%0 Generic %D 2023 %T After the ban: How to control titanium dioxide in food? %A Karlien Cheyns %A Régis Nkenda %A Sophie Van Den Neucker %A Jan Mast %B 7th Imekofoods conference %I Anses %C Paris, France %8 Oktober 2023 %G eng %0 Generic %D 2023 %T Application of silver-based biocides in face masks intended for general use requires regulatory control %A Jan Mast %A Erik Van Miert %A Lisa Siciliani %A Karlien Cheyns %A Charlotte Wouters %A Nadia Waegeneers %A Ruud Bernsen %A Christiane Vleminckx %A Joris Van Loco %A Eveline Verleysen %G eng %0 Journal Article %J Science of The Total Environment %D 2023 %T Application of silver-based biocides in face masks intended for general use requires regulatory control %A Jan Mast %A Erik Van Miert %A Lisa Siciliani %A Karlien Cheyns %A Marie-Noelle Blaude %A Charlotte Wouters %A Nadia Waegeneers %A Ruud Bernsen %A Christiane Vleminckx %A Joris Van Loco %A Eveline Verleysen %K Ag %K Biocide %K electron microscopy %K Face masks %K ICPMS %K Nanoparticle %K Physicochemical characterization %K Risk Assessment %K Silver %K textile %X

Silver-based biocides are applied in face masks because of their antimicrobial properties. The added value of biocidal silver treatment of face masks to control SARS-CoV-2 infection needs to be balanced against possible toxicity due to inhalation exposure. Direct measurement of silver (particle) release to estimate exposure is problematic. Therefore, this study optimized methodologies to characterize silver-based biocides directly in the face masks, by measuring their total silver content using ICP-MS and ICP-OES based methods, and by visualizing the type(s) and localization of silver-based biocides using electron microscopy based methods. Thirteen of 20 selected masks intended for general use contained detectable amounts of silver ranging from 3 μg to 235 mg. Four of these masks contained silver nanoparticles, of which one mask was silver coated. Comparison of the silver content with limit values derived from existing inhalation exposure limits for both silver ions and silver nanoparticles allowed to differentiate safe face masks from face masks that require a more extensive safety assessment. These findings urge for in depth characterization of the applications of silver-based biocides and for the implementation of regulatory standards, quality control and product development based on the safe-by-design principle for nanotechnology applications in face masks in general.

%B Science of The Total Environment %V 870 %8 Jan-04-2023 %G eng %R 10.1016/j.scitotenv.2023.161889 %0 Generic %D 2023 %T Characterisation of iron oxides and hydroxides applied in the food chain by electron microscopy %A Lisa Siciliani %A Eveline Verleysen %A Sara Bals %A Fanny Caputo %A Jan Mast %K E 172 %K Iron oxide %K Nanoparticles %B The 20th International Microscopy Congress (IMC20) %C Busan, South Korea %8 13 sept 2023 %G eng %0 Generic %D 2023 %T Characterisation of nanocellulose applied in the food chain by electron microscopy %A Lisa Siciliani %A Eveline Verleysen %A Sara Bals %A Francesco Fumagalli %A Francesco Cubadda %A Jan Mast %K NAMs %K nanocellulose %K nanospecific assessment %B The 20th International Microscopy Congress (IMC20) %C Busan, South Korea %8 12 sept 2023 %G eng %0 Report %D 2023 %T EFSA Project on the use of New Approach Methodologies (NAMs) for the hazard assessment of nanofibres. Lot 1, nanocellulose oral exposure: gastrointestinal digestion, nanofibres uptake and local effects %A Olimpia Vincentini %A Anne‐Louise Blier %A Alessia Bogni %A Morgane Brun %A Serena Cecchetti %A Francesca De Battistis %A Sylvain Denis %A Lucie Etienne‐Mesmin %A Francesca Ferraris %A Francesco Sirio Fumagalli %A Kevin Hogeveen %A Francesca Iacoponi %A Andrea Raggi %A Lisa Siciliani %A Stanco, Deborah %A Eveline Verleysen %A Valerie Fessard %A Jan Mast %A Stephanie Blanquet‐Diot %A Susanne Bremer‐Hoffmann %A Francesco Cubadda %K barrier crossing %K cell uptake %K hazard identification %K IATAs %K NAMs %K nanocellulose %K nanospecific assessment %X

Nanocellulose (NC) is an emerging material in the food sector with several prospective application areas. Three main types of NC exist, i.e. bacterial NC (BNC), nanofibrillated cellulose (NFC), and cellulose nanocrystals (CNC). The biological sources and processing conditions affect several physicochemical parameters of NC. In the present project, a NAM-based IATA for addressing data gaps in the assessment of potential hazards associated to NC oral exposure was considered. This IATA focused on three main pillars, i.e. (i) assessment of the uptake and potential crossing of the intestinal barrier by NC, (ii) assessment of local effects, including inflammation and genotoxicity, on the gastrointestinal epithelia, and (iii) assessment of any digestion or degradation of NC by the human microbiome. Eight NC samples belonging to the three NC types, plus a comparator in the micro-range, were selected as study materials and submitted to a thorough physicochemical characterisation. A battery of in vitro tests was used to provide insight into NC hazard and mode of action according to a tiered approach, which lead to selection of three materials belonging to the three main NC types for in depth-testing. Cell uptake of these materials was demonstrated, and such uptake was greater in a triculture model, which better simulates the barrier properties of the human intestinal epithelium, as compared to Caco-2 monolayers. Uptake was the greatest in repeated exposure conditions, in which intestinal barrier crossing was demonstrated for CNC. Pro-inflammatory responses accompanied by massive NC uptake in macrophages, indicative for potential immunotoxicological effects, and barrier function impairment were observed, whereas no indications for genotoxicity were obtained. Finally, no formation of smaller particles following colonic fermentation of NC was observed. For the integration of these results in regulatory hazard assessment of NC after oral exposure, prospective use of NC as novel food or as food additive was considered.

%B EFSA Supporting Publications %V 20 %8 20-09-2023 %G eng %N 9 %R 10.2903/sp.efsa.2023.EN-8258 %0 Generic %D 2023 %T Identification and characterization of inorganic food additives and pearlescent pigments in sprays for food decoration by STEM-EDX %A Noa Olluyn %A Eveline Verleysen %A Lisa Siciliani %A Daniel Arenas Esteban %A S. Mathioudaki %A Sara Bals %A Subhalakshmi Sharma %A Jan Mast %A Joris Van Loco %K electron microscopy %K inorganic food additives %K pearlescent pigments %K sprays %K STEM-EDX %X

Food colorants are applied in commercial products and domestic cooking to enhance the appearance of food. To obtain specific hues, inorganic food additives containing (nano)particles are mixed in varying concentrations, and pearlescent pigments consisting of mica platelets coated with a layer of titanium dioxide and/or iron oxide particles, are applied to provide glitter effects1. For control and risk assessment of multi-constituent substances and mixtures, characterisation of the fraction of small particles, including the particle size distribution, is needed for each single constituent or each component in the mixture2. This is challenging for control laboratories and only limited guidance is currently available3.
This study developed electron microscopy-based methods to identify and characterize the particles of individual components in eight commercially available food-decoration sprays of different colours, containing mixtures of food additives and pearlescent pigments. No dispersion protocols or purifications steps were applied to optimally assess the properties of the particles consumers are exposed to. Samples were prepared by spraying 5mL in a glass vial and coated on EM-grids using the grid-on-drop method. Scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray spectroscopy (EDX) allowed identifying particles of separate components, including potassium aluminium silicate-based pearlescent pigments, vegetable carbon, rutile titanium dioxide (nano)particles, iron oxide (nano)particles and aluminium containing (nano)particles by their elemental composition. Their presence and relative concentration varied between spray colours. Often the layer of titanium dioxide particles detached from the mica, and titanium dioxide particles were also observed forming near-spherical aggregates. STEM-EDX tomography allowed identifying particles of overlapping components and examining the structure of the pearlescent pigments in 3D. The presence of a fraction of nanoparticles in each component was demonstrated based on their particle size distributions. The methods and findings support regulatory bodies to assess and control possible health risks of mixtures of (nano)particles present in the food chain.

%B IMEKOFOODS 2023 %I Imekofoods %C Paris %8 22/11/2023 %G eng %0 Journal Article %J Trends in Food Science & Technology %D 2023 %T Regulatory safety assessment of nanoparticles for the food chain in Europe %A Reinhilde Schoonjans %A Jacqueline Castenmiller %A Qasim Chaudhry %A Francesco Cubadda %A Takis Daskaleros %A Roland Franz %A David Gott %A Jan Mast %A Mortensen, Alicja %A Agnes G. Oomen %A Hubert Rauscher %A Stefan Weigel %A Maria Chiara Astuto %A Irene Cattaneo %A Eric Barthelemy %A Ana Rincon %A José Tarazona %K Integrated approaches to testing and assessment (IATA) %K nanomaterial %K particulate material %K Physicochemical characterisation %K Risk Assessment %K Toxicity testing %X

Background

The risk assessment of small particles (including nanoparticles) in products used in the food chain in the EU falls within the remit of the European Food Safety Authority (EFSA) and has been under thorough scientific considerations for over a decade. Now that more experience is gained with evaluating novel foods, food contact materials, food/feed additives and pesticides, the outlines for regulatory safety assessments and data requirements are established.

Scope and approach

This paper reviews the principles underlying safety testing of small particles, referring to two recently published EFSA guidance documents. Examples and observations from assessing existing materials are provided to facilitate to a wider readership adequate implementation of the regulatory requirements.

Main findings and conclusions

The starting point for safety testing is the physicochemical characterisation of the pristine material, being an engineered nanomaterial, a nanostructured material or a conventional material that contains a fraction of small particles that may retain properties at the nanoscale. Key parameters and threshold values for establishing the presence of small particles, the techniques and methods for characterisation in complex matrices, as well as approaches for dietary exposure assessment are outlined. Where there is the likelihood of small particles remaining after gastrointestinal digestion, hazard identification and hazard characterisation are required with special provisions. In particular, certain nano-specific considerations are highlighted that have to be considered during toxicological testing with the aim to demonstrate consumer safety of products to be used in the food chain in Europe.

%B Trends in Food Science & Technology %8 Jan-02-2023 %G eng %R 10.1016/j.tifs.2023.01.017 %0 Generic %D 2023 %T Release of Nano-objects, their aggregates and agglomerates from Masks: ambitions and scientific objectives of the RENAAME project %A F.-X. Ouf %A F. Gaie-Levrel %A S. Chazelet %A G. Favre %A N. Feltin %A V. Ferré %A V. Godefert %A Jan Mast %A J. Noireaux %A S. Pacault %A X. Poisson %A C. Wouters %K airborne release %K masks %K nanomaterials %X

Le contexte pandémique a mis en avant la nécessité de disposer de masques (chirurgicaux, de protection respiratoire FFPx et à usage non sanitaire). Pour la fabrication de ces différents types de masques, l'ajout de nanomatériaux représente une voie majeure d'innovation mais est aussi source de préoccupation sanitaire en lien avec une possible exposition par inhalation aux nano-objets, leurs agrégats et agglomérats (NOAA). Ainsi, plusieurs produits commerciaux déclarant des propriétés biocides/virucides ont soulevé diverses interrogations au niveau national [1] et international [2] quant à leur innocuité, aboutissant à leur retrait dès la mise sur le marché. Plus récemment, la présence de NOAA de TiO2 dans des masques a été démontrée [3] alors qu'aucune mention n'était précisée sur leurs emballages. Ce sujet de préoccupation relativement émergent souffre cependant d'un manque de développement méthodologique en ce qui concerne l'évaluation de l'exposition par inhalation aux NOAA lors de l'utilisation de masques. Le projet RENAAME vise à développer une méthodologie d'évaluation du relargage potentiel en phase aérosol des nanomatériaux déclarés ou impliqués sans indication commerciale dans la fabrication de masques afin d'évaluer l'exposition par inhalation dans des conditions réalistes d'utilisation. L'originalité du projet RENAAME consiste à développer et valider une approche couplant analyse des matériaux constitutifs des masques et des aérosols potentiellement émis. Si des travaux prénormatifs sont en cours (ISO/TC229 et ISO/NP TS11353) sur ce sujet, le nombre d'études scientifiques dédiées s'avère très limité. La méthodologie proposée vise à quantifier la fraction mobilisable (FM) de NOAA dans les masques puis à caractériser la fraction relarguée (FR) en phase aérosol pour des conditions représentatives d'utilisation. Cette évaluation sera menée sur des masques déclarant commercialement la présence de nanomatériaux présentant un potentiel risque sanitaire (dans le cas de ce projet NOAA composés de TiO2 et d'Ag), mais également sur des masques ne mentionnant aucune utilisation explicite de NOAA. La détermination de ces deux fractions permettra in-fine d'aboutir à un classement des différents types de masques en fonction de leur pouvoir émissif (PE=FR/FM). L'objectif de cette communication est de présenter les actions visées dans le projet RENAAME ainsi que la méthodologie expérimentale associée et les étapes nécessaires à sa qualification et sa validation. Les premiers résultats, en ce qui concerne l'identification par spectroscopie de fluorescence des rayons X (XRF), la quantification par spectrométrie de masse/d'émission atomique de plasma à couplage inductif (ICP-MS/OES) et l'analyse dimensionnelle par microscopie électronique (MEB/MET) de NOAA potentiellement présents, seront présentés. Ce projet est financé par le Programme National de Recherche Environnement Santé-Travail de l'ANSES avec le soutien des ministères chargés de l'environnement, de l'agriculture et du travail (ANSES-22-EST-023). [1] https://www.anses.fr/fr/system/files/CONSO2021SA0089.pdf [2] https://recalls-rappels.canada.ca/fr/avis-rappel/masques-contenant-du-graphene [3] https://doi.org/10.1038/s41598-022-06605-w

%B Congrès Français sur les Aérosols %I - %C Paris, France %8 2023 %G eng %R 10.25576/ASFERA-CFA2023-33869 %0 Journal Article %J Sci Total Environ %D 2023 %T Release of silver and titanium from face masks traded for the general population. %A Daniela Montalvo %A Garbiel Mercier %A Jan Mast %A Karlien Cheyns %K Biocide %K Face masks %K ICPMS %K Nanoparticle %K Risk Assessment %K Silver %K textile %X

Previous assessments of a selection of face masks intended for the general population in Belgium found that silver (Ag)-based biocides were present in masks advertised for antimicrobial properties; whereas titanium dioxide (TiO) particles were detected in all the face masks in at least one layer corroborating its widespread use in the textile industry. The presence of Ag-based biocides and TiO particles in face masks raised questions on the possibility of release under normal wearing conditions, which could potentially cause a health risk to the consumers. Direct measurement of release of Ag and TiO particles during normal wearing is problematic by the lack of methodology to test release and to quantify inhaled particles. Therefore in this study, we investigated leaching experiments using artificial acid sweat as a method to evaluate the release of Ag-based biocides and TiO particles present in face masks. Leaching experiments were proposed as an alternative method to evaluate the quality of face masks, and as a higher tier method to assess face masks that are not safe-by-design. Results from leaching experiments showed that Ag was released in amounts varying from 0.03 up to 36 % of total Ag content, in four out of the eight face masks that claimed antimicrobial properties and that contained Ag. The leaching data of titanium (Ti) showed that despite TiO being detected in all face masks, only in one mask Ti was measured in detectable concentrations in artificial sweat (0.35 % of total Ti content). Comparison of leachable Ag and Ti with respective acceptable exposure limit values derived from inhalation exposure limits indicate that three face masks would need further risk assessment and could not be considered as intrinsically safe.

%B Sci Total Environ %V 901 %8 2023 Jul 19 %G eng %R 10.1016/j.scitotenv.2023.165616 %0 Journal Article %J EFSA Journal %D 2023 %T Re‐evaluation of calcium carbonate (E 170) as a food additive in foods for infants below 16 weeks of age and follow‐up of its re‐evaluation as food additive for uses in foods for all population groups %A Younes, Maged %A Gabriele Aquilina %A Laurence Castle %A Gisela Degen %A Engel, Karl‐Heinz %A Paul J Fowler %A Maria Jose Frutos Fernandez %A Peter Fürst %A Rainer Gürtler %A Trine Husøy %A Melania Manco %A Wim Mennes %A Peter Moldeus %A Sabina Passamonti %A Romina Shah %A Ine Waalkens‐Berendsen %A Wright, Matthew %A Detlef Wölfle %A Birgit Dusemund %A Mortensen, Alicja %A Dominique Turck %A Karlien Cheyns %A Eric Gaffet %A Katrin Loeschner %A Jan Mast %A Manuela Mirat %A Anna Undas %A Stefania Barmaz %A Agnieszka Mech %A Ana Maria Rincon %A Camilla Smeraldi %A Alexandra Tard %A Ursula Gundert‐Remy %B EFSA Journal %V 21 %8 Jan-07-2023 %G eng %N 7 %R 10.2903/j.efsa.2023.8106 %0 Report %D 2023 %T Silver-based biocides and titanium dioxide particles in face masks for general use. Final report of the TiO2Mask and AgMask COVID-19 projects. %A Daniela Montalvo %A Charlotte Wouters %A Lisa Siciliani %A Christiane Vleminckx %A Erik Van Miert %A Nadia Waegeneers %A Joris Van Loco %A Eveline Verleysen %A Karlien Cheyns %A Jan Mast %K Face masks %K Nanoparticles %K silver-based biocides %K titanium dioxide %I Sciensano %C Brussels, Belgium %P 48 %8 02/2023 %G eng %M D/2023.14.440/36 %0 Report %D 2023 %T Titanium analyses in food before and after ban of the food additive E171 %A Régis Nkenda %A Sophie Van Den Neucker %A Heidi Demaegdt %A Jan Mast %A Karlien Cheyns %P 16 %8 jan 2023 %G eng %0 Government Document %D 2022 %T Acid digestion of nonwoven textiles for measuring their trace element content by ICP techniques %A Gabriel Mercier %A Karlien Cheyns %A Regis Nkanda %A Ronny Machiels %A Ann Dewinne %A Nadia Waegeneers %A Jan Mast %A Daniela Montalvo %K Acid digestion %K Face mask %K microwave %K Nanoparticles %K nonwoven textile %X

We present herein a digestion method based on high-temperature (260 °C) microwave heating and strong
acidic media in order to perform a complete digestion of nonwoven textiles, which can be found in face
masks. In particular, this protocol allows the digestion of PET-, PTFE-, polyamide- and elastane-containing
textiles resulting in a complete release of metallic contents in order to analyze their elemental
contents by ICP techniques.

%B Protocol exchange %8 17-jun-2022 %G eng %R https://doi.org/10.21203/rs.3.pex-1924/v1 %0 Generic %D 2022 %T Advances in the physicochemical characterization of nanoparticles in a regulatory context. %A Jan Mast %K Nanoparticles %K Physicochemical characterization %K regulation %K transmission electron microscopy %X

 

Advances in the physicochemical characterization of nanoparticles in a regulatory context

Defining, specifying, registering and labelling, as well as characterizing particulate (nano)materials for risk assessment, require measurement of the physicochemical properties of their constituent particles. Because of its resolution covering 1-100 nm, its possibility to identify constituent particles in aggregates and agglomerates and to measure key parameters such as their minimal external dimension on a number-basis, electron microscopic measurement of (nano)particles is necessary in a regulatory context.

Approaches, selected examples and new developments of transmission electron microscopic characterization are proposed from the perspective of the Belgian national reference laboratory responsible for control of particulate (nano)materials applied in the food and feed chain, and in consumer products. The importance of optimizing sample (dispersion) and specimen (EM-grids) preparation will be demonstrated, application of conventional TEM for characterizing pristine materials will be shown, and the possibilities of STEM-EDX analyses for control purposes requiring identification and measurement of specific particles in complex food matrices and mixtures will be presented. Further, approaches to validate these EM-based methods and to determine the measurement uncertainties are given. Initiatives to make EM analyses more time- and cost-efficient by automation of STEM EDX imaging and image analysis with constituent particle detection supported machine learning are proposed.

