Fluorescence flow cytometer to determine urine particle reference intervals in doping control samples.

Background: Urine is still the matrix of choice to fight against doping, because it can be collected non-invasively during anti-doping tests. Most of the World Anti-Doping Agency's accredited laboratories have more than 20 years experience in analyzing this biological fluid and the majority of the compounds listed in the 2010 Prohibited List - International Standard are eliminated through the urinary apparatus. Storing and transporting urine samples for doping analyses does not include a specific protocol to prevent microbial and thermal degradation. The use of a rapid and reliable screening method could enable determine reference intervals for urine specimens in doping control samples and evaluate notably the prevalence of microbial contamination known to be responsible for the degradation of chemical substances in urine.Methods: The Sysmex(R) UF-500i is a recent urine flow cytometer analyzer capable of quantifying BACT and other urinary particles such as RBC, WBC, EC, DEBRIS, CAST, PATH. CAST, YLC, SRC as well as measuring urine conductivity. To determine urine anti-doping reference intervals, 501 samples received in our laboratory over a period of two months were submitted to an immediate examination. All samples were collected and then transported at room temperature. Analysis of variance was performed to test the effects of factors such as gender, test type [in-competition, out-of-competition] and delivery time.Results: The data obtained showed that most of the urine samples were highly contaminated with bacteria. The other urine particles were also very different according to the factors.Conclusions: The Sysmex(R) UF-500i was capable of providing a snapshot of urine particles present in the samples at the time of the delivery to the laboratory. These particles, BACT in particular, gave a good idea of the possible microbial degradation which had and/or could have occurred in the sample. This information could be used as the first quality control set up in WADA (World Anti-Doping Agency) accredited laboratories to determine if steroid profiles, endogenous and prohibited substances have possibly been altered. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Concentration of Airborne Staphylococcus aureus (MRSA and MSSA), Total Bacteria, and Endotoxins in Pig Farms.

Pigs are very often colonized by Staphylococcus aureus and transmission of such pig-associated S. aureus to humans can cause serious medical, hygiene, and economic problems. The transmission route of zoonotic pathogens colonizing farm animals to humans is not well established and bioaerosols could play an important role. The aim of this study was to assess the potential occupational risk of working with S. aureus-colonized pigs in Switzerland. We estimated the airborne contamination by S. aureus in 37 pig farms (20 nursery and 17 fattening units; 25 in summer, 12 in winter). Quantification of total airborne bacterial DNA, airborne Staphylococcus sp. DNA, fungi, and airborne endotoxins was also performed. In this experiment, the presence of cultivable airborne methicillin-resistant S. aureus (MRSA) CC398 in a pig farm in Switzerland was reported for the first time. Airborne methicillin-sensitive S. aureus (MSSA) was found in ~30% of farms. The average airborne concentration of DNA copy number of total bacteria and Staphylococcus sp. measured by quantitative polymerase chain reaction was very high, respectively reaching values of 75 (± 28) × 10(7) and 35 (± 9.8) × 10(5) copy numbers m(-3) in summer and 96 (± 19) × 10(8) and 40 (± 12) × 10(6) copy numbers m(-3) in winter. Total mean airborne concentrations of endotoxins (1298 units of endotoxin m(-3)) and fungi (5707 colony-forming units m(-3)) exceeded the Swiss recommended values and were higher in winter than in summer. In conclusion, Swiss pig farmers will have to tackle a new emerging occupational risk, which could also have a strong impact on public health. The need to inform pig farmers about biological occupational risks is therefore crucial.

6.2

Airborne particles can come from a variety of sources and contain variable chemical constituents. Some particles are formed by natural processes, such as volcanoes, erosion, sea spray, and forest fires, while other are formed by anthropogenic processes, such as industrial- and motor vehicle-related combustion, road-related wear, and mining. In general, larger particles (those greater than 2.5 μm) are formed by mechanical processes, while those less than 2.5 μm are formed by combustion processes. The chemical composition of particles is highly influenced by the source: for combustion-related particles, factors such as temperature of combustion, fuel type, and presence of oxygen or other gases can also have a large impact on PM composition. These differences can often be observed at a regional level, such as the greater sulphate-composition of PM in regions that burn coal for electricity production (which contains sulphur) versus regions that do not. Most countries maintain air monitoring networks, and studies based on the resulting data are the most common basis for epidemiology studies on the health effects of PM. Data from these monitoring stations can be used to evaluate the relationship between community-level exposure to ambient particles and health outcomes (i.e., morbidity or mortality from various causes). Respiratory and cardiovascular outcomes are the most commonly assessed, although studies have also considered other related specific outcomes such as diabetes and congenital heart disease. The data on particle characteristics is usually not very detailed and most often includes some combination of PM2.5, PM10, sulphate, and NO2. Other descriptors that are less commonly found include particle number (ultrafine particles), metal components of PM, local traffic intensity, and EC/OC. Measures of association are usually reported per 10 μg/m3 or interquartile range increase in pollutant concentration. As the exposure data are taken from regional monitoring stations, the measurements are not representative of an individual's exposure. Particle size is an important descriptor for understanding where in the human respiratory system the particles will deposit: as a general rule, smaller particles penetrate to deeper regions of the lungs. Initial studies on the health effects of particulate matter focused on mass of the particles, including either all particles (often termed total suspended particulate or TSP) or PM10 (all particles with an aerodynamic diameter less than 10 μm). More recently, studies have considered both PM10 and PM2.5, with the latter corresponding more directly to combustion-related processes. UFPs are a dominant source of particles in terms of PNC, yet are negligible in terms of mass. Very few epidemiology studies have measured the effect of UFPs on health; however, the numbers of studies on this topic are increasing. In addition to size, chemical composition is of importance when understanding the toxicity of particles. Some studies consider the composition of particles in addition to mass; however this is not common, in part due the cost and labour involved in such analyses.

