Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Determination and Evaluation of Alternative Methods for Managing and Controlling Highway-Related Dust Phase II—Demonstration Project, June 2005

The State of Iowa currently has approximately 69,000 miles of unpaved secondary roads. Due to the low traffic count on these unpaved o nts as ng e two dust ed d roads, paving with asphalt or Portland cement concrete is not economical. Therefore to reduce dust production, the use of dust suppressants has been utilized for decades. This study was conducted to evaluate the effectiveness of several widely used dust suppressants through quantitative field testing on two of Iowa’s most widely used secondary road surface treatments: crushed limestone rock and alluvial sand/gravel. These commercially available dust suppressants included: lignin sulfonate, calcium chloride, and soybean oil soapstock. These suppressants were applied to 1000 ft test sections on four unpaved roads in Story County, Iowa. Tduplicate field conditions, the suppressants were applied as a surface spray once in early June and again in late August or early September. The four unpaved roads included two with crushed limestone rock and two with alluvial sand/gravel surface treatmewell as high and low traffic counts. The effectiveness of the dust suppressants was evaluated by comparing the dust produced on treated and untreated test sections. Dust collection was scheduled for 1, 2, 4, 6, and 8 weeks after each application, for a total testiperiod of 16 weeks. Results of a cost analysis between annual dust suppressant application and biennial aggregate replacement indicated that the cost of the dust suppressant, its transportation, and application were relatively high when compared to that of thaggregate types. Therefore, the biennial aggregate replacement is considered more economical than annual dust suppressant application, although the application of annual dust suppressant reduced the cost of road maintenance by 75 %. Results of thecollection indicated that the lignin sulfonate suppressant outperformed calcium chloride and soybean oil soapstock on all four unpavroads, the effect of the suppressants on the alluvial sand/gravel surface treatment was less than that on the crushed limestone rock, the residual effects of all the products seem reasonably well after blading, and the combination of alluvial sand/gravel surface treatment anhigh traffic count caused dust reduction to decrease dramatically.

Transhepatic ipsilateral right portal vein embolization extended to segment IV: improving hypertrophy and resection outcomes with spherical particles and coils.

PURPOSE: To analyze outcomes after right portal vein embolization extended to segment IV (right PVE + IV) before extended right hepatectomy, including liver hypertrophy, resection rates, and complications after embolization and resection, and to assess differences in outcomes with two different particulate embolic agents. MATERIALS AND METHODS: Between 1998 and 2004, transhepatic ipsilateral right PVE + IV with particles and coils was performed in 44 patients with malignant hepatobiliary disease, including metastases (n = 24), biliary cancer (n = 14), and hepatocellular carcinoma (n = 6). Right PVE + IV was considered if the future liver remnant (FLR; segments II/III with or without I) was less than 25% of the total estimated liver volume (TELV). Tris-acryl microspheres (100-700 microm; n = 21) or polyvinyl alcohol (PVA) particles (355-1,000 microm; n = 23) were administered in a stepwise fashion. Smaller particles were used to occlude distal branches, followed by larger particles to occlude proximal branches until near-complete stasis. Coils were then placed in secondary portal branches. Computed tomographic volumetry was performed before and 3-4 weeks after right PVE + IV to assess FLR hypertrophy. Liver volumes and postembolization and postoperative outcomes were measured. RESULTS: After right PVE + IV with PVA particles, FLR volume increased 45.5% +/- 40.9% and FLR/TELV ratio increased 6.9% +/- 5.6%. After right PVE + IV with tris-acryl microspheres, FLR volume increased 69.0% +/- 30.7% and FLR/TELV ratio increased 9.7% +/- 3.3%. Differences in FLR volume (P = .0011), FLR/TELV ratio (P = .027), and resection rates (P = .02) were statistically significant. Seventy-one percent of patients underwent extended right hepatectomy (86% after receiving tris-acryl microspheres, 57% after receiving PVA). Thirteen patients (29%) did not undergo resection (extrahepatic spread [n = 9], inadequate hypertrophy [n = 3], other reasons [n = 1]). No patient developed postembolization syndrome or progressive liver insufficiency after embolization or resection. One death after resection occurred as a result of sepsis and hemorrhage. Median hospital stays were 1 day after right PVE + IV and 7 days after resection. CONCLUSION: Transhepatic ipsilateral right PVE + IV with use of particles and coils is a safe, effective method for inducing contralateral hypertrophy before extended right hepatectomy. Embolization with small spherical particles provides improved hypertrophy and resection rates compared with larger, nonspherical particles.