%B 12TH Global summit on regulatory science (GSRS22), 19 – 21 October 2022, Singapore %8 2022 October %G eng %0 Journal Article %J Nanomaterials %D 2022 %T Determination of the Transport Efficiency in spICP-MS Analysis Using Conventional Sample Introduction Systems: An Interlaboratory Comparison Study %A Otmar Geiss %A Ivana Bianchi %A Guillaume Bucher %A Eveline Verleysen %A Frederic Brassinne %A Jan Mast %A Katrin Loeschner %A Lucas Givelet %A Francesco Cubadda %A Francesca Ferraris %A Andrea Raggi %A Francesca Iacoponi %A Peters, Ruud J B %A Anna Undas %A Alexandra Müller %A Ann-Katrin Meinhardt %A Birgit Hetzer %A Volker Gräf %A Antonio R. Montoro Bustos %A Josefa Barrero-Moreno %B Nanomaterials %V 12 %8 Jan-02-2022 %G eng %N 4 %R 10.3390/nano12040725 %0 Report %D 2022 %T Evaluation of the types, efficient use and health risks of application of silver-based biocides to provide antimicrobial properties to face masks applied during the Covid-19 crisis %A Jan Mast %A Marie-Noelle Blaude %A Lisa Siciliani %A Karlien Cheyns %A Nadia Waegeneers %A Christiane Vleminckx %A Joris Van Loco %A Eveline Verleysen %K biocides %K Face masks %K Ions %K Nanoparticles %K Silver %X

In situ analysis of silver based biocides in face masks using electron microscopy and EDX, combined with total silver measurement using ICP-MS or ICP-OES demonstrated the presence of varying amounts and different types of silver-based biocides in a selection of face masks on the Belgian market and intended to be worn by the general public. Following types of silver-based biocides were demonstrated: (i) Ag+ ions, (ii) metallic Ag0 NP distributed in the matrix of the fibers, (iii) Ag NP and large silver particles at the surface of, or close to cotton fibres in face masks containing polycationic polymers binding Ag+ ions, and (iv) a coating consisting of metallic silver releasing Ag+ ions, Ag0 NP and large silver particles.

For metallic and ionic silver, an acceptable exposure level (AELmask) of 25 µg per mask was established based on occupational exposure levels and assuming an intensive exposure scenario considering subchronic exposure of the general adult population.

Comparison of the measured amount of total silver in the masks with this AELmask indicated that seven out of nine face masks, with a silver biocide based on Ag+ ions only, can be considered as safe. The two other face masks with a silver biocide based on Ag+ ions require a more refined risk evaluation.

The amount of silver in the four masks that contain Ag0 NP, Ag+ ions, and/or non-nanoparticulate silver exceeded the AELmask. Per case an in depth risk analysis needs to be undertaken to account for the different forms of silver that are potentially released from face masks treated with the applied silver-based biocides.

%C Brussels, Belgium %P 48 %8 14/07/2022 %G eng %M D/2021/14.440/100 %0 Government Document %D 2022 %T Identification and characterization of TiO2 nanoparticles in face masks by TEM %A Charlotte Wouters %A Lisa Siciliani %A Marina Ledecq %A Eveline Verleysen %A Jan Mast %K Agglomerates %K electron microscopy %K energy dispersive X-ray spectroscopy %K Face mask %K image analysis %K Nanoparticles %K particle size distribution %K STEMEDX %K TEM %K textile %K ultramicrotomy %X

Because of the possible health risks related to the use of nanoparticle technologies in face masks, a general methodology is developed for identification, localization and particle size measurement of nanoparticles in situ in face masks by conventional and analytical transmission electron microscopy (TEM). The methodology can be applied by following this protocol which includes (i) preparation of resin-embedded ultra-thin sections of face masks suitable for TEM analysis, a procedure that can be applied to any type of textile, (ii) visualization and identification of the (nano)particles inside the cross section of the textile fibres by scanning TEM (STEM) combined with energy dispersive X-ray spectroscopy (EDX), (iii) measurement of the number-based particle size distribution based on a (semi-)automatic quantitative analysis using ImageJ, and (iv) in view of risk assessment, a calculation to estimate the amount of particles available for release from the mask.

%B Protocol Exchange %I Creative Commons %C PO Box 1866, Mountain View, CA 94042 %P 17 %8 19/05/2022 %G eng %R https://doi.org/10.21203/rs.3.pex-1902/v1 %0 Journal Article %J Scientific Reports %D 2022 %T Titanium dioxide particles frequently present in face masks intended for general use require regulatory control %A Eveline Verleysen %A Ledecq, Marina %A Lisa Siciliani %A Karlien Cheyns %A Christiane Vleminckx %A Marie-Noelle Blaude %A Sandra De Vos %A Frederic Brassinne %A Frederic Van Steen %A Régis Nkenda %A Ronny Machiels %A Nadia Waegeneers %A Joris Van Loco %A Jan Mast %B Scientific Reports %V 12 %8 Jan-12-2022 %G eng %N 1 %R 10.1038/s41598-022-06605-w %0 Journal Article %J Food Control %D 2022 %T Towards a generic protocol for measuring the constituent particle size distribution of E171 in food by electron microscopy %A Eveline Verleysen %A Frederic Brassinne %A Frederic Van Steen %A Nadia Waegeneers %A Karlien Cheyns %A Ronny Machiels %A Stella Mathioudaki %A Isaac Ojea Jimenez %A Ledecq, Marina %A Jan Mast %B Food Control %V 132 %8 Jan-02-2022 %G eng %R 10.1016/j.foodcont.2021.108492 %0 Generic %D 2021 %T The Belgian Nanoregister in Figures - Trade Year 2017 %A Stella Mathioudaki %A Eveline Verleysen %A Jan Mast %K nanomaterials %K nanoregister %K Trade year 2017 %8 2021 %G eng %0 Generic %D 2021 %T The Belgian Nanoregister in Figures - Trade Year 2018 %A Stella Mathioudaki %A Eveline Verleysen %A Jan Mast %K nanomaterials %K nanoregister %K Trade year 2018 %8 2021 %G eng %0 Journal Article %J Vaccines (Basel) %D 2021 %T The Expression of Hemagglutinin by a Recombinant Newcastle Disease Virus Causes Structural Changes and Alters Innate Immune Sensing. %A Ingrao, Fiona %A Victoria Duchatel %A Rodil, Isabel Fernandez %A Mieke Steensels %A Eveline Verleysen %A Jan Mast %A Bénédicte Lambrecht %X

Recombinant Newcastle disease viruses (rNDV) have been used as bivalent vectors for vaccination against multiple economically important avian pathogens. NDV-vectored vaccines expressing the immunogenic H5 hemagglutinin (rNDV-H5) are considered attractive candidates to protect poultry from both highly pathogenic avian influenza (HPAI) and Newcastle disease (ND). However, the impact of the insertion of a recombinant protein, such as H5, on the biological characteristics of the parental NDV strain has been little investigated to date. The present study compared a rNDV-H5 vaccine and its parental NDV LaSota strain in terms of their structural and functional characteristics, as well as their recognition by the innate immune sensors. Structural analysis of the rNDV-H5 demonstrated a decreased number of fusion (F) and a higher number of hemagglutinin-neuraminidase (HN) glycoproteins compared to NDV LaSota. These structural differences were accompanied by increased hemagglutinating and neuraminidase activities of rNDV-H5. During in vitro rNDV-H5 infection, increased mRNA expression of TLR3, TLR7, MDA5, and LGP2 was observed, suggesting that the recombinant virus is recognized differently by sensors of innate immunity when compared with the parental NDV LaSota. Given the growing interest in using NDV as a vector against human and animal diseases, these data highlight the importance of thoroughly understanding the recombinant vaccines' structural organization, functional characteristics, and elicited immune responses.

%B Vaccines (Basel) %V 9 %8 2021 Jul 07 %G eng %N 7 %R 10.3390/vaccines9070758 %0 Journal Article %J Vaccines %D 2021 %T The Expression of Hemagglutinin by a Recombinant Newcastle Disease Virus Causes Structural Changes and Alters Innate Immune Sensing %A Ingrao, Fiona %A Victoria Duchatel %A Rodil, Isabel Fernandez %A Mieke Steensels %A Eveline Verleysen %A Jan Mast %A Bénédicte Lambrecht %B Vaccines %V 9 %8 Jan-07-2021 %G eng %N 7 %R 10.3390/vaccines9070758 %0 Journal Article %J EFSA Journal %D 2021 %T Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health %A Simon More %A Vasileios Bampidis %A Diane Benford %A Claude Bragard %A Halldorsson, Thorhallur %A Antonio Hernández‐Jerez %A Susanne Hougaard Bennekou %A Kostas Koutsoumanis %A Claude Lambré %A Machera, Kyriaki %A Hanspeter Naegeli %A Søren Nielsen %A Josef Schlatter %A Dieter Schrenk %A Vittorio Silano (deceased) %A Dominique Turck %A Younes Maged %A Jacqueline Castenmiller %A Qasim Chaudhry %A Francesco Cubadda %A Roland Franz %A David Gott %A Jan Mast %A Mortensen, Alicja %A Agnes G. Oomen %A Stefan Weigel %A Eric Barthelemy %A Ana Rincon %A Jose Tarazona %A Reinhilde Schoonjans %B EFSA Journal %V 19 %8 Jan-08-2021 %G eng %N 8 %R 10.2903/j.efsa.2021.6768 %0 Journal Article %J EFSA Journal %D 2021 %T Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles %A Simon More %A Vasileios Bampidis %A Diane Benford %A Claude Bragard %A Halldorsson, Thorhallur %A Antonio Hernández‐Jerez %A Bennekou, Susanne Hougaard %A Kostas Koutsoumanis %A Claude Lambré %A Machera, Kyriaki %A Hanspeter Naegeli %A Søren Nielsen %A Josef Schlatter %A Dieter Schrenk %A Vittorio Silano (deceased) %A Dominique Turck %A Younes Maged %A Jacqueline Castenmiller %A Qasim Chaudhry %A Francesco Cubadda %A Roland Franz %A David Gott %A Jan Mast %A Mortensen, Alicja %A Agnes G. Oomen %A Stefan Weigel %A Eric Barthelemy %A Ana Rincon %A Jose Tarazona %A Reinhilde Schoonjans %B EFSA Journal %V 19 %8 Jan-08-2021 %G eng %N 8 %R 10.2903/j.efsa.2021.6769 %0 Report %D 2021 %T IDENTIFICATION, PHYSICOCHEMICAL CHARACTERISATION AND PRELIMINARY RISK ANALYSIS OF TITANIUM DIOXIDE PARTICLES IN FACE MASKS Intermediate report TiO2-Mask COVID-19 project September 2021 %A Jan Mast %A Marie-Noelle Blaude %A Lisa Siciliani %A Karlien Cheyns %A Nadia Waegeneers %A Joris Van Loco %A Christiane Vleminckx %A Eveline Verleysen %X

In situ analysis of titanium dioxide (TiO2) particles in face masks demonstrated the presence of agglomerated TiO2 (nano)particles in all examined face masks that contain polyester or polyamide (nylon) fibres, or that are made of non-woven, synthetic fabrics. These particles resemble fibre-grade TiO2 particles. Because there are no methods available for measuring exposure directly, the methodology that ANSES applied to determine the professional exposure limits to titanium dioxide in its nanoform, was applied for a scenario with intensive use of face masks. Our calculations show that a health risk cannot be excluded for most of the examined face masks when intensively used. The applied approach may overestimate the health risks because of the conservative inhalation exposure assumptions. However, for some face masks the amount of titanium dioxide is so high that a health risk cannot be excluded even when only a small fraction of the titanium dioxide particles are released and inhaled. Currently, we have no indications that TiO2 particles are released in amounts which might result in public health risks, but so far, research and publications of TiO2 particles in textiles, and particularly of their release, are limited. In view of EFSA's conclusion that TiO2 cannot be considered any longer as safe to be used as a food additive because a concern for genotoxicity cannot be ruled out, it is advisable to issue precautionary standards to limit the presence of TiO2 particles in face masks.

%I Sciensano %C Elsene, Belgium %P 50 %8 sept 2021 %G eng %M D/2021/14.440/72 %R 10.25608/ba73-8j24 %0 Journal Article %J Environmental Science: Nano %D 2021 %T Identifying nanodescriptors to predict the toxicity of nanomaterials: a case study on titanium dioxide %A Sivakumar Murugadoss %A Das, Nilakash %A Godderis, Lode %A Jan Mast %A Hoet, Peter H. %A Manosij Ghosh %X

Since the evaluation of nanomaterial (NM) hazards by animal testing is expensive, time-consuming and critical from an ethical point of view, much interest is being given to the development of alternative testing strategies such as computational (predictive) models based on in vitro testing. However, the variations in in vitro experimental conditions can influence the outcome of computational modelling. In this study, we aim to identify nanodescriptor(s) and biological endpoint(s) capable of predicting the toxicity of titanium-di-oxide (TiO2) NMs, and demonstrate how experimental variations determine the outcome of modelling using three case studies. We used TiO2in vitro data from our previously published study as case study 1 and two other external case studies (case study 2 and 3) performed under different exposure conditions (presence and/or absence of serum). Firstly, we identified the nanodescriptor(s) closely associated to biological endpoints. Secondly, we determined the strength of association of the identified nanodescriptor(s) with the respective biological endpoint. The results indicate that the experimental conditions influence the outcome of the computational modelling. Agglomerate size as a nanodescriptor was well associated with biological endpoints such as DNA damage and/or cytotoxicity. We conclude that, agglomerate size is an important nanodescriptor to assess the toxicological effects of TiO2 NMs in vitro. However, the agglomeration state of NMs can be potentially influenced by in vitro exposure conditions and such influences could be just a confounder in broader contexts such as safety-by-design approaches, which require linking of material specific properties to the toxicological outcome.

%B Environmental Science: Nano %V 8 %8 Jan-02-2021 %G eng %N 2 %R 10.1039/D0EN01031F %0 Journal Article %J Measurement: Sensors %D 2021 %T METROFOOD-RI: Pilot services with physical, remote and virtual access %A Karine Vandermeiren %A Subhalakshmi Sharma %A Nastasia Belc %A Jan Mast %A Agnes Matuszczak %A karl Presser %A Eveline Verleysen %A Claudia Zoani %A Joris Van Loco %K ESFRI %K Food safety and quality %K Metrology %K research infrastructure %X

METROFOOD-RI (www.metrofood.eu) is an ESFRI research infrastructure, funded upon the EU H2020 METROFOOD-PP project for its Preparatory Phase, aiming to establish a new distributed European Research Infrastructure (RI) to promote scientific excellence and increase efficiency in food quality and food safety. It strives to provide and coordinate high-level metrological services on a European scale for researchers, laboratories, food inspection agencies and policymakers.

As part of the preparatory phase towards the legal statute of ERIC (European Research Infrastructure Consortium), a service portfolio is being set up along with its provision diagram. As a test for this model of service provision, three use cases have been defined that are representative of the different types of access that will be provided by METROFOOD-RI:

  1. physical access to a food pilot plant for demonstrating technical solutions and adaptations of food processing technology to minimize acrylamide in bakery products at the National Research & Development Institute for Food Bio-resources (IBA, Romania).
  2. remote access to the transmission electron microscope facility for physicochemical characterization of nanoparticles in food (Sciensano, Belgium).
  3. virtual access to two e-services for open data use, mainly addressed to researchers, laboratories, food inspection agencies and policy makers:

The use cases will help to evaluate the usability of the single access point of the research infrastructure and to fine-tune the access procedures and interfaces with users. This will support the elaboration of the final service chart of METROFOOD-RI including all the potential physical, electronical and integrated services that the infrastructure aims to provide to its users.

The paper will give an overview and first evaluation of the use case service provision and provide an overview of the potential METROFOOD-RI service portfolio.

%B Measurement: Sensors %V 18 %8 Jan-12-2021 %G eng %R 10.1016/j.measen.2021.100309 %0 Journal Article %J Food Control %D 2021 %T Particle size analysis of pristine food-grade titanium dioxide and E 171 in confectionery products: Interlaboratory testing of a SP-ICP-MS screening method and confirmation with transmission electron microscopy. %A Otmar Geiss %A Ivana Bianchi %A Chiara Senaldi %A Guillaume Bucher %A Eveline Verleysen %A Nadia Waegeneers %A Frederic Brassinne %A Jan Mast %A Katrin Loeschner %A Janja Vidmar %A Federica Aureli %A Francesco Cubadda %A Andrea Raggi %A Francesca Iacoponi %A Ruud Peters %A Anna Undas %A Alexandra Müller %A Ann-Katrin Meinhardt %A Elke Walz %A Volker Gräf %A Josefa Barrero-Moreno %K confectionery %K E 171 %K Food-grade titanium dioxide %K Single-particle ICP-MS %K VALIDATION %X

Titanium dioxide is a white colourant authorised as food additive E 171 in the EU, where it is used in a range of alimentary products. As these materials may contain a fraction of particulates with sizes below 100 nm and current EU regulation requires specific labelling of food ingredient to indicate the presence of engineered nanomaterials there is now a need for standardised and validated methods to appropriately size and quantify (nano)particles in food matrices. A single-particle inductively coupled plasma mass spectrometry (spICP-MS) screening method for the determination of the size distribution and concentration of titanium dioxide particles in sugar-coated confectionery and pristine food-grade titanium dioxide was developed. Special emphasis was placed on the sample preparation procedure, crucial to reproducibly disperse the particles before analysis. The transferability of this method was tested in an interlaboratory comparison study among seven experienced European food control and food research laboratories equipped with various ICP-MS instruments and using different software packages. The assessed measurands included the particle mean diameter, the most frequent diameter, the percentage of particles (in number) with a diameter below 100 nm, the particles' number concentration and a number of cumulative particle size distribution parameters (D0, D10, D50, D99.5, D99.8 and D100). The evaluated method's performance characteristics were, the within-laboratory precision, expressed as the relative repeatability standard deviation (RSDr), and the between-laboratory precision, expressed as the relative reproducibility standard deviation (RSDR). Transmission electron microscopy (TEM) was used as a confirmatory technique and served as the basis for bias estimation. The optimisation of the sample preparation step showed that when this protocol was applied to the relatively simple sample food matrices used in this study, bath sonication turned out to be sufficient to reach the highest, achievable degree of dispersed constituent particles. For the pristine material, probe sonication was required. Repeatability and reproducibility were below 10% and 25% respectively for most measurands except for the lower (D0) and the upper (D100) bound of the particle size distribution and the particle number concentration. The broader distribution of the lower and the upper bounds could be attributed to instrument-specific settings/setups (e.g. the timing parameters, the transport efficiency, type of mass-spectrometer) and software-specific data treatment algorithms. Differences in the upper bound were identified as being due to the non-harmonised application of the upper counting limit. Reporting D99.5 or D99.8 instead of the effectively largest particle diameter (D100) excluded isolated large particles and considerably improved the reproducibility. The particle number-concentration was found to be influenced by small differences in the sample preparation procedure. The comparison of these results with those obtained using electron microscopy showed that the mean and median particle diameter was, in all cases, higher when using spICP-MS. The main reason for this was the higher size detection limit for spICP-MS plus the fact that some of the analysed particles remained agglomerated/aggregated after sonication. Single particle ICP-MS is a powerful screening technique, which in many cases provides sufficient evidence to confirm the need to label a food product as containing (engineered) titanium dioxide nanomaterial according to the current EU regulatory requirements. The overall positive outcome of the method performance evaluation and the current lack of alternative standardised procedures, would indicate this method as being a promising candidate for a full validation study.