Causal Warrant for Realism about Particle Physics

While scientific realism generally assumes that successful scientific explanations yield information about reality, realists also have to admit that not all information acquired in this way is equally well warranted. Some versions of scientific realism do this by saying that explanatory posits with which we have established some kind of causal contact are better warranted than those that merely appear in theoretical hypotheses. I first explicate this distinction by considering some general criteria that permit us to distinguish causal warrant from theoretical warrant. I then apply these criteria to a specific case from particle physics, claiming that scientific realism has to incorporate the distinction between causal and theoretical warrant if it is to be an adequate stance in the philosophy of particle physics.

Nano-particle vaccination combined with TLR-7 and -9 ligands triggers memory and effector CD8⁺ T-cell responses in melanoma patients.

Optimal vaccine strategies must be identified for improving T-cell vaccination against infectious and malignant diseases. MelQbG10 is a virus-like nano-particle loaded with A-type CpG-oligonucleotides (CpG-ODN) and coupled to peptide(16-35) derived from Melan-A/MART-1. In this phase IIa clinical study, four groups of stage III-IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan-A/MART-1-specific T-cell responses. T-cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T-cell responses in all (11/11) patients, with predominant generation of effector-memory-phenotype cells. In turn, Imiquimod induced higher proportions of central-memory-phenotype cells and increased percentages of CD127(+) (IL-7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T-cell frequencies, associated with lower proportions of memory and effector-phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG-ODN induced combined memory and effector CD8(+) T-cell responses.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Sensitive photonic system to measure oxidative potential of airborne nanoparticles and ROS levels in exhaled air

A photonic system has been developed that enables sensitive quantitative determination of reactive oxygen species (ROS) - mainly hydrogen peroxide (H2O2) - in aerosol samples such as airborne nanoparticles and exhaled air from patients. The detection principle relies on the amplification of the absorbance under multiple scattering conditions due to optical path lengthening [1] and [2]. In this study, the presence of cellulose membrane that acts as random medium into the glass optical cell considerably improved the sensitivity of the detection based on colorimetric FOX assay (FeII/orange xylenol). Despite the loss of assay volume (cellulose occupies 75% of cell volume) the limit of detection is enhanced by one order of magnitude reaching the value of 9 nM (H2O2 equivalents). Spectral analysis is performed automatically with a periodicity of 5 to 15 s, giving rise to real-time ROS measurements. Moreover, the elution of air sample into the collection chamber via a micro-diffuser (impinger) enables quantitative determination of ROS contained in or generated from airborne samples. As proof-of-concept the photonic ROS detection system was used in the determination of both ROS generated from traffic pollution and ROS contained in the exhaled breath as lung inflammation biomarkers.

The composition of airborne and grain-dust fungal communities is shaped at regional scale by plant genotypes and farming practices

Chronic exposure to airborne fungi has been associated with different respiratory symptoms and pathologies in occupational populations, such as grain workers. However, the homogeneity in the fungal species composition of these bioaerosols on a large geographical scale and the different drivers that shape these fungal communities remain unclear. In this study, the diversity of fungi in grain dust and in the aerosols released during harvesting was determined across 96 sites at a geographical scale of 560 km(2) along an elevation gradient of 500 m by tag-encoded 454-pyrosequencing of the internal transcribed spacer (ITS) sequences. Associations between the structure of fungal communities in the grain dust and different abiotic (farming system, soil characteristics, geographic and climatic parameters) and biotic (wheat cultivar, previous crop culture) factors were explored. These analyses revealed a strong relationship between the airborne and grain dust fungal communities and showed the presence of allergenic and mycotoxigenic species in most samples, which highlights the potential contribution of these fungal species to work-related respiratory symptoms of grain workers. The farming system was the major driver of the alpha and beta phylogenetic diversity of fungal communities. In addition, elevation and soil CaCO3 concentrations shaped the alpha diversity whereas wheat cultivar, cropping history and the number of freezing days per year shaped the taxonomic beta diversity of these communities.