Virus-like particles induce robust human T-helper cell responses.

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qβ-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qβ. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Respiratory effects of an exposure to grain dust among grain workers in the Vaud region (Switzerland)

Introduction: Bioaerosols such as grain dust (GD) elicit direct immunological reactions within the human respiratory system. Workplace-dependent exposure to GD may induce asthma, chronic bronchitis, and hypersensitivity pneumonitis. Aims: To assess the clinical impact of occupational exposure to GD and to determine quantitative biological markers of bioaerosol exposure in grain workers. Methods: This longitudinal study has been conducted from summer 2012 to summer 2013, comprising 6 groups of 30 active workers with different GD exposure patterns (4 groups of grain workers, 2 control groups). Two evaluations at high- and low-exposing seasons take place, during which an occupational and a medical history are questionnaire-assessed, lung function is evaluated by spirometry, airway inflammation is measured by exhaled nitric oxide (eNO) and specific blood IgG and IgE are titrated. Results: The preliminary results are those of 2 of the 4 exposed groups, (harvesters and mill workers), compared to the control groups, at first assessment (n=100). Mean age is 38.4 [years]; 98% are male. Exposed groups differ from controls (p<0.05) in daily contact with animals (57% vs. 40%) and active smoking (39% vs. 11%). Grain workers have more respiratory (50%), nasal (57%), ocular (45%) and dermatologic (36%) occupational symptoms than controls (6.4%, 19%, 16%, 6.4% respectively, p<0.05). Lower mean peak-expiratory-flow (PEF) values (96.1 ± 18.9 vs. 108.2 ± 17.4 [% of predicted], p<0.05) and eNO values (13.9 ± 9.6 vs. 20.5 ± 14.7 [ppm], p<0.05) are observed in the exposed groups. Conclusion: Preliminary results show a higher prevalence of clinical symptoms and a lower mean PEF value in the groups exposed to GD.

Reduction of connexin36 content by ICER-1 contributes to insulin-secreting cells apoptosis induced by oxidized LDL particles.

Connexin36 (Cx36), a trans-membrane protein that forms gap junctions between insulin-secreting beta-cells in the Langerhans islets, contributes to the proper control of insulin secretion and beta-cell survival. Hypercholesterolemia and pro-atherogenic low density lipoproteins (LDL) contribute to beta-cell dysfunction and apoptosis in the context of Type 2 diabetes. We investigated the impact of LDL-cholesterol on Cx36 levels in beta-cells. As compared to WT mice, the Cx36 content was reduced in islets from hypercholesterolemic ApoE-/- mice. Prolonged exposure to human native (nLDL) or oxidized LDL (oxLDL) particles decreased the expression of Cx36 in insulin secreting cell-lines and isolated rodent islets. Cx36 down-regulation was associated with overexpression of the inducible cAMP early repressor (ICER-1) and the selective disruption of ICER-1 prevented the effects of oxLDL on Cx36 expression. Oil red O staining and Plin1 expression levels suggested that oxLDL were less stored as neutral lipid droplets than nLDL in INS-1E cells. The lipid beta-oxidation inhibitor etomoxir enhanced oxLDL-induced apoptosis whereas the ceramide synthesis inhibitor myriocin partially protected INS-1E cells, suggesting that oxLDL toxicity was due to impaired metabolism of the lipids. ICER-1 and Cx36 expressions were closely correlated with oxLDL toxicity. Cx36 knock-down in INS-1E cells or knock-out in primary islets sensitized beta-cells to oxLDL-induced apoptosis. In contrast, overexpression of Cx36 partially protected INS-1E cells against apoptosis. These data demonstrate that the reduction of Cx36 content in beta-cells by oxLDL particles is mediated by ICER-1 and contributes to oxLDL-induced beta-cell apoptosis.

In vitro assessment of the pulmonary toxicity and gastric availability of lead-rich particles from a lead recycling plant

Epidemiological studies in urban areas have linked increasing respiratory and cardiovascular pathologies with atmospheric particulate matter (PM) from anthropic activities. However, the biological fate of metal-rich PM industrial emissions in urban areas of developed countries remains understudied. Lead toxicity and bioaccessibility assessments were therefore performed on emissions from a lead recycling plant, using complementary chemical acellular tests and toxicological assays, as a function of PM size (PM(10-2.5), PM(2.5-1) and PM(1)) and origin (furnace, refining and channeled emissions). Process PM displayed differences in metal content, granulometry, and percentage of inhalable fraction as a function of their origin. Lead gastric bioaccessibility was relatively low (maximum 25%) versus previous studies; although, because of high total lead concentrations, significant metal quantities were solubilized in simulated gastrointestinal fluids. Regardless of origin, the finest PM(1) particles induced the most significant pro-inflammatory response in human bronchial epithelial cells. Moreover, this biological response correlated with pro-oxidant potential assay results, suggesting some biological predictive value for acellular tests. Pulmonary effects from lead-rich PM could be driven by thiol complexation with either lead ions or directly on the particulate surface. Finally, health concern of PM was discussed on the basis of pro-inflammatory effects, accellular test results, and PM size distribution.

Rectal and vaginal immunization of mice with human papillomavirus L1 virus-like particles.

Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.

Comparative testing of a miniature diffusion size classifier to assess airborne ultrafine particles under field conditions

Miniature diffusion size classifiers (miniDiSC) are novel handheld devices to measure ultrafine particles (UFP). UFP have been linked to the development of cardiovascular and pulmonary diseases; thus, detection and quantification of these particles are important for evaluating their potential health hazards. As part of the UFP exposure assessments of highwaymaintenance workers in western Switzerland, we compared a miniDiSC with a portable condensation particle counter (P-TRAK). In addition, we performed stationary measurements with a miniDiSC and a scanning mobility particle sizer (SMPS) at a site immediately adjacent to a highway. Measurements with miniDiSC and P-TRAK correlated well (correlation of r = 0.84) but average particle numbers of the miniDiSC were 30%âeuro"60% higher. This difference was significantly increased for mean particle diameters below 40 nm. The correlation between theminiDiSC and the SMPSduring stationary measurements was very high (r = 0.98) although particle numbers from the miniDiSC were 30% lower. Differences between the three devices were attributed to the different cutoff diameters for detection. Correction for this size dependent effect led to very similar results across all counters.We did not observe any significant influence of other particle characteristics. Our results suggest that the miniDiSC provides accurate particle number concentrations and geometric mean diameters at traffic-influenced sites, making it a useful tool for personal exposure assessment in such settings.

The policy relevance of wear emissions from road transport, now and in the future : an international workshop report and consensus statement

Road transport emissions are a major contributor to ambient particulate matter concentrations and have been associated with adverse health effects. Therefore, these emissions are targeted through increasingly stringent European emission standards. These policies succeed in reducing exhaust emissions, but do not address "nonexhaust" emissions from brake wear, tire wear, road wear, and suspension in air of road dust. Is this a problem? To what extent do nonexhaust emissions contribute to ambient concentrations of PM10 or PM2.5? In the near future, wear emissions may dominate the remaining traffic-related PM10 emissions in Europe, mostly due to the steep decrease in PM exhaust emissions. This underlines the need to determine the relevance of the wear emissions as a contribution to the existing ambient PM concentrations, and the need to assess the health risks related to wear particles, which has not yet received much attention. During a workshop in 2011, available knowledge was reported and evaluated so as to draw conclusions on the relevance of traffic-related wear emissions for air quality policy development. On the basis of available evidence, which is briefly presented in this paper, it was concluded that nonexhaust emissions and in particular suspension in air of road dust are major contributors to exceedances at street locations of the PM10 air quality standards in various European cities. Furthermore, wear-related PM emissions that contain high concentrations of metals may (despite their limited contribution to the mass of nonexhaust emissions) cause significant health risks for the population, especially those living near intensely trafficked locations. To quantify the existing health risks, targeted research is required on wear emissions, their dispersion in urban areas, population exposure, and its effects on health. Such information will be crucial for environmental policymakers as an input for discussions on the need to develop control strategies.

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]