%B Food Control %V 120 %8 2021 Feb %G eng %R 10.1016/j.foodcont.2020.107550 %0 Journal Article %J EFSA Supporting Publications %D 2021 %T Physicochemical characterization of nanoparticles in food additives in the context of risk identification %A Eveline Verleysen %A Nadia Waegeneers %A Sandra De Vos %A Frederic Brassinne %A Ledecq, Marina %A Frederic Van Steen %A Mirjana Andjelkovic %A Raphael Janssens %A Stella Mathioudaki %A Lotte Delfosse %A Ronny Machiels %A Karlien Cheyns %A Jan Mast %B EFSA Supporting Publications %V 18 %8 Jan-06-2021 %G eng %N 6 %R 10.2903/sp.efsa.2021.EN-6678 %0 Journal Article %J EFSA J %D 2021 %T Safety assessment of titanium dioxide (E171) as a food additive. %A Younes Maged %A Gabriele Aquilina %A Laurence Castle %A Karl-Heinz Engel %A Paul Fowler %A Maria Jose Frutos Fernandez %A Peter Fürst %A Ursula Gundert-Remy %A Rainer Gürtler %A Trine Husøy %A Melania Manco %A Wim Mennes %A Peter Moldeus %A Sabina Passamonti %A Romina Shah %A Ine Waalkens-Berendsen %A Detlef Wölfle %A Emanuela Corsini %A Francesco Cubadda %A Didima De Groot %A Rex FitzGerald %A Sara Gunnare %A Arno Christian Gutleb %A Jan Mast %A Mortensen, Alicja %A Agnes Oomen %A Aldert Piersma %A Veronika Plichta %A Beate Ulbrich %A Henk van Loveren %A Diane Benford %A Margherita Bignami %A Claudia Bolognesi %A Riccardo Crebelli %A Maria Dusinska %A Francesca Marcon %A Elsa Nielsen %A Josef Schlatter %A Christiane Vleminckx %A Stefania Barmaz %A Maria Carfí %A Consuelo Civitella %A Alessandra Giarola %A Ana Maria Rincon %A Rositsa Serafimova %A Camilla Smeraldi %A Jose Tarazona %A Alexandra Tard %A Wright, Matthew %K CAS No 13463-67-7 %K E 171 %K titanium dioxide %X

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO nanoparticles (NPs) and data from an extended one-generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico-chemical properties of TiO particles and the outcome of either or genotoxicity assays. A concern for genotoxicity of TiO particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut-off value for TiO particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.

%B EFSA J %V 19 %8 2021 May %G eng %N 5 %R 10.2903/j.efsa.2021.6585 %0 Journal Article %J EFSA Journal %D 2021 %T Safety assessment of titanium dioxide (E171) as a food additive %A Younes Maged %A Gabriele Aquilina %A Laurence Castle %A Karl‐Heinz Engel %A Paul Fowler %A Maria Jose Frutos Fernandez %A Peter Fürst %A Ursula Gundert‐Remy %A Rainer Gürtler %A Trine Husøy %A Melania Manco %A Wim Mennes %A Peter Moldeus %A Sabina Passamonti %A Romina Shah %A Ine Waalkens‐Berendsen %A Detlef Wölfle %A Emanuela Corsini %A Francesco Cubadda %A Didima De Groot %A Rex FitzGerald %A Sara Gunnare %A Arno Christian Gutleb %A Jan Mast %A Mortensen, Alicja %A Agnes Oomen %A Aldert Piersma %A Veronika Plichta %A Beate Ulbrich %A Henk van Loveren %A Diane Benford %A Margherita Bignami %A Claudia Bolognesi %A Riccardo Crebelli %A Maria Dusinska %A Francesca Marcon %A Elsa Nielsen %A Josef Schlatter %A Christiane Vleminckx %A Stefania Barmaz %A Maria Carfí %A Consuelo Civitella %A Alessandra Giarola %A Ana Maria Rincon %A Rositsa Serafimova %A Camilla Smeraldi %A Jose Tarazona %A Alexandra Tard %A Wright, Matthew %K CAS No 13463-67-7 %K E 171 %K titanium dioxide %X

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO2 nanoparticles (NPs) and data from an extended one-generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO2 NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO2 particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO2 NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO2 NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO2 particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico-chemical properties of TiO2 particles and the outcome of either in vitro or in vivo genotoxicity assays. A concern for genotoxicity of TiO2 particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO2 particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut-off value for TiO2 particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO2 NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.

%B EFSA Journal %V 19 %8 Jan-05-2021 %G eng %N 5 %R 10.2903/j.efsa.2021.6585 %0 Journal Article %J Particle and Fibre Toxicology %D 2020 %T Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo %A Sivakumar Murugadoss %A Noham Sebaihi %A Frederic Brassinne %A Jasmine Petry %A Stevan M. Cokic %A Kirsten L. Van Landuy %A Lode Godderis %A Jan Mast %A Dominique Lison %A Peter H. Hoet %A Sybille van den Brule %K Agglomerates %K Biological responses %K nanomaterials %K titanium dioxide %K toxicity %X

Background: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. Results: We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1β increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. Conclusion: Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

%B Particle and Fibre Toxicology %8 2020 %G eng %0 Journal Article %J Particle and Fibre Toxicology %D 2020 %T Is aggregated synthetic amorphous silica toxicologically relevant? %A Sivakumar Murugadoss %A van den Brûle, Sybille %A Frederic Brassinne %A Sebaihi, Noham %A Jorge Mejia %A Stéphane Lucas %A Jasmine Petry %A Godderis, Lode %A Jan Mast %A Lison, Dominique %A Hoet, Peter H. %K Aggregates %K Biological activity %K In vitro toxicity %K nanomaterials %K synthetic amorphous silica %X

Background:

The regulatory definition(s) of nanomaterials (NMs) frequently uses the term ‘agglomerates and aggregates’ (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective.This knowledge gap greatly afects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nanosized particles (1–100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes.


Results:

We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR.

Conclusion:

Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

%B Particle and Fibre Toxicology %V 17 %8 Jan-12-2020 %G eng %N 1 %R 10.1186/s12989-019-0331-3 %0 Book %B Characterization of nanomaterials by transmission electron microscopy: Measurement procedures %D 2020 %T Characterization of Nanoparticles: Measurement Processes for Nanoparticles : Characterization of nanomaterials by transmission electron microscopy: Measurement procedures %A Jan Mast %A Eveline Verleysen %A Vasile-Dan Hodoroaba %A Ralf Kaegi %K Characterization %K nanomaterials %K transmission electron microscopy %X

Description

Characterization of Nanoparticles: Measurement Processes for Nanoparticles surveys this fast growing field, including established methods for the physical and chemical characterization of nanoparticles. The book focuses on sample preparation issues (including potential pitfalls), with measurement procedures described in detail. In addition, the book explores data reduction, including the quantitative evaluation of the final result and its uncertainty of measurement. The results of published inter-laboratory comparisons are referred to, along with the availability of reference materials necessary for instrument calibration and method validation. The application of these methods are illustrated with practical examples on what is routine and what remains a challenge.

In addition, this book summarizes promising methods still under development and analyzes the need for complementary methods to enhance the quality of nanoparticle characterization with solutions already in operation.

Key Features

Helps readers decide which nanocharacterization method is best for each measurement problem, including limitations, advantages and disadvantages

Shows which nanocharacterization methods are best for different classes of nanomaterial

Demonstrates the practical use of a method based on selected case studies

Readership

Materials scientists, chemists and chemical engineers who are looking to learn more about nanocharacterization for materials selection at the nanoparticle level

Paperback ISBN: 9780128141823

eBook ISBN: 9780128141830

%B Characterization of nanomaterials by transmission electron microscopy: Measurement procedures %I Elsevier %8 2020 %@ 978-0-12-814182-3 %G eng %) Vasile-Dan Hodoroaba Wolfgang Unger Alexander Shard %0 Journal Article %J Retrovirology %D 2020 %T Correction to: HIV-1 IN/Pol recruits LEDGF/p75 into viral particles. %A Desimmie, Belete Ayele %A Weydert, Caroline %A Schrijvers, Rik %A Vets, Sofie %A Demeulemeester, Jonas %A Proost, Paul %A Paron, Igor %A De Rijck, Jan %A Jan Mast %A Bannert, Norbert %A Gijsbers, Rik %A Christ, Frauke %A Debyser, Zeger %X

An amendment to this paper has been published and can be accessed via the original article.

%B Retrovirology %V 17 %8 2020 Jul 29 %G eng %N 1 %R 10.1186/s12977-020-00531-3 %0 Report %D 2020 %T EFSA guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles %A Simon More %A Vasileos Bampidis %A Diane Benford %A Claude Bragard %A Thorhallur Halldorsson %A Antonio Hernández-Jerez %A Susanne Hougaard Bennekou %A Kostas Koutsoumanis %A Kyriaki Machera %A Hanspeter Naegeli %A Søren Nielsen %A Josef Schlatter %A Dieter Schrenk %A Vittorio Silano %A Dominique Turck %A Younes Maged %A Jacqueline Castenmiller %A Qasim Chaudhry %A Francesco Cubadda %A Roland Franz %A David Gott %A Jan Mast %A Alicja Mortensen %A Agnes G. Oomen %A Stefan Weigel %A Jose Tarazona %A Reinhilde Schoonjans %K dissolution rate %K electron microscopy. %K feed additives %K Food Additives %K food flavourings %K Food Supplements %K Minerals %K Nanoparticles %K nanoscale %K novel foods %K particle size distribution %K particles %K sample dispersion protocol %K Solubility %K Vitamins %X

Following a mandate from the European Commission, the European Food Safety Authority has developed a Guidance on Technical Requirements (Guidance on Particle-TR), setting out information requirements for applications in the regulated food and feed product areas, and establishing criteria for assessing the presence of a fraction of small particles. These requirements apply to particles requiring specific assessment at the nanoscale in conventional materials that do not meet the definition of engineered nanomaterial as set out in the Novel Food Regulation (EU) 2015/2283. The guidance outlines appraisal criteria grouped in three sections, to confirm whether or not the conventional risk assessment should be complemented with nanospecific considerations.. The first group addresses solubility and dissolution rate as key physicochemical properties to assess whether consumers will be exposed to particles. The second group establishes the information requirements for assessing whether the conventional material contains a fraction or consists of small particles, and its characterisation. The third group describes the information to be presented for existing safety studies to demonstrate that the fraction of small particles, including particles at the nanoscale, has been properly evaluated. In addition, in order to guide the appraisal of existing safety studies, recommendations for fulfilling the data gaps while minimising the need for conducting new animal studies are provided. This Guidance on Particle-TR complements the Guidance on Nanoscience and Nanotechnology adopted by the EFSA Scientific Committee in 2018. Applicants are advised to consult both guidance documents before conducting new studies.

%8 2020 %G eng %0 Journal Article %J Environmental Toxicology and Pharmacology %D 2020 %T Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice %A Barfod, Kenneth Klingenberg %A Katja Maria Bendtsen %A Trine Berthing %A Antti Joonas Koivisto %A Sarah S Poulsen %A Ester Segal %A Eveline Verleysen %A Jan Mast %A Andreas Holländer %A Jensen, Keld Alstrup %A Hougaard, Karin S %A Vogel, Ulla %K Acute phase response %K Airway exposure %K Comet Assay %K Halloysite nanotubes %K High aspect ratio nanomaterial (HARN) %X

The toxicological potential of halloysite nanotubes (HNTs) and variants after functional alterations to surface area are not clear. We assessed the toxicological response to HNTs (NaturalNano (NN)) before and after surface etching (NN-etched). Potential cytotoxicity of the two HNTs was screened in vitro in MutaTMMouse lung epithelial cells. Lung inflammation, acute phase response and genotoxicity were assessed 1, 3, and 28 days after a single intratracheal instillation of adult female C57BL/6 J BomTac mice. The doses were 6, 18 or 54 μg of HNTs, compared to vehicle controls and the Carbon black NP (Printex 90) of 162 μg/mouse. The cellular composition of bronchoalveolar lavage (BAL) fluid was determined as a measure of lung inflammation. The pulmonary and hepatic acute phase responses were assessed by Serumamyloida mRNA levels in lung and liver tissue by real-time quantitative PCR. Pulmonary and systemic genotoxicity were analyzed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The etched HNT (NN-etched) had 4–5 times larger BET surface area than the unmodified HNT (NN). Instillation of NN-etched at the highest dose induced influx of neutrophils into the lungs at all time points and increased Saa3 mRNA levels in lung tissue on day 1 and 3 after exposure. No genotoxicity was observed at any time point. In conclusion, functionalization by etching increased BET surface area of the studied NN and enhanced pulmonary inflammatory toxicity in mice.

%B Environmental Toxicology and Pharmacology %V 73 %8 Jan-01-2020 %G eng %R 10.1016/j.etap.2019.103266 %0 Report %D 2020 %T NanoRegister Evaluation: "Compliance control and substantive evaluation of the registration of substances produced in nanoparticulate state according to Royal Decree of 27 May 2014” - Trade Year 2017 %A Stella Mathioudaki %A Eveline Verleysen %A Jan Mast %K compliance %K nanomaterials %K Nanoregister database %K Royal Degree of 27 May 2014 %I Sciensano/ SPF DG5 %8 2020 %G eng %0 Report %D 2020 %T NanoRegister Evaluation: "Compliance control and substantive evaluation of the registration of substances produced in nanoparticulate state according to Royal Decree of 27 May 2014” - Trade Year 2018 %A Stella Mathioudaki %A Eveline Verleysen %A Jan Mast %K EVALUATION %K nanomaterials %K nanoregister %K Royal Decree of 27th May 2014 %I Sciensano %C Brussels, Belgium %P 49 %8 05/2020 %G eng %M %0 Journal Article %J Food Addit Contam Part A Chem Anal Control Expo Risk Assess %D 2020 %T Physico-chemical characterisation of the fraction of silver (nano)particles in pristine food additive E174 and in E174-containing confectionery. %A Sandra De Vos %A Nadia Waegeneers %A Eveline Verleysen %A Karen Smeets %A Jan Mast %K E174 %K particle size distribution %K Silver %K Single particle ICP-MS %K TEM %X

Silver (E174) is authorised as a food additive in the EU. The unknown particle size distribution of E174 is a specific concern for the E174 risk assessment. This study characterised the fraction of silver (nano)particles in 10 commercially available pristine E174 food additives and 10 E174-containing products by transmission electron microscopy (TEM) and single-particle inductively coupled plasma-mass spectrometry (spICP-MS). TEM analysis showed that all samples contained micrometre-sized flakes and also a fraction of (nano)particles. Energy-dispersive X-ray spectroscopy (EDX) and electron diffraction confirmed that the (nano)particles and micrometre-sized flakes consisted of silver. A higher amount of (nano)particles was observed in the products than in the food additives. In addition, the surface of the micrometre-sized flakes was rougher in products. The median of the minimum external dimension, assessed as minimal Feret diameter, of the fraction of (nano)particles determined by quantitative TEM analysis was 11 ± 4 nm and 18 ± 7 nm (overall mean ± standard deviation), for food additives and products, respectively. Similar size distributions were obtained by spICP-MS and TEM, considering the limit of detection of spICP-MS. The median of the equivalent spherical diameter of the fraction of (nano)particles determined by spICP-MS was 19 ± 4 nm and 21 ± 2 nm (overall mean ± standard deviation), for food additives and products, respectively. In all samples, independent of the choice of technique, the nano-sized particles represented more than 97% (by number) of the silver particles, even though the largest mass of silver was present as flakes.

%B Food Addit Contam Part A Chem Anal Control Expo Risk Assess %8 2020 Sep 18 %G eng %R 10.1080/19440049.2020.1809719 %0 Journal Article %J Food Additives & Contaminants: Part A %D 2020 %T Physico-chemical characterisation of the fraction of silver (nano)particles in pristine food additive E174 and in E174-containing confectionery %A Sandra De Vos %A Nadia Waegeneers %A Eveline Verleysen %A Karen Smeets %A Jan Mast %K E174 %K food additive %K silverparticle size distribution %K spICP-MS %K TEM %X

Silver (E174) is authorised as a food additive in the EU. The unknown particle size distribution of E174 is a specific concern for the E174 risk assessment. This study characterised the fraction of silver (nano)particles in 10 commercially available pristine E174 food additives and 10 E174-containing products by transmission electron microscopy (TEM) and single-particle inductively coupled plasma-mass spectrometry (spICP-MS). TEM analysis showed that all samples contained micrometre-sized flakes and also a fraction of (nano)particles. Energy-dispersive X-ray spectroscopy (EDX) and electron diffraction confirmed that the (nano)particles and micrometre-sized flakes consisted of silver. A higher amount of (nano)particles was observed in the products than in the food additives. In addition, the surface of the micrometre-sized flakes was rougher in products. The median of the minimum external dimension, assessed as minimal Feret diameter, of the fraction of (nano)particles determined by quantitative TEM analysis was 11 ± 4 nm and 18 ± 7 nm (overall mean ± standard deviation), for food additives and products, respectively. Similar size distributions were obtained by spICP-MS and TEM, considering the limit of detection of spICP-MS. The median of the equivalent spherical diameter of the fraction of (nano)particles determined by spICP-MS was 19 ± 4 nm and 21 ± 2 nm (overall mean ± standard deviation), for food additives and products, respectively. In all samples, independent of the choice of technique, the nano-sized particles represented more than 97% (by number) of the silver particles, even though the largest mass of silver was present as flakes.

%B Food Additives & Contaminants: Part A %V 37 %8 sep 2020 %G eng %N 11 %R 10.1080/19440049.2020.1809719 %0 Journal Article %J Nanomaterials (Basel) %D 2020 %T Physicochemical Characterization of the Pristine E171 Food Additive by Standardized and Validated Methods. %A Eveline Verleysen %A Nadia Waegeneers %A Frederic Brassinne %A Sandra De Vos %A Isaac Ojea Jimenez %A Stella Mathioudaki %A Jan Mast %K E171 %K food additive %K Single particle ICP-MS %K transmission electron microscopy %X

E171 (titanium dioxide) is a food additive that has been authorized for use as a food colorant in the European Union. The application of E171 in food has become an issue of debate, since there are indications that it may alter the intestinal barrier. This work applied standardized and validated methodologies to characterize representative samples of 15 pristine E171 materials based on transmission electron microscopy (TEM) and single-particle inductively coupled plasma mass spectrometry (spICP-MS). The evaluation of selected sample preparation protocols allowed identifying and optimizing the critical factors that determine the measurement of the particle size distribution by TEM. By combining optimized sample preparation with method validation, a significant variation in the particle size and shape distributions, the crystallographic structure (rutile versus anatase), and the physicochemical form (pearlescent pigments versus anatase and rutile E171) was demonstrated among the representative samples. These results are important for risk assessment of the E171 food additive and can contribute to the implementation of the European Food Safety Authority (EFSA) guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain.

%B Nanomaterials (Basel) %V 10 %8 2020 Mar 24 %G eng %N 3 %R 10.3390/nano10030592 %0 Report %D 2019 %T BR/154/A4 To²DeNano Towards a toxicologically relevant definition of nanomaterials final report %A Peter Hoet %A Godderis, Lode %A Sivakumar Murugadoss %A Jan Mast %A Frederic Brassinne %A Lison, Dominique %A van den Brûle, Sybille %A Jasmine Petry %A Sebaihi, Noham %K aggregation/agglomeration %K EU definition %K exposure %K hazard %K nanomaterials %8 2019 %G eng %0 Generic %D 2019 %T Characterization of the nano-sized fraction of silver particles in food additive E174 by EM and sp-ICP-MS %A Sandra De Vos %A Eveline Verleysen %A M. Ledecq %A Nadia Waegeneers %A Jan Mast %B IMEKOFOODS 4 %8 sep 2019 %G eng %0 Generic %D 2019 %T Combining TEM with single particle ICP-MS: assessing the size of individual nanoparticles in food additives by multiple techniques %A Nadia Waegeneers %A Delfosse,L. %A Sandra De Vos %A Eveline Verleysen %A Jan Mast %K Single particle ICP-MS %K transmission electron microscopy %B Workshop: Physicochemical characterisation of nano-sized particles in food %I Sciensano %C Brussels, Belgium %8 2019 %G eng %N Sciensano %0 Journal Article %J Materials (Basel) %D 2019 %T Estimation of the Uncertainties Related to the Measurement of the Size and Quantities of Individual Silver Nanoparticles in Confectionery. %A Nadia Waegeneers %A Sandra De Vos %A Eveline Verleysen %A Ann Ruttens %A Jan Mast %K Single particle ICP-MS %X

E174 (silver) is a food additive that may contain silver nanoparticles (AgNP). Validated methods are needed to size and quantify these particles in a regulatory context. However, no validations have yet been performed with food additives or real samples containing food additives requiring a sample preparation step prior to analysis. A single-particle inductively coupled plasma mass spectrometry (spICP-MS) method was developed and validated for sizing and quantifying the fraction of AgNP in E174 and in products containing E174, and associated uncertainties related to sample preparation, analysis and data interpretation were unraveled. The expanded measurement uncertainty for AgNP sizing was calculated to be 16% in E174-containing food products and increased up to 23% in E174 itself. The E174 food additives showed a large silver background concentration combined with a relatively low number of nanoparticles, making data interpretation more challenging than in the products. The standard uncertainties related to sample preparation, analysis, and challenging data interpretation were respectively 4.7%, 6.5%, and 6.0% for triplicate performances. For a single replicate sample, the uncertainty related to sample preparation increased to 6.8%. The expanded measurement uncertainty related to the concentration determination was 25-45% in these complex samples, without a clear distinction between additives and products. Overall, the validation parameters obtained for spICP-MS seem to be fit for the purpose of characterizing AgNP in E174 or E174-containing products.

%B Materials (Basel) %V 12 %8 2019 Aug 22 %G eng %N 17 %& 2677 %R 10.3390/ma12172677 %0 Journal Article %J Materials (Basel) %D 2019 %T Evaluation of a TEM based Approach for Size Measurement of Particulate (Nano)materials. %A Eveline Verleysen %A Thorsten Wagner %A Hans-Gerd Lipinski %A Ralf Kägi %A Robert Koeber %A Ana Boix-Sanfeliu %A Pieter-Jan De Temmerman %A Jan Mast %X

An approach for the size measurement of particulate (nano)materials by transmission electron microscopy was evaluated. The approach combines standard operating procedures for specimen preparation, imaging, and image analysis, and it was evaluated on a series of certified reference materials and representative test materials with varying physical properties, including particle size, shape, and agglomeration state. The measurement of the median value of the minimal external particle diameter distribution was intra-laboratory validated. The validation study included an assessment of the limit of detection, working range, selectivity, precision, trueness, robustness, and ruggedness. An uncertainty that was associated to intermediate precision in the range of 1-7% and an expanded measurement uncertainty in the range of 7-20% were obtained, depending on the material and image analysis mode. No bias was observed when assessing the trueness of the approach on the certified reference materials ERM-FD100 and ERM-FD304. The image analysis method was validated in an inter-laboratory study by 19 laboratories, which resulted in a within-laboratory precision in the range of 2-8% and a between-laboratory precision of between 2% and 14%. The automation and standardization of the proposed approach significantly improves labour and cost efficiency for the accurate and precise size measurement of the particulate materials. The approach is shown to be implementable in many other electron microscopy laboratories.

%B Materials (Basel) %V 12 %8 2019 Jul 15 %G eng %N 14 %R 10.3390/ma12142274 %0 Journal Article %J Materials %D 2019 %T Evaluation of a TEM based Approach for Size Measurement of Particulate (Nano)materials %A Eveline Verleysen %A Thorsten Wagner %A Hans-Gerd Lipinski %A Ralf Kägi %A Robert Koeber %A Ana Boix-Sanfeliu %A Pieter-Jan De Temmerman %A Jan Mast %K electron microscopy %K European Commission’s Recommendation for a definition of a nanomaterial %K image analysis %K Method validation %K nanomaterial %K particulate material %X

An approach for the size measurement of particulate (nano)materials by transmission electron microscopy was evaluated. The approach combines standard operating procedures for specimen preparation, imaging, and image analysis, and it was evaluated on a series of certified reference materials and representative test materials with varying physical properties, including particle size, shape, and agglomeration state. The measurement of the median value of the minimal external particle diameter distribution was intra-laboratory validated. The validation study included an assessment of the limit of detection, working range, selectivity, precision, trueness, robustness, and ruggedness. An uncertainty that was associated to intermediate precision in the range of 1–7% and an expanded measurement uncertainty in the range of 7–20% were obtained, depending on the material and image analysis mode. No bias was observed when assessing the trueness of the approach on the certified reference materials ERM-FD100 and ERM-FD304. The image analysis method was validated in an inter-laboratory study by 19 laboratories, which resulted in a within-laboratory precision in the range of 2–8% and a between-laboratory precision of between 2% and 14%. The automation and standardization of the proposed approach significantly improves labour and cost efficiency for the accurate and precise size measurement of the particulate materials. The approach is shown to be implementable in many other electron microscopy laboratories.

%B Materials %V 12 %8 2019 July 15 %G eng %N 14 %& 2274 %R 10.3390/ma12142274 %0 Generic %D 2019 %T Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice %A Kenneth Klingenberg Barfod %A Katja Maria Bendtsen %A Trine Berthing %A Antti Joonas Koivisto %A Sarah Søs Poulsen %A Ester Segal %A Eveline Verleysen %A Jan Mast %A Andreas Holländer %A Keld Alstrup Jensen %A Karin Sørig Hougaard %A Ulla Vogel %K halloysite %K nanotubes %K pulmonary exposure %X

presentation at the Annual meeting in Danish Society for Pharmacology and Toxicology 2019, Sønderborg, Denmark

%B Annual meeting in Danish Society for Pharmacology and Toxicology %C Sønderborg, Denmark %8 06/11/2019 %G eng %U https://dstf.dk/annual-meeting-2019/ %0 Generic %D 2019 %T Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice %A Kenneth Klingenberg Barfod %A Katja Maria Bendtsen %A Trine Berthing %A Antti Joonas Koivisto %A Sarah Søs Poulsen %A Ester Segal %A Eveline Verleysen %A Jan Mast %A Andreas Holländer %A Keld Alstrup Jensen %A Karin Sørig Hougaard %A Vogel, Ulla %B Nanosafety Cluster Week. Building confidence in risk assessment and governance of Nanomaterial Innovation %C Copenhagen, Denmark %8 07/10/2019 %G eng %0 Report %D 2019 %T Nanopack Deliverable 6.2: Title: Manuscript with physico-chemical characterisation of HNTs and HNT hazard characterization %A Karin Sørig Hougaard %A Kenneth Klingenberg Barfod %A Katja Maria Bendtsen %A Trine Berthing %A Antti Joonas Koivisto %A Sarah Søs Poulsen %A Ester Segal %A Eveline Verleysen %A Jan Mast %A Andreas Holländer %A Keld Alstrup Jensen %A Vogel, Ulla %K hallosite nanotubes %K hazard characterisation %K Nanoparticles %K physico-chemical characterization %X

report for Manuscript with physico-chemical characterisation of HNTs and HNT hazard characterization

%8 2019 %G eng %0 Generic %D 2019 %T Open Lab Application for the Characterization of Nanomaterials by Transmission Electron Microscopy %A Karine Vandermeiren %A Eveline Verleysen %A Jan Mast %A Joris Van Loco %K Food Analysis %K Metrofood-RI %K nanomaterials %K open laboratory %K physico-chemical characterization %X

OPEN LAB APPLICATION FOR THE CHARACTERIZATION OF NANOMATERIALS BY TRANSMISSION ELECTRON MICROSCOPY

Karine Vandermeiren(1), Eveline Verleysen(1), Jan Mast(1), Joris Van Loco*(1)

1) Sciensano, Belgium

*Corresponding author - E-mail: Joris.VanLoco@sciensano.be

Applications of nanotechnologies in the food sector are rapidly growing in several areas such as food processing, packaging, nutraceutical delivery, quality control, and functional foods to even the use of nanosensors to assure food quality and safety. Since it is widely expected that more and more nanotechnology based products will become available in the European Union over the coming years, the European Commission (EC) has developed a Recommendation that provides a basis to determine whether a material should be considered as a nanomaterial (NM) for legislative and policy purposes in the EU (2011/696/EU). The aim is to further apply and adapt this current Recommendation to sector specific needs such as food and food contact materials, biocidal products, cosmetics and medical devices. As a consequence there is a growing need for validated characterization methods and for certified materials facilitating the implementation of the EC Recommendation and sector-specific regulations. The physical and chemical characterization of NM in complex matrices like food is an extremely challenging task requiring expert knowledge and modern instrumentation. The application of electron microscopy (EM) for the characterization of NM is advised in several international guidelines, including guidelines of the European Food Safety Authority (EFSA) and the Scientific Committee on emerging and Newly Identified Health Risks (SCENIHR). However, EM based methodologies are cost- and labor-intensive and such dedicated infrastructures remain limited to specialized research institutions. Over the last years the National Reference Laboratory for Nanomaterials in Food of Sciensano (Belgium) has acquired high level expertise and instrumentation to measure the size, morphology, crystallographic structure and chemical composition of a wide range of NM by EM. Identification and measurement of particles can be performed in complex matrices such as food, cosmetics, medicines and environmental samples. A high degree of automation of the EM imaging and image analysis was recently developed to facilitate the measurement of particle size and shape distributions. Within the context of the METROFOOD-RI “Infrastructure for promoting Metrology in Food and Nutrition” this facility will be further developed as a test case for an open laboratory application to be shared with interested universities, research institutes or companies, ultimately allowing remote operation of the EM and monitoring via VPN-connection.

%B Recent Advances in Food Analysis (RAFA) %I University of Chemistry and Technology, Prague, Czech Republic %C Prague, Czech Republic %8 2019 %@ 978-80-7592-055-3 %G eng %U http://www.rafa2019.eu/pdf/RAFA2019_BoA_web.pdf %0 Generic %D 2019 %T Optimization and validation of quantitative TEM analysis of pristine titanium dioxide powders in a regulatory context %A Frederic Brassinne %A Sandra De Vos %A Eveline Verleysen %A M. Ledecq %A Jan Mast %B IMEKOFOODS 4 %8 sep 2019 %G eng %0 Generic %D 2019 %T Our new analytical electron microscope is way better than our conventional one %A Frederic Brassinne %A Sandra De Vos %A Eveline Verleysen %A Daisy Tysmans %A Jan Mast %B Inauguration Talos analytical electron microscope %8 23/05/2019 %G eng %N Sciensano - Trace Elements and nanomaterials %0 Generic %D 2019 %T Physicochemical characterisation of several types of the E171 food additive %A Eveline Verleysen %A M. Ledecq %A Sandra De Vos %A I. Ojea Jimenez %A Frederic Brassinne %A Nadia Waegeneers %A Jan Mast %K Characterization %K E171 %K electron microscopy %K particle size distributions. %K single particle inductively coupled plasma mass spectroscopy %K titanium oxide %X

The application of E171 (titanium dioxide) as a food additive has been an issue of debate in the European Union. A detailed physicochemical characterization of the E171 particles can objectify the discussions and is essential in the context of risk analysis.
This work focuses on the physicochemical characterization of 15 pristine E171 materials by transmission electron microscopy (TEM) and single particle inductively coupled plasma mass spectroscopy (sp-ICP-MS) following CEN/TC 352 guidelines. The E171 samples were purchased on the Belgian market or were obtained from European producers.
In optimized conditions, representative TEM micrographs could be recorded and the ParticleSizer image analysis software succeeded in applying noise reduction and background subtraction, allowing robust automatic thresholding and constituent particle detection. The large majority of constituent particles, confirmed to be TiO2 by energy dispersive X-ray spectroscopy (EDX), were reliably detected and measured by the software. The measurement uncertainty budgets of particle sizing by TEM and sp-ICP-MS are in the order of 10% and 16 % (Ucx, k=2), respectively, based on validation studies of a series of representative test materials. The phase of the E171 particles was determined by powder electron diffraction.
Several types of TiO2 particles were found in pristine E171. These types were shown to be applied as well in food products containing E171. All examined E171 food additives contained a significant amount of nanoparticles. In the most dispersed state, the particle size measurements by TEM and sp-ICP-MS agreed well. Eleven E171 materials consisted of anatase. Three materials consisted of smaller rutile TiO2 particles (20-40 nm) coated on mica. One material contained a mixture of anatase and rutile particles.
In future research, the methodology will be implemented in a systematic and larger scale study of E171 food additives and food items containing E171, available on the market.

%B IMEKOFOODS 4 %8 sep 2019 %G eng %U https://www.imekofoods4.be/ %0 Generic %D 2019 %T Physicochemical Characterisation of the E171 Food Additive %A Eveline Verleysen %A Marina Ledecq %A Sandra De Vos %A Isaac Ojea Jimenez %A Nadia Waegeneers %A Frederic Brassinne %A Jan Mast %K E171 %K particle size distribution %K Single particle ICP-MS %K titanium dioxide %K transmission electron microscopy %X

The application of E171 (titanium dioxide) as a food additive has been an issue of debate in the European Union. A detailed physicochemical characterization of the E171 particles can objectify the discussions and is essential in the context of risk analysis. This work focuses on the physicochemical characterization of 15 pristine E171 materials by transmission electron microscopy (TEM) and single particle inductively coupled plasma mass spectroscopy (sp-ICP-MS) following CEN/TC 352 guidelines. The E171 samples were purchased on the Belgian market or were obtained from European producers. To measure the minimal external dimension of the constituent particles of E171, sample preparation protocols influencing particle dispersion (pH, probe sonication and centrifugation) were tested and optimized. In optimized conditions, representative TEM micrographs could be recorded, and it was demonstrated that all examined E171 food additives contained a significant amount of nanoparticles. The large majority of constituent particles, confirmed to be TiO2 by energy dispersive X-ray spectroscopy (EDX), were reliably detected and measured using the ParticleSizer software. In the most dispersed state, the particle size measurements by TEM and sp-ICP-MS agreed well. The measurement uncertainty budgets of particle sizing by TEM and sp-ICP-MS are in the order of 10% and 16 % (Ucx, k=2), respectively, based on validation studies of a series of representative test materials. Electron diffraction demonstrated that both anatase and rutile TiO2 particles were found in pristine E171. Eleven E171 materials consisted of anatase. Three materials consisted of smaller rutile TiO2 particles (20-40 nm) coated on mica. One material contained a mixture of anatase and rutile particles. In future research, the methodology will be implemented in a systematic and larger scale study of E171 food additives and food items containing E171, available on the market.

%B Recent Advances in Food Analysis (RAFA) %I University of Chemistry and Technology, Prague, Czech Republic %C Prague, Czech Republic %8 2019 %@ 978-80-7592-055-3 %G eng %U http://www.rafa2019.eu/pdf/RAFA2019_BoA_web.pdf %0 Generic %D 2019 %T Physicochemical characterization of nanoparticles in a regulatory framework %A Jan Mast %8 2019 %G eng %0 Conference Proceedings %D 2019 %T Physicochemical characterization of nanoparticles with TEM in a regulatory context %A Jan Mast %8 2019 %0 Generic %D 2019 %T Possible solutions to analytical challenges %A Frederic Brassinne %A Eveline Verleysen %A Jan Mast %K Nanoparticles %B Workshop: Physicochemical characterisation of nano-sized particles in food %8 09/2019 %G eng %0 Report %D 2019 %T RF 16/6306 Implementation and validation of an analytical methodology to assess engineered nanomaterials in food additives Nanofood@ %A Eveline Verleysen %A Sandra De Vos %A Nadia Waegeneers %A Frederic Brassinne %A Stella Mathioudaki %A Marina Ledecq %A Lotte Delfosse %A Jan Mast %8 2019 %G eng %0 Journal Article %J EFSA Journal %D 2019 %T Scientific opinion on the proposed amendment of the EU specifications for titanium dioxide (E 171) with respect to the inclusion of additional parameters related to its particle size distribution %A Younes Maged %A Gabriele Aquilina %A Laurence Castle %A Karl‐Heinz Engel %A Paul Fowler %A Maria Jose Frutos Fernandez %A Rainer Gürtler %A Ursula Gundert‐Remy %A Trine Husøy %A Wim Mennes %A Peter Moldeus %A Agneta Oskarsson %A Sandra Rainieri %A Romina Shah %A Ine Waalkens‐Berendsen %A Detlef Wölfle %A Eric Gaffet %A Jan Mast %A Ruud Peters %A Ana Maria Rincon %A Peter Fürst %K E171 %K food additive %K Particle Size %K specifications %K titanium dioxide %X

The present opinion deals with the assessment of the data provided by interested business operators in support of an amendment of the EU specifications for titanium dioxide (E 171) with respect to the inclusion of additional parameters related to its particle size distribution. Titanium dioxide which is used as a food additive E 171 in food undergoes no surface treatment and is not coated. It consists of anatase or rutile generally containing small amounts of the other phase (rutile or anatase, < 2% m/m) and it may also contain small quantities (< 0.5%) of constituent particle growth and crystal phase control agents (alumina, sodium or potassium in combination with phosphate). Particle size analyses, by TEM, SEM, XDC or DC, have been carried out on five commercial brands of anatase E 171 and one of rutile E 171 manufactured by the only three EU manufacturers that, according to information submitted by interested business operators, produce food‐grade titanium dioxide. Interested business operators proposed to introduce in the EU specifications for E 171 a specification of more than 100 nm for median Feret min diameter and less than 50% of the number of constituent particles below 100 nm; measured by EM in both cases. The Panel, after reviewing the data, concluded that a specification of more than 100 nm for median minimal external dimension, equivalent to less than 50% of the number of constituent particles with a median minimal external dimension below 100 nm, should be inserted in the current EU specifications. The Panel considered that the conclusions made, and the uncertainties identified, in the previous EFSA assessments on E 171 remain valid. The Panel reiterates the need for the further research as recommended in the previous opinions in order to decrease the level of uncertainty and acknowledged that additional studies with characterised E 171 are being carried out by interested business operators.

%B EFSA Journal %V 17 %8 2019 July 12 %G eng %N 7 %R 10.2903/j.efsa.2019.5760 %0 Government Document %D 2019 %T Syllabus of the Interactive infosession on the methodologies and reporting of the characterisation of nanoparticles in food additives and novel food (FAVV/AFSCA) %A Jan Mast %A Eveline Verleysen %8 2019 %G eng %0 Government Document %D 2019 %T Syllabus of the workshop physicochemical characterization of nano-sized particles in food %A Eveline Verleysen %A Ralf Kaegi %A Nadia Waegeneers %A Stella Mathioudaki %A Frederic Brassinne %A Jan Mast %K Nanoparticles %K physico-chemical characterization %K transmission electron microscopy %X

Scope of this workshop

%8 2019 %G eng %0 Generic %D 2019 %T Validation of single particle ICP-MS for routine sizing and quantification of the fraction of silver nanoparticles in E174 food addititves and confectionery products %A Nadia Waegeneers %A Sandra De Vos %A Eveline Verleysen %A Jan Mast %K Nanomaterials; Food additives; E174; Silver %X

Silver (Ag) is a food additive (E174) approved by the European Commission to be used for the external coating of confectionery, for decoration of chocolates, and in liqueurs [1]. It is commercially distributed in its pristine powder and sheet form, and in confectionery products. Due to its nature, E174 may contain silver nanoparticles, which implies a need for validated methods to size and quantify these particles. Single particle inductively coupled plasma-mass spectrometry (spICP-MS) is thereby a promising technique as it is capable of sizing and counting particles at the same time.
A spICP-MS method was developed and validated for sizing and quantifying the fraction of silver nanoparticles in E174 food additives and in products containing E174. The samples were prepared for analysis according to a slightly modified version of the method of Jensen et al. [2]. The E174 food additives showed a large silver background concentration combined with a relatively low number of nanoparticles, making the quantification of the nanoparticles more challenging than in the products containing E174. Validation of the method showed good performance with respect for the size distribution compared to the size distribution obtained from transmission electron microscopy. Depending on the sample and the background silver concentration, particles with an equivalent spherical diameter (ESD) down to 11 nm could be detected. The performance in terms of repeatability (size 4-11%, concentration 16-29%), and intermediate precision (size 2-8%, concentration 18-31%) depended on the type of sample. The large repeatability compared to the intermediate precision demonstrates the need to analyze multiple independent replicates under routine conditions. When analyzing three replicates, the extended measurement uncertainty (k = 2) on the mean ESD is 20% for E174 food additives and 11% for products containing E174. The quantification of the mass and number concentration is more challenging with extended measurement uncertainties up to 45%.

%B IMEKOFOODS 4 %8 sep 2019 %G eng %N IMEKO %0 Generic %D 2018 %T Characterization of the TiO2 E171 food additive %A Frederic Brassinne %A Sandra De Vos %A Eveline Verleysen %A Pieter-Jan De Temmerman %A M. Ledecq %A Jan Mast %B Toxicology Letters %V 295 %8 Jan-10-2018 %G eng %R 10.1016/j.toxlet.2018.06.909 %0 Journal Article %J Sci Adv %D 2018 %T Multivalent binding of herpesvirus to living cells is tightly regulated during infection. %A Martin Delguste %A Zeippen, Caroline %A Machiels, Bénédicte %A Jan Mast %A Gillet, Laurent %A David Alsteens %X

Viral infection, initiated by the landing of a virion on a cellular surface, is largely defined by the preliminary interactions established between viral particles and their receptors at the cell surface. While multiple parallel interactions would allow strong virus attachment, a low number of bonds could be preferred to allow lateral diffusion toward specific receptors and to promote efficient release of progeny virions from the cell surface. However, so far, the molecular mechanisms underlying the regulation of the multivalency in virus attachment to receptors are poorly understood. We introduce a new method to force-probe multivalent attachment directly on living cells, and we show, for the first time, direct evidence of a new mechanism by which a herpesvirus surface glycoprotein acts as a key negative regulator in the first step of herpesvirus binding. Using atomic force microscopy, we probe at the single-virion level the number and the strength of the bonds established with heparan sulfate both on model surfaces and on living cells. Our biophysical results, correlated with other techniques, show that the major envelope glycoprotein functions as a regulator of binding valency during both attachment and release steps, determining the binding, diffusion, and release potential of virions at the cellular surface.

%B Sci Adv %V 4 %8 2018 Aug %G eng %N 8 %R 10.1126/sciadv.aat1273 %0 Report %D 2018 %T Physico-chemical characterization of the fraction of engineered nanomaterials in silver food additives (E174) in the context of risk assessment (nanoAg@) %A Eveline Verleysen %A Sandra De Vos %A Nadia Waegeneers %A Lotte Delfosse %A Marina Ledecq %A Jan Mast %X

report of project nanoAg@

%8 2018 %G eng %U https://www.sciensano.be/en/projects/physico-chemical-characterization-fraction-engineered-nanomaterials-silver-e174-food-additives %0 Generic %D 2018 %T Screening for nanoparticles in complex matrices within a regulatory framework: are we there yet? %A Nadia Waegeneers %A Delfosse,L. %A Sandra De Vos %A Jan Mast %K Nanoparticles %X

Silver (Ag) is a food additive (E174) approved by the European Commission to be used for the external coating of confectionery, for decoration of chocolates, and in liqueurs. It is commercially distributed in its pristine powder and sheet form, and in confectionary products. The presented work is part of a larger project that aims at a systematic examination of silver (E174) food additives and food items containing silver, available on the market in line with Regulation (EU) N° 1169/2011. Ten pristine samples of E174 and ten food items containing E174 were investigated for their number-based size distribution of Ag particles. The food items were as well investigated for their total Ag content. Single particle inductively coupled plasma – mass spectrometry (spICP-MS) was studied as a screening tool to determine the number-based size distribution of Ag particles, their particle number and mass concentration. The number-based size distribution of Ag particles was verified by transmission electron microscopy. Sample preparation, which included separation of the particles from the matrix in case of food products and preparation of a stable particle dispersion, proved to be a challenging step. The precision (repeatability and intermediate precision) of the selected methodology was determined for both the pristine E174 food additives and food samples containing E174. The determination of total Ag in the food samples by means of inductively coupled plasma – optical emission spectrometry (ICP-OES) after acid microwave digestion was hampered by matrix effects, but could be solved by internal calibration. The applied methodology was able to demonstrate the presence of silver nanoparticles in E174 and confectionery products within the framework of Regulation (EU) N° 1169/2011.

Acknowledgement: This research was funded by the Belgian Federal Public Service of Health, Food Chain Safety and Environment. The authors wish to thank R. Machiels and F. Van Steen for their technical assistance.

%B 40th International Conference on Environmental & Food Monitoring (ISEAC-40) %I ISEAC-40 %C Santiago de Compostela, Spain %8 2018 %G eng %0 Journal Article %J Toxicology Reports %D 2018 %T Short-term biodistribution and clearance of intravenously administered silica nanoparticles %A Nadia Waegeneers %A Anne Brasseur %A Van Doren, Elke %A Van der Heyden, Sara %A Pieter-Jan Serreyn %A Pussemier, Luc %A Jan Mast %A Schneider, Yves-Jacques %A Ann Ruttens %A S. Roels %K distribution %K in vivo %K intravenous exposure %K nanomaterials %K synthetic amorphous silica %X Recently, concerns have been raised about potential adverse effects of synthetic amorphous silica, commonly used as food additive (E551), since silica nanoparticles have been detected in food containing E551. We examined the biodistribution and excretion in female Sprague-Dawley rats of NM-200, a well characterized nanostructured silica representative for food applications. A single intravenous injection of NM-200 was applied at a dose of 20 mg/kgbw, followed by autopsy after 6 and 24 hours. The main organs where silicon accumulated were liver and spleen. The silicon concentration significantly decreased in spleen between 6 and 24 hours. In liver the tendency was the same but the effect was not significant. This could be due to clearance of the spleen to the liver via the splenic vein, while liver clearance takes more time due to hepatic processing and biliary excretion. In treated animals the liver showed in addition a prominent increase of macrophages between both evaluation moments. Within the first 24 hours, silicon was mainly excreted through urine. Further studies are necessary to evaluate the toxicokinetics of different types of silica nanomaterials at lower exposure doses in order to be able to predict kinetics and toxicity of silica nanoparticles depending on their physicochemical characteristics. %B Toxicology Reports %V 5 %8 23/05/2018 %G eng %& 632 %R https://doi.org/10.1016/j.toxrep.2018.05.004 %0 Journal Article %J Toxicology Reports %D 2018 %T Short-term biodistribution and clearance of intravenously administered silica nanoparticles %A Nadia Waegeneers %A Anne Brasseur %A Van Doren, Elke %A Van der Heyden, Sara %A Pieter-Jan Serreyn %A Pussemier, Luc %A Jan Mast %A Schneider, Yves-Jacques %A Ann Ruttens %A S. Roels %B Toxicology Reports %V 5 %8 Jan-01-2018 %G eng %R 10.1016/j.toxrep.2018.05.004 %0 Journal Article %J Vaccine %D 2018 %T Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design. %A Lagoutte, Priscillia %A Mignon, Charlotte %A Stadthagen, Gustavo %A Supanee Potisopon %A Donnat, Stéphanie %A Jan Mast %A Lugari, Adrien %A Werle, Bettina %X

In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24 nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery.

%B Vaccine %V 36 %8 2018 06 14 %G eng %N 25 %R 10.1016/j.vaccine.2018.05.034 %0 Journal Article %J PLoS One %D 2018 %T Y-box-binding protein 1 supports the early and late steps of HIV replication. %A Weydert, Caroline %A Bart van Heertum %A Lieve Dirix %A Stéphanie De Houwer %A Flore De Wit %A Jan Mast %A Steven J Husson %A Katrien Busschots %A Renate König %A Gijsbers, Rik %A De Rijck, Jan %A Debyser, Zeger %X

The human immunodeficiency virus (HIV) depends on cellular proteins, so-called cofactors, to complete its replication cycle. In search for new therapeutic targets we identified the DNA and RNA binding protein Y-box-binding Protein 1 (YB-1) as a cofactor supporting early and late steps of HIV replication. YB-1 depletion resulted in a 10-fold decrease in HIV-1 replication in different cell lines. Dissection of the replication defects revealed that knockdown of YB-1 is associated with a 2- to 5-fold decrease in virion production due to interference with the viral RNA metabolism. Using single-round virus infection experiments we demonstrated that early HIV-1 replication also depends on the cellular YB-1 levels. More precisely, using quantitative PCR and an in vivo nuclear import assay with fluorescently labeled viral particles, we showed that YB-1 knockdown leads to a block between reverse transcription and nuclear import of HIV-1. Interaction studies revealed that YB-1 associates with integrase, although a direct interaction with HIV integrase could not be unambiguously proven. In conclusion, our results indicate that YB-1 affects multiple stages of HIV replication. Future research on the interaction between YB-1 and the virus will reveal whether this protein qualifies as a new antiviral target.

%B PLoS One %V 13 %8 2018 %G eng %N 7 %R 10.1371/journal.pone.0200080 %0 Generic %D 2017 %T Human absorption of silver ENM: through particles or ions? %A Nadia Waegeneers %A A Brasseur %A Van Der Heyden,S %A Jan Mast %A S. Roels %K Absorption %K Nanoparticles %K Silver %X

One of the many questions in the human risk assessment of metallic engineered nanomaterials (ENM) such as silver ENM is whether these ENM dissolve in the gastrointestinal (GI) system or are absorbed and translocated to organs and tissues as intact nanoparticles. It is difficult to elucidate this question as in situ measurement of silver ENM is still hampered by technical difficulties, but also because silver nanoparticles can be formed in vivo after exposure to silver salts. To tackle these difficulties, we compared the tissue distribution of orally administered silver ENM with the tissue distribution pattern of silver ENM and a silver salt that were administered by intravenous (IV) injection.

Female rats were exposed to a single dose of NM-300K, consisting of silver nanoparticles with a mean diameter <20 nm, or to AgNO3. The rats were treated either by oral gavage or by IV injection. During a 24-hour period, urine and feces were collected. After 24 hours, selected tissues were sampled and total Ag concentrations were measured by ICP-MS.

After treatment by IV injection with the silver salt, largest Ag concentrations were found in the liver, spleen and pancreas, and Ag was largely excreted (32% of administered dose) via urine and feces. After IV injection with NM-300K, largest Ag concentrations were found in the spleen, and <1% of the administered dose was excreted via urine and feces. This demonstrates that NM-300K, once present systemically, is circulated as particles and solubilization is limited.

After treatment of the animals by oral gavage there was a low absorption of Ag, the absorption after treatment with NM-300K being 10-fold lower than after treatment with AgNO3. As there is a certain amount of soluble Ag present in the administered NM-300K dispersion (~3%), the tissue concentrations were normalized for the soluble Ag dose. After this normalization, the Ag concentrations in tissues after exposure to NM-300K were up to 17 times larger than after exposure to AgNO3. This might indicate that either an additional fraction of NM-300K has been solubilized in the GI tract, or that NM-300K is partially taken up as intact nanoparticles. The Ag distribution pattern in the tissues after treatment with NM-300K resembled, however, more that of ionic Ag after IV injection than that of NM-300K after IV injection. This suggests that it is more likely that NM-300K is partially dissolved in the GI tract and subsequently absorbed and excreted via urine and feces.

%B SETAC Brussels %I SETAC %C Brussel, België %8 2017 %G eng %U https://brussels.setac.org/ %0 Generic %D 2017 %T Influence of dispersion method on silica nanoparticle size distribution/aggregation and their chronic toxicity in-vitro %A Sivakumar Murugadoss %A van den Brûle, Sybille %A Sebaihi, Noham %A Frederic Brassinne %A Jan Mast %A Godderis, Lode %A Lison, Dominique %A Hoet, Peter H. %8 2018 %G eng %0 Journal Article %J Toxicology Letters %D 2017 %T Toxicity and biological responses (in vitro) influenced by aggregation and agglomeration of manufactured nanomaterials %A Sivakumar Murugadoss %A van den Brûle, Sybille %A Sebaihi, Noham %A Frederic Brassinne %A Jan Mast %A Godderis, Lode %A Lison, Dominique %A Peter Hoet %B Toxicology Letters %V 280 %8 Jan-10-2017 %G eng %R 10.1016/j.toxlet.2017.07.535 %0 Journal Article %J Archives of Toxicology %D 2017 %T Toxicology of silica nanoparticles: an update %A Sivakumar Murugadoss %A Lison, Dominique %A Godderis, Lode %A van den Brûle, Sybille %A Jan Mast %A Frederic Brassinne %A Sebaihi, Noham %A Hoet, Peter H. %K Amorphous silica nanoparticles %K Oxidative Stress %K Pyrogenic %K Stöber %K toxicity %X

Large-scale production and use of amorphous silica nanoparticles (SiNPs) have increased the risk of human exposure to SiNPs, while their health effects remain unclear. In this review, scientific papers from 2010 to 2016 were systematically selected and sorted based on in vitro and in vivo studies: to provide an update on SiNPs toxicity and to address the knowledge gaps indicated in the review of Napierska (Part Fibre Toxicol 7:39, 2010). Toxicity of SiNPs in vitro is size, dose, and cell type dependent. SiNPs synthesized by wet route exhibited noticeably different biological effects compared to thermal route-based SiNPs. Amorphous SiNPs (particularly colloidal and stöber) induced toxicity via mechanisms similar to crystalline silica. In vivo, route of administration and physico-chemical properties of SiNPs influences the toxicokinetics. Adverse effects were mainly observed in acutely exposed animals, while no significant signs of toxicity were noted in chronically dosed animals. The correlation between in vitro and in vivo toxicity remains less well established mainly due to improper-unrealistic-dosing both in vitro and in vivo. In conclusion, notwithstanding the multiple studies published in recent years, unambiguous linking of physico-chemical properties of SiNPs types to toxicity, bioavailability, or human health effects is not yet possible.

%B Archives of Toxicology %V 91 %8 1 June 2017 %G eng %& 2967 %R 10.1007/s00204-017-1993-y %0 Journal Article %J J Nanopart Res %D 2016 %T Challenges in the size analysis of a silica nanoparticle mixture as candidate certified reference material. %A Kestens, Vikram %A Roebben, Gert %A Herrmann, Jan %A Jämting, Åsa %A Coleman, Victoria %A Minelli, Caterina %A Clifford, Charles %A Pieter-Jan De Temmerman %A Jan Mast %A Junjie, Liu %A Babick, Frank %A Cölfen, Helmut %A Emons, Hendrik %X

A new certified reference material for quality control of nanoparticle size analysis methods has been developed and produced by the Institute for Reference Materials and Measurements of the European Commission's Joint Research Centre. The material, ERM-FD102, consists of an aqueous suspension of a mixture of silica nanoparticle populations of distinct particle size and origin. The characterisation relied on an interlaboratory comparison study in which 30 laboratories of demonstrated competence participated with a variety of techniques for particle size analysis. After scrutinising the received datasets, certified and indicative values for different method-defined equivalent diameters that are specific for dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), particle tracking analysis (PTA) and asymmetrical-flow field-flow fractionation (AF4) were assigned. The value assignment was a particular challenge because metrological concepts were not always interpreted uniformly across all participating laboratories. This paper presents the main elements and results of the ERM-FD102 characterisation study and discusses in particular the key issues of measurand definition and the estimation of measurement uncertainty.

%B J Nanopart Res %V 18 %P 171 %8 2016 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/27441027?dopt=Abstract %R 10.1007/s11051-016-3474-2 %0 Journal Article %J Viruses %D 2016 %T Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis. %A de Brogniez, Alix %A Jan Mast %A Willems, Luc %K Animals %K Cattle %K Cell Membrane %K Glycosylation %K Leukemia Virus, Bovine %K Protein Interaction Domains and Motifs %K Protein Subunits %K Viral Envelope Proteins %K Viral Fusion Proteins %K Virus Attachment %K Virus Internalization %K Virus Replication %X

Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis.

%B Viruses %V 8 %P 88 %8 2016 Mar 24 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/27023592?dopt=Abstract %R 10.3390/v8040088 %0 Journal Article %J Avian Dis %D 2016 %T The Enrichment of Histomonas meleagridis and Its Pathogen-Specific Protein Analysis: A First Step to Shed Light on Its Virulence. %A Pham, Anh Dao Nguyen %A Jan Mast %A Magez, Stefan %A Goddeeris, Bruno Maria %A Carpentier, Sebastien C %K Animals %K Cecum %K Electrophoresis, Gel, Two-Dimensional %K Poultry Diseases %K Protozoan Infections, Animal %K Protozoan Proteins %K Trichomonadida %K Turkeys %K virulence %X

Since the discovery of Histomonas meleagridis in 1893, the necessity of isolating pure H. meleagridis has been highlighted over the years in the battle against histomonosis. Insights into the molecular characteristics of this protozoon open possibilities to proper treatment. Axenization of H. meleagridis in vitro cultures cocultured with bacteria has been unsuccessful. Numerous unsuccessful attempts at culturing H. meleagridis axenically have reinforced the assumption that the protozoa had an obligate relationship with certain bacteria originating from the host ceca. Within these perspectives, we enriched H. meleagridis cells from a mono-eukaryotic culture copropagated with host cecal bacteria by flow cytometry. The enrichment of histomonads was confirmed through transmission electron microscopy and two-dimensional gel electrophoresis. For the first time several protein spots were successfully identified. The majority of spots were annotated as cytoskeletal proteins. Actin microfilaments are known to be a key player in cell spreading, cell adhesion, phagocytosis, signal transduction, and several other processes. Together with the identification of superoxide dismutase, the information generated from protein analysis of H. meleagridis may serve as a very first step toward understanding its pathogenesis and virulence.

%B Avian Dis %V 60 %P 628-36 %8 2016 Sep %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/27610722?dopt=Abstract %R 10.1637/11389-021016-Reg.1 %0 Journal Article %J Avian Diseases %D 2016 %T The Enrichment of Histomonas meleagridis and Its Pathogen-Specific Protein Analysis: A First Step to Shed Light on Its Virulence %A Pham, Anh Dao Nguyen %A Jan Mast %A Magez, Stefan %A Goddeeris, Bruno Maria %A Sebastien C Carpentier %B Avian Diseases %V 60 %8 Jan-09-2016 %G eng %N 3 %R 10.1637/11389-021016-Reg.1 %0 Journal Article %J J Virol Methods %D 2016 %T Scalable chromatography-based purification of virus-like particle carrier for epitope based influenza A vaccine produced in Escherichia coli. %A Lagoutte, Priscillia %A Mignon, Charlotte %A Donnat, Stéphanie %A Stadthagen, Gustavo %A Jan Mast %A Sodoyer, Régis %A Lugari, Adrien %A Werle, Bettina %K Animals %K Chromatography, Gel %K Epitopes %K Escherichia coli %K Influenza A virus %K Influenza Vaccines %K Levivirus %K Orthomyxoviridae Infections %K Vaccines, Synthetic %K Vaccines, Virus-Like Particle %K Veterinary Medicine %K Viral Matrix Proteins %X

Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price.

%B J Virol Methods %V 232 %P 8-11 %8 2016 Jun %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26947397?dopt=Abstract %R 10.1016/j.jviromet.2016.02.011 %0 Journal Article %J Avian Dis %D 2016 %T Stronger Interference of Avian Influenza Virus-Specific Than Newcastle Disease Virus-Specific Maternally Derived Antibodies with a Recombinant NDV-H5 Vaccine. %A Lardinois, Amélyne %A Vandersleyen, Olivier %A Mieke Steensels %A Desloges, Nathalie %A Jan Mast %A Thierry van den Berg %A Bénédicte Lambrecht %K Animals %K Antibodies, Viral %K Chickens %K Female %K Immunity, Maternally-Acquired %K Influenza A Virus, H5N1 Subtype %K Influenza in Birds %K Influenza Vaccines %K Male %K Newcastle Disease %K Newcastle disease virus %K Poultry Diseases %K Vaccines, Combined %K Viral Vaccines %X

Maternally derived antibodies (MDA) are known to provide early protection from disease but also to interfere with vaccination efficacy of young chicks. This interference phenomenon is well described in the literature for viral diseases such as infectious bursal disease, Newcastle disease (ND), and avian influenza (AI). The goal of this work was to investigate the impact of H5 MDA and/or ND virus (NDV) MDA on the vaccine efficacy of a recombinant NDV-H5-vectored vaccine (rNDV-H5) against two antigenically divergent highly pathogenic AI (HPAI) H5N1 challenges. In chickens with both H5 and NDV MDA, a strong interference was observed with reduced clinical protection when compared to vaccinated specific-pathogen-free (SPF) chickens. In contrast, in chickens from commercial suppliers with NDV MDA only, a beneficial impact on the vaccine efficacy was observed with full protection and reduced viral excretion in comparison with rNDV-H5-vaccinated SPF chickens. To distinguish between the respective effects of the H5 and NDV MDA, an SPF model where passive immunity had been artificially induced by inoculations of H5 and NDV hyperimmunized polysera, respectively, was used. In the presence of H5 artificial MDA, a strong interference reflected by a reduction in vaccine protection was demonstrated whereas no interference and even an enhancing protective effect was confirmed in presence of NDV MDA. The present work suggests that H5 and NDV MDA interact differently with the rNDV-H5 vaccine with different consequences on its efficacy, the mechanisms of which require further investigations.

%B Avian Dis %V 60 %P 191-201 %8 2016 May %G eng %N 1 Suppl %1 http://www.ncbi.nlm.nih.gov/pubmed/27309055?dopt=Abstract %R 10.1637/11133-050815-Reg %0 Journal Article %J J Virol %D 2015 %T Deletion of Murid Herpesvirus 4 ORF63 Affects the Trafficking of Incoming Capsids toward the Nucleus. %A Latif, Muhammad Bilal %A Machiels, Bénédicte %A Xiao, Xue %A Jan Mast %A Vanderplasschen, Alain %A Gillet, Laurent %K Animals %K Biological Transport %K Capsid %K Cell Line %K Cricetinae %K Female %K Gene Deletion %K Herpesviridae Infections %K Histocytochemistry %K Lung %K Mice, Inbred BALB C %K Rhadinovirus %K Viral Proteins %K Virus Internalization %X

UNLABELLED: Gammaherpesviruses are important human and animal pathogens. Despite the fact that they display the classical architecture of herpesviruses, the function of most of their structural proteins is still poorly defined. This is especially true for tegument proteins. Interestingly, a potential role in immune evasion has recently been proposed for the tegument protein encoded by Kaposi's sarcoma-associated herpesvirus open reading frame 63 (ORF63). To gain insight about the roles of ORF63 in the life cycle of a gammaherpesvirus, we generated null mutations in the ORF63 gene of murid herpesvirus 4 (MuHV-4). We showed that disruption of ORF63 was associated with a severe MuHV-4 growth deficit both in vitro and in vivo. The latter deficit was mainly associated with a defect of replication in the lung but did not affect the establishment of latency in the spleen. From a functional point of view, inhibition of caspase-1 or the inflammasome did not restore the growth of the ORF63-deficient mutant, suggesting that the observed deficit was not associated with the immune evasion mechanism identified previously. Moreover, this growth deficit was also not associated with a defect in virion egress from the infected cells. In contrast, it appeared that MuHV-4 ORF63-deficient mutants failed to address most of their capsids to the nucleus during entry into the host cell, suggesting that ORF63 plays a role in capsid movement. In the future, ORF63 could therefore be considered a target to block gammaherpesvirus infection at a very early stage of the infection.

IMPORTANCE: The important diseases caused by gammaherpesviruses in human and animal populations justify a better understanding of their life cycle. In particular, the role of most of their tegument proteins is still largely unknown. In this study, we used murid herpesvirus 4, a gammaherpesvirus infecting mice, to decipher the role of the protein encoded by the viral ORF63 gene. We showed that the absence of this protein is associated with a severe growth deficit both in vitro and in vivo that was mainly due to impaired migration of viral capsids toward the nucleus during entry. Together, our results provide new insights about the life cycle of gammaherpesviruses and could allow the development of new antiviral strategies aimed at blocking gammaherpesvirus infection at the very early stages.

%B J Virol %V 90 %P 2455-72 %8 2015 Dec 16 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/26676769?dopt=Abstract %R 10.1128/JVI.02942-15 %0 Journal Article %J Sci Adv %D 2015 %T Engineering a nanopore with co-chaperonin function. %A Ho, Ching-Wen %A Van Meervelt, Veerle %A Tsai, Keng-Chang %A Pieter-Jan De Temmerman %A Jan Mast %A Maglia, Giovanni %X

The emergence of an enzymatic function can reveal functional insights and allows the engineering of biological systems with enhanced properties. We engineered an alpha hemolysin nanopore to function as GroES, a protein that, in complex with GroEL, forms a two-stroke protein-folding nanomachine. The transmembrane co-chaperonin was prepared by recombination of GroES functional elements with the nanopore, suggesting that emergent functions in molecular machines can be added bottom-up by incorporating modular elements into preexisting protein scaffolds. The binding of a single-ring version of GroEL to individual GroES nanopores prompted large changes to the unitary nanopore current, most likely reflecting the allosteric transitions of the chaperonin apical domains. One of the GroEL-induced current levels showed fast fluctuations (<1 ms), a characteristic that might be instrumental for efficient substrate encapsulation or folding. In the presence of unfolded proteins, the pattern of current transitions changed, suggesting a possible mechanism in which the free energy of adenosine triphosphate binding and hydrolysis is expended only when substrate proteins are occupied.

%B Sci Adv %V 1 %P e1500905 %8 2015 Dec %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/26824063?dopt=Abstract %R 10.1126/sciadv.1500905 %0 Journal Article %J J Virol %D 2015 %T The Genome of a Tortoise Herpesvirus (Testudinid Herpesvirus 3) Has a Novel Structure and Contains a Large Region That Is Not Required for Replication In Vitro or Virulence In Vivo. %A Gandar, Frédéric %A Wilkie, Gavin S %A Gatherer, Derek %A Kerr, Karen %A Marlier, Didier %A Diez, Marianne %A Marschang, Rachel E %A Jan Mast %A Dewals, Benjamin G %A Davison, Andrew J %A Vanderplasschen, Alain F C %K Alphaherpesvirinae %K Animals %K Base Sequence %K brain %K Cell Line %K Chromosome Mapping %K DNA, Viral %K Genome, Viral %K Herpesviridae Infections %K Interleukin-10 %K Molecular Sequence Data %K Phylogeny %K Semaphorins %K Sequence Analysis, DNA %K Sequence Deletion %K Turtles %K Viral Vaccines %X

UNLABELLED: Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it.

IMPORTANCE: Testudinid herpesvirus 3 (TeHV-3) causes a lethal disease in tortoises, several species of which are endangered. We have characterized the viral genome and used this information to take steps toward developing an attenuated vaccine. We have sequenced the genomes of two strains (1976 and 4295), compared their growth in vitro, and investigated the pathogenesis of strain 4295, which consists of three deletion mutants. The major findings are that (i) TeHV-3 has a novel genome structure, (ii) its closest relative is a turtle herpesvirus, (iii) it contains interleukin-10 and semaphorin genes (the first time these have been reported in an alphaherpesvirus), (iv) a sizeable region of the genome is not required for viral replication in vitro or virulence in vivo, and (v) one of the components of strain 4295, which has a deletion of 22.4 kb, exhibits properties indicating that it may serve as the starting point for an attenuated vaccine.

%B J Virol %V 89 %P 11438-56 %8 2015 Nov %G eng %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/26339050?dopt=Abstract %R 10.1128/JVI.01794-15 %0 Journal Article %J Diabetologia %D 2015 %T Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability. %A Pachera, Nathalie %A Papin, Julien %A Zummo, Francesco P %A Rahier, Jacques %A Jan Mast %A Meyerovich, Kira %A Cardozo, Alessandra K %A Herchuelz, André %K Animals %K Cell Membrane %K Cell Proliferation %K Cell Survival %K Glucose %K Glucose Tolerance Test %K Insulin %K Insulin-Secreting Cells %K mice %K Plasma Membrane Calcium-Transporting ATPases %K Sodium-Calcium Exchanger %X

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function.

METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo.

RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed.

CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

%B Diabetologia %V 58 %P 2843-50 %8 2015 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/26362865?dopt=Abstract %R 10.1007/s00125-015-3745-y %0 Journal Article %J Retrovirology %D 2015 %T HIV-1 IN/Pol recruits LEDGF/p75 into viral particles. %A Desimmie, Belete Ayele %A Weydert, Caroline %A Schrijvers, Rik %A Vets, Sofie %A Demeulemeester, Jonas %A Proost, Paul %A Paron, Igor %A De Rijck, Jan %A Jan Mast %A Bannert, Norbert %A Gijsbers, Rik %A Christ, Frauke %A Debyser, Zeger %K Adaptor Proteins, Signal Transducing %K HIV Protease %K Hiv-1 %K Host-Pathogen Interactions %K Humans %K pol Gene Products, Human Immunodeficiency Virus %K Proteolysis %K Transcription Factors %K Virus Integration %K Virus Replication %X

BACKGROUND: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication.

RESULTS: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency.

CONCLUSION: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation.

%B Retrovirology %V 12 %P 16 %8 2015 Feb 12 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25809198?dopt=Abstract %R 10.1186/s12977-014-0134-4 %0 Book %B Physical characterization of nanomaterials in dispersion by transmission electron microscopy in a regulatory framework %D 2015 %T Physical Characterization of Nanomaterials in Dispersion by Transmission Electron Microscopy in a Regulatory Framework %A Jan Mast %A Eveline Verleysen %A Pieter-Jan De Temmerman %E Francis Leonard Deepak %E Alvaro Mayoral %E Raul Arenal %X

TEM is one of the few techniques that can identify nanoparticles according to the current definitions. This chapter focuses on the different steps required to analyze dispersed nanomaterials by TEM. Methodologies to obtain homogeneous and stable dispersions of colloidal nanomaterials and powders are presented. The preparation of TEM specimens to obtain a representative distribution of particles on the grid is discussed. The application of TEM imaging methods, electron diffraction and analytical TEM to obtain complementary information on the size, morphology, crystallographic structure, electronic structure and composition of nanomaterials is reviewed.

In a qualitative TEM analysis the key properties of the physical form of the nanomaterial under which it is exposed to in vitro and in vivo test systems are described based on TEM micrographs. Subsequently, a quantitative analysis which includes detection, classification and measurement of primary particle properties, and validation of the measurement results can be performed. The possibility to extract 3D information by fractal analysis of electron micrographs of aggregated nanomaterials with a fractal-like structure is explored

%B Physical characterization of nanomaterials in dispersion by transmission electron microscopy in a regulatory framework %7 1 %I Springer International Publishing %C Cham %V Advanced Transmission Electron Microscopy. Applications to Nanomaterials %P 21 %8 2015 %@ 978-3-319-15176-2 %G eng %N 8 %9 Advanced Transmission Electron Microscopy - Applications to Nanomaterials %6 1 %) Deepak, Francis Leonard, Mayoral, Alvaro, Arenal, Raul %& 249 %R 10.1007/978-3-319-15177-910.1007/978-3-319-15177-9_8 %0 Journal Article %J J Agric Food Chem %D 2015 %T TEM and SP-ICP-MS analysis of the release of silver nanoparticles from decoration of pastry. %A Eveline Verleysen %A Van Doren, E %A Nadia Waegeneers %A Pieter-Jan De Temmerman %A M Abi Daoud Francisco %A Jan Mast %K Food Additives %K Mass Spectrometry %K Metal Nanoparticles %K Microscopy, Electron, Transmission %K Particle Size %K Silver %K X-Ray Diffraction %X Metallic silver is an EU approved food additive referred to as E174. It is generally assumed that silver is only present in bulk form in the food chain. This work demonstrates that a simple treatment with water of "silver pearls", meant for decoration of pastry, results in the release of a subfraction of silver nanoparticles. The number-based size and shape distributions of the single, aggregated, and/or agglomerated particles released from the silver pearls were determined by combining conventional bright-field TEM imaging with semiautomatic particle detection and analysis. In addition, the crystal structure of the particles was studied by electron diffraction and chemical information was obtained by combining HAADF-STEM imaging with EDX spectroscopy and mapping. The TEM results were confirmed by SP-ICP-MS. The representative Ag test nanomaterial NM-300 K was used as a positive control to determine the uncertainty on the measurement of the size and shape of the particles. %B J Agric Food Chem %V 63 %P 3570-8 %8 2015 Apr 08 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/25768118?dopt=Abstract %R 10.1021/acs.jafc.5b00578 %0 Journal Article %J PLoS One %D 2014 %T Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii. %A Merabishvili, Maia %A Vandenheuvel, Dieter %A Kropinski, Andrew M %A Jan Mast %A De Vos, Daniel %A Verbeken, Gilbert %A Noben, Jean-Paul %A Lavigne, Rob %A Vaneechoutte, Mario %A Pirnay, Jean-Paul %K Acinetobacter baumannii %K Bacteriophages %K Culture Techniques %K Genome, Viral %K Host Specificity %K Molecular Sequence Annotation %K Phenotype %K Proteomics %X

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.

%B PLoS One %V 9 %P e104853 %8 2014 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/25111143?dopt=Abstract %R 10.1371/journal.pone.0104853 %0 Journal Article %J Toxicol In Vitro %D 2014 %T Genotoxicity evaluation of nanosized titanium dioxide, synthetic amorphous silica and multi-walled carbon nanotubes in human lymphocytes. %A Tavares, Ana M %A Louro, Henriqueta %A Antunes, Susana %A Quarré, Stephanie %A Simar, Sophie %A Pieter-Jan De Temmerman %A Eveline Verleysen %A Jan Mast %A Jensen, Keld A %A Norppa, Hannu %A Nesslany, Fabrice %A Silva, Maria João %K Adult %K Female %K Humans %K In Vitro Techniques %K Light %K Lymphocytes %K Male %K Micronucleus Tests %K Microscopy, Electron, Transmission %K Mutagenicity Tests %K Nanostructures %K Nanotubes, Carbon %K Particle Size %K Scattering, Radiation %K Silicon Dioxide %K Titanium %X

Toxicological characterization of manufactured nanomaterials (NMs) is essential for safety assessment, while keeping pace with innovation from their development and application in consumer products. The specific physicochemical properties of NMs, including size and morphology, might influence their toxicity and have impact on human health. The present work aimed to evaluate the genotoxicity of nanosized titanium dioxide (TiO2), synthetic amorphous silica (SAS) and multiwalled carbon nanotubes (MWCNTs), in human lymphocytes. The morphology and size of those NMs were characterized by transmission electron microscopy, while the hydrodynamic particle size-distributions were determined by dynamic light scattering. Using a standardized procedure to ensure the dispersion of the NMs and the cytokinesis-block micronucleus assay (without metabolic activation), we observed significant increases in the frequencies of micronucleated binucleated cells (MNBCs) for some TiO2 NMs and for two MWCNTs, although no clear dose-response relationships could be disclosed. In contrast, all forms of SAS analyzed in this study were unable to induce micronuclei. The present findings increase the weight of evidence towards a genotoxic effect of some forms of TiO2 and some MWCNTs. Regarding safety assessment, the differential genotoxicity observed for closely related NMs highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs.

%B Toxicol In Vitro %V 28 %P 60-9 %8 2014 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23811260?dopt=Abstract %R 10.1016/j.tiv.2013.06.009 %0 Journal Article %J Journal of Nanoparticle Research %D 2014 %T Measurement uncertainties of size, shape, and surface measurements using transmission electron microscopy of near-monodisperse, near-spherical nanoparticles %A Pieter-Jan De Temmerman %A Lammertyn, Jeroen %A De Ketelaere, Bart %A Kestens, Vikram %A Roebben, Gert %A Eveline Verleysen %A Jan Mast %K Transmission electron microscopy Method validation Reference material Silica nanoparticles Measurement uncertainty Nanometrology %B Journal of Nanoparticle Research %V 16492008221027594757410103241512943 %8 Jan-01-2014 %G eng %N 133624524212031 %R 10.1007/s11051-013-2177-1 %0 Journal Article %J Journal of Virology %D 2014 %T Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1 %A Schmeiser, S. %A Jan Mast %A Thiel, H.-J. %A Konig, M. %A Sandri-Goldin, R. M. %K electron microscopy %K morphogenisis %K Pestivirus %K ultrastructure %B Journal of Virology %V 88 %8 Jan-03-2014 %G eng %N 5 %R 10.1128/JVI.03237-13 %0 Journal Article %J Powder Technology %D 2014 %T Quantitative characterization of aggregated and agglomerated titanium dioxide nanomaterials by transmission electron microscopy %A Eveline Verleysen %A Pieter-Jan De Temmerman %A Van Doren, E. %A M. Abi Daoud Francisco %A Jan Mast %K Titanium dioxide Nanoparticles Size distribution Shape distribution Transmission electron microscopy Dispersion protocol %B Powder Technology %V 258 %8 Jan-05-2014 %G eng %R 10.1016/j.powtec.2014.03.010 %0 Journal Article %J Powder Technology %D 2014 %T Semi-automatic size measurement of primary particles in aggregated nanomaterials by transmission electron microscopy %A Pieter-Jan De Temmerman %A Eveline Verleysen %A Lammertyn, Jeroen %A Jan Mast %K Transmission electron microscopy Image analysis Aggregate Nanomaterial Primary particles Fractal analysis %B Powder Technology %V 261 %8 Jan-07-2014 %G eng %R 10.1016/j.powtec.2014.04.040 %0 Journal Article %J Journal of Nanoparticle Research %D 2014 %T Size measurement uncertainties of near-monodisperse, near-spherical nanoparticles using transmission electron microscopy and particle-tracking analysis %A Pieter-Jan De Temmerman %A Eveline Verleysen %A Lammertyn, Jeroen %A Jan Mast %K Transmission electron microscopy Particle-tracking analysis Colloidal gold Polystyrene beads Method validation Measurement uncertainty Nanometrology %B Journal of Nanoparticle Research %V 16 %8 Jan-10-2014 %G eng %N 10 %R 10.1007/s11051-014-2628-3 %0 Journal Article %J Reprod Toxicol %D 2013 %T Effects of lung exposure to carbon nanotubes on female fertility and pregnancy. A study in mice. %A Hougaard, Karin S %A Jackson, Petra %A Kyjovska, Zdenka O %A Birkedal, Renie K %A Pieter-Jan De Temmerman %A Brunelli, Andrea %A Eveline Verleysen %A Madsen, Anne Mette %A Saber, Anne T %A Pojana, Giulio %A Jan Mast %A Marcomini, Antonio %A Jensen, Keld A %A Wallin, Håkan %A Szarek, Józef %A Mortensen, Alicja %A Vogel, Ulla %K Animals %K Bronchoalveolar Lavage Fluid %K Female %K Fertility %K Liver %K Lung %K Male %K mice %K Mice, Inbred C57BL %K Motor Activity %K Nanotubes, Carbon %K Pneumonia %K Pregnancy %K Reflex, Startle %K Spermatogenesis %X

We studied the effects of preconceptional exposure to multiwalled carbon nanotubes (MWCNTs): mature, female C57BL/6J mice were intratracheally instilled with 67μg NM-400 MWCNT, and the following day co-housed with mature males, in breeding pairs. Time to delivery of the first litter, litter parameters, maternal inflammation and histopathology of lung and liver were recorded. In male offspring, locomotor activity, startle response, and daily sperm production (DSP) were assessed. In the dams, lung and liver bore evidence of MWCNT exposure when assessed 6 weeks and 4 months after exposure. A short delay in the delivery of the first litter was observed in exposed females. Litter parameters, behavior and DSP were similar in control and exposed groups. In conclusion, instillation of a single dose of MWCNT induced long lasting pathological changes in dam lung and liver. Theoretically, lung inflammation due to particle exposure could interfere with female reproductive parameters. Whether the observed lag in delivery of a first litter was in fact caused by exposure to MWCNT should be addressed in a study designed specifically to elucidate effects on the early processes involved in establishment of pregnancy. Exposure was not associated with changes in the assessed gestational or offspring parameters.

%B Reprod Toxicol %V 41 %P 86-97 %8 2013 Nov %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/23714338?dopt=Abstract %R 10.1016/j.reprotox.2013.05.006 %0 Journal Article %J Parasitology %D 2013 %T Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp. %A Pham, Anh Dao Nguyen %A Jan Mast %A Jeroen Koen De Gussem %A McDougald, Larry R %A Goddeeris, Bruno Maria %K Animals %K Blastocystis %K Culture Techniques %K Male %K Microscopy, Electron, Transmission %K polymerase chain reaction %K Poultry Diseases %K Protozoan Infections, Animal %K Trichomonadida %K Turkeys %X

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

%B Parasitology %V 140 %P 1266-74 %8 2013 Sep %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/23790160?dopt=Abstract %R 10.1017/S0031182013000723 %0 Journal Article %J Microbes Infect %D 2013 %T Infectivity of rabies virus-exposed macrophages. %A Nazé, Florence %A Vanessa Suin %A Lamoral, Sophie %A Aurélie Francart %A Bernard Brochier %A S. Roels %A Jan Mast %A Kalai, Michael %A Steven Van Gucht %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Antibody Formation %K Antigens, Viral %K Bone Marrow %K brain %K Cell Death %K Immunity, Humoral %K Injections, Intramuscular %K Macrophages %K mice %K Mice, Inbred C57BL %K Microscopy, Electron %K Nose %K Rabies %K Rabies virus %K RNA, Viral %K Spleen %K Viral Load %K Virus Cultivation %X

Rabies virus distributes widely in infected mice, including lymphoid tissues and spleen macrophages. The infection characteristics in murine macrophages and the infectivity of virus-exposed macrophages were examined upon inoculation in mice. In vitro, Mf4/4 spleen macrophages supported mild virus production (10(4)-fold less than neuroblastoma), with formation of typical virions. Bone marrow-derived macrophages (BMM) were most efficient to capture virus, but new virus production was not detected. Virus-induced cell death was significantly stronger in BMM, which might have eliminated BMM with productive infection. Still, viral RNA remained detectable in the remaining BMM for at least 4 weeks. Injection of in vitro-infected Mf4/4 in the nose or brain proved efficient to propagate infection in mice, even when cells were pre-incubated with neutralizing antibodies. Surprisingly, injection of ex-vivo-infected BMM in the brain also led to lethal infection in 8 out of 12 mice. Injection of infected Mf4/4 in the muscle mostly favoured a protective antibody response. Despite that macrophages are less fit to support virus production, they can still act as a source of infectious virus upon transfer in mice. This may be relevant for screening donor organs/cells, for which RT-PCR should be preferred over the traditional antigen or virus isolation assays.

%B Microbes Infect %V 15 %P 115-25 %8 2013 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/23159243?dopt=Abstract %R 10.1016/j.micinf.2012.10.018 %0 Journal Article %J PLoS One %D 2013 %T Proteomic characterization of murid herpesvirus 4 extracellular virions. %A Vidick, Sarah %A Leroy, Baptiste %A Palmeira, Leonor %A Machiels, Bénédicte %A Jan Mast %A François, Sylvie %A Wattiez, Ruddy %A Vanderplasschen, Alain %A Gillet, Laurent %K Animals %K Capsid %K Cell Line %K Cricetinae %K Extracellular Space %K Glycosylation %K Mass Spectrometry %K Proteomics %K Rhadinovirus %K Viral Proteins %K Virion %X

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

%B PLoS One %V 8 %P e83842 %8 2013 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/24386290?dopt=Abstract %R 10.1371/journal.pone.0083842 %0 Journal Article %J PLoS ONE %D 2013 %T Stability of Staphylococcus aureus Phage ISP after Freeze-Drying (Lyophilization) %A Merabishvili, Maia %A Vervaet, Chris %A Pirnay, Jean-Paul %A De Vos, Daniel %A Verbeken, Gilbert %A Jan Mast %A Chanishvili, Nino %A Vaneechoutte, Mario %E Lin Baochuan %K Bacteriophage %K stability %K Staphylococcus aureus %B PLoS ONE %V 8199143038972110100728167116741661024203583810547741612519243 %8 Feb-07-2013 %G eng %N 7 %R 10.1371/journal.pone.006879710.1371/journal.pone.0068797.g00110.1371/journal.pone.0068797.g00210.1371/journal.pone.0068797.g00310.1371/journal.pone.0068797.g00410.1371/journal.pone.0068797.t001 %0 Journal Article %J ACS Nano %D 2012 %T Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure. %A van der Zande, Meike %A Vandebriel, Rob J %A Van Doren, Elke %A Kramer, Evelien %A Herrera Rivera, Zahira %A Serrano-Rojero, Cecilia S %A Gremmer, Eric R %A Jan Mast %A Peters, Ruud J B %A Hollman, Peter C H %A Hendriksen, Peter J M %A Marvin, Hans J P %A Peijnenburg, Ad A C M %A Bouwmeester, Hans %K Administration, Oral %K Animals %K Ions %K Male %K Metabolic Clearance Rate %K Metal Nanoparticles %K Organ Specificity %K rats %K Rats, Sprague-Dawley %K Silver %K Tissue Distribution %X

We report the results of a 28-day oral exposure study in rats, exposed to <20 nm noncoated, or <15 nm PVP-coated silver nanoparticles ([Ag] = 90 mg/kg body weight (bw)), or AgNO(3) ([Ag] = 9 mg/kg bw), or carrier solution only. Dissection was performed at day 29, and after a wash-out period of 1 or 8 weeks. Silver was present in all examined organs with the highest levels in the liver and spleen for all silver treatments. Silver concentrations in the organs were highly correlated to the amount of Ag(+) in the silver nanoparticle suspension, indicating that mainly Ag(+), and to a much lesser extent silver nanoparticles, passed the intestines in the silver nanoparticle exposed rats. In all groups silver was cleared from most organs after 8 weeks postdosing, but remarkably not from the brain and testis. Using single particle inductively coupled plasma mass spectrometry, silver nanoparticles were detected in silver nanoparticle exposed rats, but, remarkably also in AgNO(3) exposed rats, hereby demonstrating the formation of nanoparticles from Ag(+)in vivo that are probably composed of silver salts. Biochemical markers and antibody levels in blood, lymphocyte proliferation and cytokine release, and NK-cell activity did not reveal hepatotoxicity or immunotoxicity of the silver exposure. In conclusion, oral exposure to silver nanoparticles appears to be very similar to exposure to silver salts. However, the consequences of in vivo formation of silver nanoparticles, and of the long retention of silver in brain and testis should be considered in a risk assessment of silver nanoparticles.

%B ACS Nano %V 6 %P 7427-42 %8 2012 Aug 28 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/22857815?dopt=Abstract %R 10.1021/nn302649p %0 Journal Article %J Vet Res %D 2012 %T Feeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa. %A Fournier, Guillaume %A Boutier, Maxime %A Stalin Raj, Victor %A Jan Mast %A Parmentier, Eric %A Vanderwalle, Pierre %A Peeters, Dominique %A Lieffrig, François %A Farnir, Frédéric %A Gillet, Laurent %A Vanderplasschen, Alain %K Animals %K DNA Virus Infections %K DNA Viruses %K Fish Diseases %K Luminescent Measurements %K Mucous Membrane %K Pharynx %K SKIN %X

Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection.

%B Vet Res %V 43 %P 6 %8 2012 Jan 25 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/22276598?dopt=Abstract %R 10.1186/1297-9716-43-6 %0 Journal Article %J J Virol %D 2012 %T Proteomic characterization of bovine herpesvirus 4 extracellular virions. %A Lété, Céline %A Palmeira, Leonor %A Leroy, Baptiste %A Jan Mast %A Machiels, Bénédicte %A Wattiez, Ruddy %A Vanderplasschen, Alain %A Gillet, Laurent %K Animals %K Cattle %K Cell Line %K Glycoproteins %K Herpesvirus 4, Bovine %K Mass Spectrometry %K Proteome %K Viral Proteins %K Virion %X

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host's antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi's sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.

%B J Virol %V 86 %8 2012 Nov %G eng %N 21 %R 10.1128/JVI.00456-12 %0 Journal Article %J J Nanobiotechnology %D 2012 %T Quantitative characterization of agglomerates and aggregates of pyrogenic and precipitated amorphous silica nanomaterials by transmission electron microscopy. %A Pieter-Jan De Temmerman %A Van Doren, Elke %A Eveline Verleysen %A Yves Van der Stede %A Michel Abi Daoud Francisco %A Jan Mast %K Chemical Precipitation %K Microscopy, Electron, Transmission %K Nanostructures %K Particle Size %K Principal Component Analysis %K Silicon Dioxide %K SOFTWARE %K Sonication %K Temperature %X

BACKGROUND: The interaction of a nanomaterial (NM) with a biological system depends not only on the size of its primary particles but also on the size, shape and surface topology of its aggregates and agglomerates. A method based on transmission electron microscopy (TEM), to visualize the NM and on image analysis, to measure detected features quantitatively, was assessed for its capacity to characterize the aggregates and agglomerates of precipitated and pyrogenic synthetic amorphous silicon dioxide (SAS), or silica, NM.

RESULTS: Bright field (BF) TEM combined with systematic random imaging and semi-automatic image analysis allows measuring the properties of SAS NM quantitatively. Automation allows measuring multiple and arithmetically complex parameters simultaneously on high numbers of detected particles. This reduces operator-induced bias and assures a statistically relevant number of measurements, avoiding the tedious repetitive task of manual measurements. Access to multiple parameters further allows selecting the optimal parameter in function of a specific purpose.Using principle component analysis (PCA), twenty-three measured parameters were classified into three classes containing measures for size, shape and surface topology of the NM.

CONCLUSION: The presented method allows a detailed quantitative characterization of NM, like dispersions of precipitated and pyrogenic SAS based on the number-based distributions of their mean diameter, sphericity and shape factor.

%B J Nanobiotechnology %V 10 %P 24 %8 2012 Jun 18 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/22709926?dopt=Abstract %R 10.1186/1477-3155-10-24 %0 Journal Article %J PLoS One %D 2012 %T Selection and characterization of a candidate therapeutic bacteriophage that lyses the Escherichia coli O104:H4 strain from the 2011 outbreak in Germany. %A Merabishvili, Maia %A De Vos, Daniel %A Verbeken, Gilbert %A Kropinski, Andrew M %A Vandenheuvel, Dieter %A Lavigne, Rob %A P Wattiau %A Jan Mast %A Ragimbeau, Catherine %A Mossong, Joel %A Scheres, Jacques %A Chanishvili, Nina %A Vaneechoutte, Mario %A Pirnay, Jean-Paul %K Bacteriolysis %K Bacteriophages %K Disease Outbreaks %K Escherichia coli Infections %K Germany %K Hemolytic-Uremic Syndrome %K Humans %K Open Reading Frames %K Shiga-Toxigenic Escherichia coli %X

In 2011, a novel strain of O104:H4 Escherichia coli caused a serious outbreak of foodborne hemolytic uremic syndrome and bloody diarrhea in Germany. Antibiotics were of questionable use and 54 deaths occurred. Candidate therapeutic bacteriophages that efficiently lyse the E. coli O104:H4 outbreak strain could be selected rather easily from a phage bank or isolated from the environment. It is argued that phage therapy should be more considered as a potential armament against the growing threat of (resistant) bacterial infections.

%B PLoS One %V 7 %P e52709 %8 2012 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/23285164?dopt=Abstract %R 10.1371/journal.pone.0052709 %0 Journal Article %J PLoS Pathog %D 2011 %T Antibody evasion by a gammaherpesvirus O-glycan shield. %A Machiels, Bénédicte %A Lété, Céline %A Guillaume, Antoine %A Jan Mast %A Stevenson, Philip G %A Vanderplasschen, Alain %A Gillet, Laurent %K Amino Acid Sequence %K Animals %K Epitopes %K Glycosylation %K Herpesvirus 4, Bovine %K Membrane Glycoproteins %K Molecular Sequence Data %K Neutralization Tests %K Rabbits %K Sequence Alignment %K Viral Envelope Proteins %K Virion %K Virus Replication %X

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog--gp180--contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target.

%B PLoS Pathog %V 7 %P e1002387 %8 2011 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/22114560?dopt=Abstract %R 10.1371/journal.ppat.1002387 %0 Journal Article %J J Virol %D 2011 %T The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects. %A Machiels, Bénédicte %A Lété, Céline %A de Fays, Katalin %A Jan Mast %A Dewals, Benjamin %A Stevenson, Philip G %A Vanderplasschen, Alain %A Gillet, Laurent %K Animals %K Cattle %K Cell Line %K Epithelial Cells %K Gene Deletion %K Herpesvirus 4, Bovine %K Molecular Weight %K Proteome %K Viral Envelope Proteins %K Viral Tropism %K Virion %K Virus Attachment %X

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.

%B J Virol %V 85 %P 1011-24 %8 2011 Jan %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/21068242?dopt=Abstract %R 10.1128/JVI.01092-10 %0 Journal Article %J J Nanobiotechnology %D 2011 %T Determination of the volume-specific surface area by using transmission electron tomography for characterization and definition of nanomaterials. %A Van Doren, Elke A F %A Pieter-Jan De Temmerman %A Michel Abi Daoud Francisco %A Jan Mast %K Electron Microscope Tomography %K Gold %K Image Processing, Computer-Assisted %K Imaging, Three-Dimensional %K Metal Nanoparticles %K Silicon Dioxide %K Surface Properties %K Suspensions %X

BACKGROUND: Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

RESULTS: The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m(2)/cm(3). The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

CONCLUSION: The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.

%B J Nanobiotechnology %V 9 %8 2011 May 11 %G eng %R 10.1186/1477-3155-9-17 %0 Journal Article %J Diabetes %D 2011 %T Heterozygous inactivation of the Na/Ca exchanger increases glucose-induced insulin release, β-cell proliferation, and mass. %A Nguidjoe, Evrard %A Sokolow, Sophie %A Bigabwa, Serge %A Pachera, Nathalie %A D'Amico, Eva %A Allagnat, Florent %A Vanderwinden, Jean-Marie %A Sener, Abdullah %A Manto, Mario %A Depreter, Marianne %A Jan Mast %A Joanny, Geraldine %A Montanya, Eduard %A Rahier, Jacques %A Cardozo, Alessandra K %A Eizirik, Décio L %A Schurmans, Stéphane %A Herchuelz, André %K Animals %K Blood Glucose %K Calcium %K Cell Proliferation %K Diabetes Mellitus, Experimental %K Female %K Glucose %K Insulin %K Insulin-Secreting Cells %K Islets of Langerhans Transplantation %K Male %K mice %K Sodium-Calcium Exchanger %X

OBJECTIVE: We have previously shown that overexpression of the Na-Ca exchanger (NCX1), a protein responsible for Ca(2+) extrusion from cells, increases β-cell programmed cell death (apoptosis) and reduces β-cell proliferation. To further characterize the role of NCX1 in β-cells under in vivo conditions, we developed and characterized mice deficient for NCX1.

RESEARCH DESIGN AND METHODS: Biologic and morphologic methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release, and point counting morphometry) were used to assess β-cell function in vitro. Blood glucose and insulin levels were measured to assess glucose metabolism and insulin sensitivity in vivo. Islets were transplanted under the kidney capsule to assess their performance to revert diabetes in alloxan-diabetic mice.

RESULTS: Heterozygous inactivation of Ncx1 in mice induced an increase in glucose-induced insulin release, with a major enhancement of its first and second phase. This was paralleled by an increase in β-cell proliferation and mass. The mutation also increased β-cell insulin content, proinsulin immunostaining, glucose-induced Ca(2+) uptake, and β-cell resistance to hypoxia. In addition, Ncx1(+/-) islets showed a two- to four-times higher rate of diabetes cure than Ncx1(+/+) islets when transplanted into diabetic animals.

CONCLUSIONS: Downregulation of the Na/Ca exchanger leads to an increase in β-cell function, proliferation, mass, and resistance to physiologic stress, namely to various changes in β-cell function that are opposite to the major abnormalities seen in type 2 diabetes. This provides a unique model for the prevention and treatment of β-cell dysfunction in type 2 diabetes and after islet transplantation.

%B Diabetes %V 60 %P 2076-85 %8 2011 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/21659499?dopt=Abstract %R 10.2337/db10-0924 %0 Journal Article %J J Clin Microbiol %D 2011 %T Original findings associated with two cases of bovine papular stomatitis. %A dal Pozzo, F %A Martinelle, L %A Gallina, L %A Jan Mast %A Sarradin, P %A Thiry, E %A Scagliarini, A %A Büttner, M %A Saegerman, C %K Amino Acid Sequence %K Animals %K Cattle %K Cattle Diseases %K Cluster Analysis %K DNA, Viral %K Female %K Parapoxvirus %K Phylogeny %K Poxviridae Infections %K Sequence Analysis, DNA %K Sequence Homology %K Stomatitis %K Viral Proteins %X

Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

%B J Clin Microbiol %V 49 %P 4397-400 %8 2011 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/21976753?dopt=Abstract %R 10.1128/JCM.05281-11 %0 Journal Article %J Vet Res %D 2011 %T Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells. %A Raj, Victor Stalin %A Fournier, Guillaume %A Rakus, Krzysztof %A Ronsmans, Maygane %A Ouyang, Ping %A Michel, Benjamin %A Delforges, Cédric %A Costes, Bérénice %A Farnir, Frédéric %A Leroy, Baptiste %A Wattiez, Ruddy %A Melard, Charles %A Jan Mast %A Lieffrig, François %A Vanderplasschen, Alain %K Animals %K Carps %K Cells, Cultured %K DNA Virus Infections %K DNA Viruses %K Epidermis %K Fish Diseases %K Immunity, Innate %K Luminescent Measurements %K Microscopy, Electron, Transmission %K Mucus %K Virus Attachment %X

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.

%B Vet Res %V 42 %P 92 %8 2011 Aug 04 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/21816061?dopt=Abstract %R 10.1186/1297-9716-42-92 %0 Journal Article %J Appl Environ Microbiol %D 2010 %T Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox. %A Vlaeminck, Siegfried E %A Terada, Akihiko %A Smets, Barth F %A De Clippeleir, Haydée %A Schaubroeck, Thomas %A Bolca, Selin %A Demeestere, Lien %A Jan Mast %A Boon, Nico %A Carballa, Marta %A Verstraete, Willy %K Aerobiosis %K Anaerobiosis %K Autotrophic Processes %K Bacteria, Aerobic %K Biodegradation, Environmental %K Bioreactors %K Catalysis %K DNA, Ribosomal %K Hydrogen-Ion Concentration %K In Situ Hybridization, Fluorescence %K Nitrates %K Nitrites %K Oxygen %K Phylogeny %K Quaternary Ammonium Compounds %K RNA, Ribosomal, 16S %K Temperature %K Waste Disposal, Fluid %K Water Microbiology %K Water Pollutants, Chemical %K Water Purification %X

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

%B Appl Environ Microbiol %V 76 %P 900-9 %8 2010 Feb %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/19948857?dopt=Abstract %R 10.1128/AEM.02337-09 %0 Journal Article %J Emerg Infect Dis %D 2010 %T Brucella ceti infection in harbor porpoise (Phocoena phocoena). %A Jauniaux, Thierry P %A Brenez, Cecile %A David Fretin %A Godfroid, Jacques %A Haelters, Jan %A Jacques, Thierry %A Kerckhof, Francis %A Jan Mast %A Sarlet, Michael %A Coignoul, Freddy L %K Animals %K Belgium %K Brucella %K Brucellosis %K Fatal Outcome %K Female %K Phocoena %X

We describe Brucella sp. infection and associated lesions in a harbor porpoise (Phocoena phocoena) found on the coast of Belgium. The infection was diagnosed by immunohistochemistry, transmission electron microscopy, and bacteriology, and the organism was identified as B. ceti. The infection's location in the porpoise raises questions of abortion and zoonotic risks.

%B Emerg Infect Dis %V 16 %P 1966-8 %8 2010 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/21122233?dopt=Abstract %R 10.3201/eid1612.101008 %0 Journal Article %J Environ Sci Technol %D 2010 %T Concomitant microbial generation of palladium nanoparticles and hydrogen to immobilize chromate. %A Chidambaram, Dev %A Hennebel, Tom %A Taghavi, Safiyh %A Jan Mast %A Boon, Nico %A Verstraete, Willy %A van der Lelie, Daniel %A Fitts, Jeffrey P %K Biocatalysis %K Chromates %K Clostridium %K Hydrogen %K Metal Nanoparticles %K Microscopy, Electron, Scanning %K Microscopy, Electron, Transmission %K Oxidation-Reduction %K Palladium %X

The catalytic properties of various metal nanoparticles have led to their use in environmental remediation. Our aim is to develop and apply an efficient bioremediation method based on in situ biosynthesis of bio-Pd nanoparticles and hydrogen. C. pasteurianum BC1 was used to reduce Pd(II) ions to form Pd nanoparticles (bio-Pd) that primarily precipitated on the cell wall and in the cytoplasm. C. pasteurianum BC1 cells, loaded with bio-Pd nanoparticle in the presence of glucose, were subsequently used to fermentatively produce hydrogen and to effectively catalyze the removal of soluble Cr(VI) via reductive transformation to insoluble Cr(III) species. Batch and aquifer microcosm experiments using C. pasteurianum BC1 cells loaded with bio-Pd showed efficient reductive Cr(VI) removal, while in control experiments with killed or viable but Pd-free bacterial cultures no reductive Cr(VI) removal was observed. Our results suggest a novel process where the in situ microbial production of hydrogen is directly coupled to the catalytic bio-Pd mediated reduction of chromate. This process offers significant advantages over the current groundwater treatment technologies that rely on introducing preformed catalytic nanoparticles into groundwater treatment zones and the costly addition of molecular hydrogen to above ground pump and treat systems.

