Alveolar Macrophage Interaction With Air Pollution Particulates

We applied flow cytometric analysis to characterize the in vitro response of alveolar macrophages (AM) to air pollution particulates. Normal hamster AM were incubated with varying concentrations of residual oil fly ash (ROFA) or concentrated ambient air particulates (CAP). We found a dose-dependent increase in AM-associated right angle light scatter (RAS) after uptake of ROFA (e.g., mean channel number 149.4 ± 6.5, 102.5 ± 4.1, 75.8 ± 3.5, and 61.0 ± 4.6 at 200, 100, 50, and 25 mg/ml, respectively) or CAP. A role for scavenger-type receptors (SR) in AM uptake of components of ROFA and CAP was identified by marked inhibition of RAS increases in AM pretreated with the specific SR inhibitor polyinosinic acid. We combined measurement of particle uptake (RAS) with flow cytometric analysis of intracellular oxidation of dichlorofluorescin. Both ROFA and CAP caused a dose-related intracellular oxidant stress within AM, comparable to that seen with phorbol myristate acetate (PMA) (e.g., fold increase over control, 6.6 ± 0.4, 3.6 ± 0.4, 4.6 ± 0.5, 200 mg/ml ROFA, 100 mg/ml ROFA, and $10^{-7}$ M PMA, respectively). We conclude that flow cytometry of RAS increases provides a useful relative measurement of AM uptake of complex particulates within ROFA and CAP. Both ROFA and CAP cause substantial intracellular oxidant stress within AM, which may contribute to subsequent cell activation and production of proinflammatory mediators

Hot wire chemical vapor deposition: limits and opportunities of protecting the tungsten catalyzer from silicide with a cavity

Hot Wire Chemical Vapor Deposition (HW-CVD) is one of the most promising techniques for depositing the intrinsic microcrystalline silicon layer for the production of micro-morph solar cells. However, the silicide formation at the colder ends of the tungsten wire drastically reduces the lifetime of the catalyzer, thus limiting its industrial exploitation. A simple but interesting strategy to decrease the silicide formation is to hide the electrical contacts of the catalyzer in a long narrow cavity which reduces the probability of the silane molecules to reach the colder ends of the wire. In this paper, the working mechanism of the cavity is elucidated. Measurements of the thickness profile of the silicon deposited in the internal walls of the cavity have been compared with those predicted using a simple diffusion model based on the assumption of Knudsen flow. A lifetime study of the protected and unprotected wires has been carried out. The different mechanisms which determine the deterioration of the catalyzer have been identified and discussed

Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid

The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas aeruginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame

Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells

BACKGROUND: Pancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix. METHODS: We tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2. RESULTS: EGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells. CONCLUSION: An inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells

Effects of acutely inhibiting PI3K isoforms and mTOR on regulation of glucose metabolism in vivo

In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O2 consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism

T-cell epitope polymorphisms of the Plasmodium falciparum circumsporozoite protein among field isolates from Sierra Leone: age-dependent haplotype distribution?

<p>Abstract</p> <p>Background</p> <p>In the context of the development of a successful malaria vaccine, understanding the polymorphisms exhibited by malaria antigens in natural parasite populations is crucial for proper vaccine design. Recent observations have indicated that sequence polymorphisms in the C-terminal T-cell epitopes of the <it>Plasmodium falciparum </it>circumsporozoite protein (Pf<it>csp</it>) are rather low and apparently stable in low endemic areas. This study sought to assess the pattern in a malaria endemic setting in Africa, using samples from Freetown, Sierra Leone.</p> <p>Methods</p> <p>Filter-paper blood samples were collected from subjects at a teaching hospital in Freetown during September–October 2006 and in April–May 2007. The C-terminal portion of the Pf<it>csp </it>gene spanning the Th2R and Th3R epitopes was amplified and directly sequenced; sequences were analysed with subject parameters and polymorphism patterns in Freetown were compared to that in other malaria endemic areas.</p> <p>Results and Discussion</p> <p>Overall, the genetic diversity in Freetown was high. From a total of 99 sequences, 42 haplotypes were identified with at least three accounting for 44.4% (44/99): the 3D7-type (19.2%), a novel type, P-01 (17.2%), and E12 (8.1%). Interestingly, all were unique to the African sub-region and there appeared to be predilection for certain haplotypes to distribute in certain age-groups: the 3D7 type was detected mainly in hospitalized children under 15 years of age, while the P-01 type was common in adult antenatal females (Pearson Chi-square = 48.750, degrees of freedom = 34, <it>P </it>= 0.049). In contrast, the single-haplotype predominance (proportion > 50%) pattern previously identified in Asia was not detected in Freetown.</p> <p>Conclusion</p> <p>Haplotype distribution of the T-cell epitopes of Pf<it>csp </it>in Freetown appeared to vary with age in the study population, and the polymorphism patterns were similar to that observed in neighbouring Gambia, but differed significantly at the sequence level from that observed in Asia. The findings further emphasize the role of local factors in generating polymorphisms in the T-cell epitopes of the <it>P. falciparum </it>circumsporozoite protein.</p

Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis

In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10-8 M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7%±4.4 vs. control 22.6%±2.4; p1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10-8 M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6±6.6 vs. 28.0±6.6; p=0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection

Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein