To do this, we consider the experimental data on the concentration of FMD virus genomes in the blood (viral load). In this experiment, 12 pigs were inoculated with one of three different doses of FMD virus: low; medium; or high. Measurements of the viral load were recorded over a time course of approximately 11 days for every 8 hours. The model is a set of deterministic differential equations with the following variables: viral load; virus in the interstitial space; and the proportion of epithelial cells available for infection, infected and uninfected. The model was fitted to the data for each animal individually and also simultaneously over all animals varying only the initial dose. We show that the general trend in the data can be explained by varying only the initial dose. The higher the initial dose the earlier the development of a detectable viral load.     

Derivation of accurate glass transition temperatures by differential scanning calorimetry

Glass transition temperatures, T_g, cannot be directly determined from differential scanning calorimetry (d.s.c.) curves because of kinetic effects which are especially serious for well annealed samples. Using an anionic polystyrene as an example it is shown how d.s.c. data can be transformed to enthalpy curves which give T_g with an accuracy of ±1K. As the rate of cooling through the glassy region is lowered T_g is found to decrease by 2.2 K per decade decrease in cooling rate. Quantitative data are given for the specific heat increment at T_g.     

Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases

Programmed cell death invariably requires the activation of proteolytic cascades that are not yet well defined but are initiated after apical caspase activation. We provide evidence that calpains and the proteasome function synergistically downstream of caspases to assist the constitutive apoptotic program of aging neutrophils, which plays an important role in resolution of inflammatory responses. Inhibitor studies indicated that "tethering" of preapoptotic senescent neutrophils to human macrophages required caspase activity. However, the development of morphological features characteristic of apoptosis, including nuclear morphology, PS exposure, surface protein shedding, and the capacity to be ingested by macrophages, required the downstream action of either calpains or the proteasome. Calpain activities were constitutively active in freshly isolated neutrophils and responsible for rearrangements in the protein composition and structure of the plasmalemmal cytoskeleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for increased susceptibility to cleavage, and a loss in immunodetectable alpha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a physiologically important form of constitutive apoptosis.     

Visual and auditory attention in patient monitoring: a formative analysis

We investigate the effectiveness of sonification (continuous auditory display) for supporting patient monitoring while reducing visual attentional workload in the operating theatre. Non-anaesthetist participants performed a simple continuous arithmetic task while monitoring the status of a simulated anaesthetised patient, reporting the status of vital signs when asked. Patient data were available either on a monitoring screen behind the participant, or were partially or completely sonified. Video captured when, how often and for how long participants turned to look at the screen. Participants gave the most accurate responses with visual displays, the fastest responses with sonification and the slowest responses when sonification was added to visual displays. A formative analysis identifying the constraints under which participants timeshare the arithmetic and monitoring tasks provided a context for interpreting the video data. It is evident from the pattern of their visual attention that participants are sensitive to events with different but overlapping temporal rhythms.     

Comparison of multiplex PCR with conventional biochemical methods for the identification of Listeria spp. isolates from food and clinical samples in Queensland, Australia

Listeria monocytogenes is an important foodborne pathogen with high mortality. L. monocytogenes and five other Listeria species can frequently be found in the same sample. To identify Listeria isolates found in foods to the species level, two multiplex PCRs were designed. The PCR and conventional biochemical methods were compared for the identification of 456 Listeria isolates collected from routine food quality monitoring schemes between June 2004 and February 2006 and for 62 L. monocytogenes isolates from patients between 1999 and 2005. The results showed that the PCR and biochemical methods had 100% agreement in Listeria identification. The distribution of Listeria species from foods was as follows: L. monocytogenes, 50.4%; L. innocua, 33.8%; L. welshimeri, 14.9%; L. seeligeri, 0.7%; L. grayi, 0.2%; and L. ivanovii, 0.0%. Additional analyses were performed to identify the major serotypes (1/2a, 1/2b, 1/2c, and 4b) and the three lineages of L. monocytogenes isolates from foods and patients, with 1/2a (69.6%) and 1/2b (21.7%) dominating the food isolates and 1/2b (54.8%) and 4b (30.7%) dominating the patient isolates. The lineage results showed that isolates of 1/2a and 1/2c belonged to lineage II and that isolates of 1/2b and 4b belonged to lineage I. The multiplex PCRs for Listeria identification that have been established provide an accurate and rapid method for food quality control. This study has provided the basic knowledge of distribution of Listeria species and L. monocytogenes serotypes in Queensland, Australia, which is useful for epidemiological investigations of listeriosis.     

Cytokines promote glomerular mesangial cell survival in vitro by stimulus-dependent inhibition of apoptosis

Resolution of glomerular inflammation requires the removal of proliferating resident glomerular mesangial cells, but excessive loss of glomerular cells is a feature of postinflammatory scarring. Because apoptosis regulates mesangial cell number in glomerular inflammation, we have studied the exogenous control of apoptosis triggered in cultured mesangial cells by stimuli likely to be important in vivo. Apoptosis could be induced by serum deprivation to model decreased availability of survival factors, by etoposide as an example of DNA-damaging agents, by ligation of mesangial cell Fas, and by protein synthesis inhibition by cycloheximide. Insulin-like growth factor I (IGF-I), IGF-II, and basic fibroblast growth factor were each able to suppress apoptosis induced by serum deprivation, whereas TGF-beta 1, epidermal growth factor, and platelet-derived growth factor had no effect. IGF-I and IGF-II (but not basic fibroblast growth factor) were also able to protect cells from apoptosis induced by etoposide or cycloheximide. However, Fas-mediated apoptosis was resistant to suppression by all three cytokines. None of the cytokines tested caused a change in the levels of expression of Bcl-2, Bax, Bcl-x, or Bak proteins. The survival-promoting properties of serum-free medium conditioned by mesangial cells was abrogated by neutralizing IGF-I Ab. These experiments are the first to define cytokines that inhibit apoptosis and thereby promote survival of mesangial cells, and the data indicate a paracrine survival signaling role for IGF-I. Finally, the data show that Fas ligation can override cytokine survival signaling, emphasizing a candidate role for this molecule in the undesirable apoptotic loss of mesangial cells during the progression of glomerular scarring.