%B Environ Sci Technol %V 44 %8 2010 Oct 01 %G eng %N 19 %R 10.1021/es101559r %0 Journal Article %J J Gen Virol %D 2010 %T The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions. %A Michel, B %A Leroy, B %A V Stalin Raj %A Lieffrig, F %A Jan Mast %A Wattiez, R %A Vanderplasschen, A F %A Costes, B %K Animals %K Carps %K Cells, Cultured %K DNA Virus Infections %K Fish Diseases %K Genome, Viral %K Host-Pathogen Interactions %K Molecular Sequence Data %K Viral Structural Proteins %K Virion %K Viruses %X

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

%B J Gen Virol %V 91 %P 452-62 %8 2010 Feb %G eng %N Pt 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/19846671?dopt=Abstract %R 10.1099/vir.0.015198-0 %0 Journal Article %J Food Microbiol %D 2010 %T Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment. %A Garcia-Gonzalez, L %A Geeraerd, A H %A Jan Mast %A Briers, Y %A Elst, K %A Van Ginneken, L %A Van Impe, J F %A Devlieghere, F %K Carbon Dioxide %K Cell Membrane Permeability %K Coloring Agents %K Escherichia coli %K Hydrostatic Pressure %K Listeria monocytogenes %K Microbial Viability %K Propidium %K Saccharomyces cerevisiae %K Spectrometry, Fluorescence %K Temperature %K Time Factors %X

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35-45 degrees C), pressure (10.5-21.0 MPa) and treatment time (0-60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.

%B Food Microbiol %V 27 %8 2010 Jun %G eng %N 4 %R 10.1016/j.fm.2009.12.004 %0 Journal Article %J Vet Res %D 2009 %T Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity. %A Gillet, Laurent %A Schroeder, Hélène %A Jan Mast %A Thirion, Muriel %A Renauld, Jean-Christophe %A Dewals, Benjamin %A Vanderplasschen, Alain %K Animals %K Antibodies, Monoclonal %K Cats %K Cattle %K Cell Adhesion %K Cell Line %K Complement Inactivator Proteins %K Complement System Proteins %K Dogs %K Gene Expression Regulation %K Ixodes %K Rabbits %K Saliva %X

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

%B Vet Res %V 40 %P 51 %8 2009 Sep-Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/19531344?dopt=Abstract %R 10.1051/vetres/2009034 %0 Journal Article %J Diagn Pathol %D 2009 %T Electron tomography of negatively stained complex viruses: application in their diagnosis. %A Jan Mast %A Demeestere, Lien %K eletron tomography %K negative staining %K paramyxovirus %K Parapoxvirus %X

BACKGROUND: Electron tomographic analysis can be combined with the simple and rapid negative staining technique used in electron microscopy based virus diagnosis.

METHODS: Standard negative staining of representative examples of parapoxviruses and paramyxoviruses was combined with electron tomographic analysis.

RESULTS: Digital sectioning of reconstructions of these viruses at a selected height demonstrated the viral ultrastructure in detail, including the characteristic diagnostic features like the surface threads on C-particles of a parapoxvirus and individual glycoproteins and the internal nucleoprotein strand of Newcastle disease virus. For both viruses, deformation and flattening were observed.

CONCLUSION: The combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents.

%B Diagn Pathol %V 4 %8 2009 Feb 10 %G eng %R 10.1186/1746-1596-4-5 %0 Journal Article %J Vet Microbiol %D 2009 %T Epidemiological study of bovine norovirus infection by RT-PCR and a VLP-based antibody ELISA. %A Mauroy, Axel %A Scipioni, Alexandra %A Elisabeth Mathijs %A Saegerman, Claude %A Jan Mast %A Bridger, Janice C %A Ziant, Dominique %A Thys, Christine %A Thiry, Etienne %K Animals %K Antibodies, Viral %K Belgium %K Caliciviridae Infections %K Cattle %K Cattle Diseases %K Enzyme-Linked Immunosorbent Assay %K Feces %K Gastroenteritis %K Norovirus %K Phylogeny %K Reverse Transcriptase Polymerase Chain Reaction %K Seroepidemiologic Studies %X

Noroviruses, belonging to the family Caliciviridae, have been identified in human beings and in several animal species including cattle. The distribution of bovine norovirus infections was investigated by both RT-PCR to detect norovirus genomes and a virus-like particles-based ELISA to detect genotype 2 bovine norovirus antibodies. During a 1-year systematic study, a virus prevalence of 7.5% (CI 95%: [3.7; 13.4%]) (10 out of 133 samples) was found in stool samples from diarrhoeic calves screened by RT-PCR. Nucleotide sequencing performed on the polymerase region classified all the norovirus amplicons in the bovine norovirus genotype 2. Rather surprisingly, some rotavirus sequences were also detected. On the basis of the polymerase region, genotype 1 bovine norovirus was not identified. Other enteropathogens were found in all samples. By ELISA, a genotype 2 seroprevalence of 93.2% (CI 95%: [90.4; 95.3%]) was found from calves and adult cattle. Antibody levels against genotype 2 bovine noroviruses rose in the first 6 months of life and were maintained in adults. Together the results of virus prevalence and seroprevalence studies suggest that bovine norovirus infection occurs early in life and that re-infection with serologically related bovine noroviruses strains could occur in adult cattle as reported for rotaviruses. The antibody rise against genotype 2 bovine noroviruses in the adult cattle also suggests a short lived and/or strain specific immunity as already shown in human noroviruses. Genotype 2 bovine noroviruses are endemic in the region investigated.

%B Vet Microbiol %V 137 %P 243-51 %8 2009 Jun 12 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19232845?dopt=Abstract %R 10.1016/j.vetmic.2009.01.031 %0 Journal Article %J Appl Microbiol Biotechnol %D 2009 %T Lactic acid bacteria as reducing and capping agent for the fast and efficient production of silver nanoparticles. %A Sintubin, Liesje %A De Windt, Wim %A Dick, Jan %A Jan Mast %A van der Ha, David %A Verstraete, Willy %A Boon, Nico %K Gram-Negative Bacteria %K Gram-Positive Bacteria %K Lactic Acid %K Microscopy, Electron, Transmission %K Nanoparticles %K Oxidation-Reduction %K Silver %X

There is a growing demand for silver-based biocides, including both ionic silver forms and metallic nanosilver. The use of metallic nanosilver, typically chemically produced, faces challenges including particle agglomeration, high costs, and upscaling difficulties . Additionally, there exists a need for the development of a more eco-friendly production of nanosilver. In this study, Gram-positive and Gram-negative bacteria were utilized in the non-enzymatic production of silver nanoparticles via the interaction of silver ions and organic compounds present on the bacterial cell. Only lactic acid bacteria, Lactobacillus spp., Pediococcus pentosaceus, Enterococcus faecium, and Lactococcus garvieae, were able to reduce silver. The nanoparticles of the five best producing Lactobacillus spp. were examined more into detail with transmission electron microscopy. Particle localization inside the cell, the mean particle size, and size distribution were species dependent, with Lactobacillus fermentum having the smallest mean particle size of 11.2 nm, the most narrow size distribution, and most nanoparticles associated with the outside of the cells. Furthermore, influence of pH on the reduction process was investigated. With increasing pH, silver recovery increased as well as the reduction rate as indicated by UV-VIS analyses. This study demonstrated that Lactobacillus spp. can be used for a rapid and efficient production of silver nanoparticles.

%B Appl Microbiol Biotechnol %V 84 %P 741-9 %8 2009 Sep %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19488750?dopt=Abstract %R 10.1007/s00253-009-2032-6 %0 Journal Article %J J Virol %D 2009 %T The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin. %A Costes, B %A V Stalin Raj %A Michel, B %A Fournier, G %A Thirion, M %A Gillet, L %A Jan Mast %A Lieffrig, F %A Bremont, M %A Vanderplasschen, A %K Animals %K Carps %K Fish Diseases %K Genes, Reporter %K Herpesviridae %K Herpesviridae Infections %K Luminescent Proteins %K SKIN %K Whole Body Imaging %X

Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed "U-tube," to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

%B J Virol %V 83 %P 2819-30 %8 2009 Apr %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/19153228?dopt=Abstract %R 10.1128/JVI.02305-08 %0 Journal Article %J Trop Anim Health Prod %D 2009 %T Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe. %A Madoroba, Evelyn %A Van Driessche, Edilbert %A De Greve, Henri %A Jan Mast %A Ncube, Ignatious %A Read, John %A Beeckmans, Sonia %K Agglutination Tests %K Animals %K DNA Primers %K Enterotoxins %K Escherichia coli %K Escherichia coli Infections %K Fimbriae, Bacterial %K Genes, Bacterial %K Genotype %K Microscopy, Electron, Transmission %K polymerase chain reaction %K prevalence %K Sus scrofa %K Swine %K Swine Diseases %K Zimbabwe %X

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.

%B Trop Anim Health Prod %V 41 %P 1539-47 %8 2009 Oct %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/19347597?dopt=Abstract %R 10.1007/s11250-009-9345-4 %0 Journal Article %J PLoS One %D 2009 %T Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. %A Merabishvili, Maya %A Pirnay, Jean-Paul %A Verbeken, Gilbert %A Chanishvili, Nina %A Tediashvili, Marina %A Lashkhi, Nino %A Glonti, Thea %A Krylov, Victor %A Jan Mast %A Van Parys, Luc %A Lavigne, Rob %A Volckaert, Guido %A Wesley Mattheus %A Verween, Gunther %A De Corte, Peter %A Rose, Thomas %A Jennes, Serge %A Zizi, Martin %A De Vos, Daniel %A Vaneechoutte, Mario %K Bacteriophages %K Burns %K Clinical Trials as Topic %K Genome, Viral %K Humans %K Proteome %K Pseudomonas aeruginosa %K Pseudomonas Infections %K Staphylococcal Infections %K Staphylococcus aureus %K wound infection %X

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.

%B PLoS One %V 4 %8 2009 %G eng %N 3 %R 10.1371/journal.pone.0004944 %0 Journal Article %J Microb Ecol %D 2008 %T Evidence for the presence of Legionella bacteriophages in environmental water samples. %A Lammertyn, Elke %A Johan Vande Voorde %A Meyen, Eef %A Maes, Liesbeth %A Jan Mast %A Anné, Jozef %K Bacteriophages %K Culture Media %K DNA, Viral %K Fresh Water %K Humans %K Legionella %K Legionella pneumophila %K Lysogeny %K Myoviridae %K Species Specificity %X

The existence and preliminary characterization of bacteriophages active against the Gram-negative human pathogen Legionella pneumophila, the causative agent of a very severe form of pneumonia, are reported. Four phages belonging to the family of the Myoviridae were isolated from various fresh water environments, and preliminary characterization showed that these crude preparations infect exclusively bacteria belonging to the genus Legionella. Standard phage amplification, purification, and characterization procedures were, however, not efficiently applicable making more research into these novel phages and their mechanism of infection necessary. The existence of Legionella bacteriophages is very promising for future applications such as the development of novel molecular tools, the design of new detection and typing methods, and the bioremediation of this environmental pathogen.

%B Microb Ecol %V 56 %8 2008 Jul %G eng %N 1 %R 10.1007/s00248-007-9325-z %0 Journal Article %J Int J Syst Evol Microbiol %D 2008 %T Helicobacter baculiformis sp. nov., isolated from feline stomach mucosa. %A Baele, M %A Decostere, A %A Vandamme, P %A Van den Bulck, K %A Gruntar, I %A Mehle, J %A Jan Mast %A Ducatelle, R %A Haesebrouck, F %K Animals %K Bacterial Typing Techniques %K Cat Diseases %K Cats %K Chaperonin 60 %K DNA, Bacterial %K Gastric Mucosa %K Genes, rRNA %K Helicobacter %K Helicobacter Infections %K Molecular Sequence Data %K Phenotype %K Phylogeny %K RNA, Ribosomal, 16S %K Sequence Analysis, DNA %K Species Specificity %K urease %X

A Gram-negative, microaerophilic slender rod, measuring approximately 10 mum long and approximately 1 microm wide, isolated from the gastric mucosa of a cat and designated strain M50(T), was subjected to a polyphasic taxonomic study. Despite its apparent lack of helical coils, the organism showed a corkscrew-like motion by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril coiled around the body. Strain M50(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed about 98 to 99 % sequence similarity to Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, Helicobacter cynogastricus and 'Candidatus Helicobacter heilmannii', five highly related species previously detected in the feline or canine gastric mucosa. Protein profiling of strain M50(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin. Additionally, the urease and HSP60 gene sequences of strain M50(T) were different from those of H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus and 'Ca. H. heilmannii'. It is thus proposed that strain M50(T) (=LMG 23839(T)=CCUG 53816(T)) represents a novel species within this genus, for which the name Helicobacter baculiformis sp. nov. is proposed.

%B Int J Syst Evol Microbiol %V 58 %P 357-64 %8 2008 Feb %G eng %N Pt 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/18218931?dopt=Abstract %R 10.1099/ijs.0.65152-0 %0 Journal Article %J Int J Syst Evol Microbiol %D 2008 %T Isolation and characterization of Helicobacter suis sp. nov. from pig stomachs. %A Baele, M %A Decostere, A %A Vandamme, P %A Ceelen, L %A Hellemans, A %A Jan Mast %A Chiers, K %A Ducatelle, R %A Haesebrouck, F %K Animals %K Bacterial Proteins %K Bacterial Typing Techniques %K Bacteriological Techniques %K Chaperonin 60 %K Culture Media %K DNA, Bacterial %K Electrophoresis, Polyacrylamide Gel %K Female %K Gastric Mucosa %K Gastritis %K Helicobacter %K Helicobacter Infections %K Humans %K Molecular Sequence Data %K RNA, Ribosomal, 16S %K RNA, Ribosomal, 23S %K Sequence Analysis, DNA %K Species Specificity %K Swine %K Swine Diseases %K urease %X

A new cultivation method was successfully applied for the in vitro isolation of a hitherto uncultured spiral Helicobacter species associated with ulceration of the non-glandular stomach and gastritis in pigs and formerly described as 'Candidatus Helicobacter suis'. Three isolates, HS1(T), HS2 and HS3, were subcultured from the stomach mucosa of three pigs after slaughter and were analysed using a polyphasic taxonomic approach. The novel isolates grew on biphasic culture plates or very moist agar bases in microaerobic conditions and exhibited urease, oxidase and catalase activities. Sequencing of the 16S rRNA gene, the 23S rRNA gene, the partial hsp60 gene and partial ureAB genes confirmed that the strains present in the gastric mucosa of pigs constituted a separate taxon, corresponding to 'Helicobacter heilmannii' type 1 strains as detected in the gastric mucosa of humans and other primates. For all genes sequenced, the highest sequence similarities were obtained with Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, Helicobacter species isolated from the gastric mucosa of dogs and cats, which have also been detected in the human gastric mucosa and which are commonly referred to as 'Helicobacter heilmannii' type 2. SDS-PAGE of whole-cell proteins of strains HS1(T), HS2 and HS3 differentiated them from other Helicobacter species of gastric origin. The results of the polyphasic taxonomic analysis confirmed that the novel isolates constitute a novel taxon corresponding to 'Helicobacter heilmannii' type 1 strains from humans and to 'Candidatus H. suis' from pigs. The name Helicobacter suis sp. nov. is proposed for the novel isolates with the type strain HS1(T) (=LMG 23995(T)=DSM 19735(T)).

%B Int J Syst Evol Microbiol %V 58 %P 1350-8 %8 2008 Jun %G eng %N Pt 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/18523177?dopt=Abstract %R 10.1099/ijs.0.65133-0 %0 Journal Article %J Vaccine %D 2008 %T The polymeric stability of the Escherichia coli F4 (K88) fimbriae enhances its mucosal immunogenicity following oral immunization. %A Verdonck, Frank %A Joensuu, Jussi Joonas %A Stuyven, Edith %A De Meyer, Julie %A Muilu, Mikko %A Pirhonen, Minna %A Goddeeris, Bruno Maria %A Jan Mast %A Niklander-Teeri, Viola %A Cox, Eric %K Adhesins, Escherichia coli %K Administration, Oral %K Animals %K Animals, Suckling %K Enterotoxigenic Escherichia coli %K Escherichia coli Infections %K Escherichia coli Vaccines %K Fimbriae, Bacterial %K Immunity, Mucosal %K Immunization %K Intestinal Mucosa %K Mutation %K Polymers %K Swine %K Swine Diseases %X

Only a few vaccines are commercially available against intestinal infections since the induction of a protective intestinal immune response is difficult to achieve. For instance, oral administration of most proteins results in oral tolerance instead of an antigen-specific immune response. We have shown before that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. In the present study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. These data suggest that the mucosal immunogenicity of soluble virulence factors can be increased by the construction of stable polymeric structures and therefore help in the development of effective mucosal vaccines.

%B Vaccine %V 26 %P 5728-35 %8 2008 Oct 23 %G eng %N 45 %1 http://www.ncbi.nlm.nih.gov/pubmed/18762221?dopt=Abstract %R 10.1016/j.vaccine.2008.08.017 %0 Journal Article %J Int J Syst Evol Microbiol %D 2007 %T Helicobacter equorum sp. nov., a urease-negative Helicobacter species isolated from horse faeces. %A Moyaert, H %A Decostere, A %A Vandamme, P %A Debruyne, L %A Jan Mast %A Baele, M %A Ceelen, L %A Ducatelle, R %A Haesebrouck, F %K Aerobiosis %K Animals %K Anti-Bacterial Agents %K Bacterial Proteins %K Chaperonin 60 %K DNA, Bacterial %K DNA, Ribosomal %K Electrophoresis, Polyacrylamide Gel %K Enzymes %K Feces %K Flagella %K Genes, rRNA %K Helicobacter %K Horses %K Molecular Sequence Data %K movement %K Nitrates %K Nitrites %K Phylogeny %K Proteome %K RNA, Bacterial %K RNA, Ribosomal, 16S %K RNA, Ribosomal, 23S %K Sequence Analysis, DNA %K Sequence Homology, Nucleic Acid %K Temperature %X

Gram-negative, curved, motile bacteria (strains EqF1T and EqF2) were isolated from faecal samples from two clinically healthy horses. Both strains possessed a single, monopolar, sheathed flagellum and were urease-negative. The novel strains grew at 37 degrees C under microaerobic conditions and were positive for oxidase, catalase and alkaline phosphatase activities. The isolates reduced nitrate to nitrite, but gamma-glutamyl transpeptidase activity was not detected. The novel isolates did not grow at 42 degrees C or on media containing 1 % glycine. They were resistant to cephalotin and nalidixic acid and susceptible to metronidazole. Analysis of the 16S and 23S rRNA gene sequences of the two novel strains identified them as representing a single species within the genus Helicobacter. In terms of 16S rRNA gene sequence similarity, Helicobacter pullorum and Helicobacter canadensis were the most closely related species (98 % similarity). 23S rRNA gene sequence analysis also classified strains EqF1T and EqF2 within the enterohepatic division of the genus Helicobacter, but only 94 % similarity was detected with H. pullorum and H. canadensis, which are helicobacters with unsheathed flagella. The most closely related species in terms of 23S rRNA gene sequence similarity was Helicobacter canis (95 %). Numerical analysis of whole-cell protein extracts by SDS-PAGE was performed and the novel isolates were clearly differentiated from H. pullorum, H. canadensis, H. canis and other species of the genus Helicobacter. This finding was also confirmed by sequence analysis of the hsp60 gene. On the basis of these genetic, biochemical and protein data, the isolates are classified as representing a novel species, for which the name Helicobacter equorum sp. nov. is proposed (type strain EqF1T=LMG 23362T=CCUG 52199T).

%B Int J Syst Evol Microbiol %V 57 %P 213-8 %8 2007 Feb %G eng %N Pt 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/17267952?dopt=Abstract %R 10.1099/ijs.0.64279-0 %0 Journal Article %J Eur J Pharm Sci %D 2007 %T An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells. %A des Rieux, Anne %A Fievez, Virginie %A Théate, Ivan %A Jan Mast %A Préat, Véronique %A Schneider, Yves-Jacques %K Amiloride %K B-Lymphocytes %K Caco-2 Cells %K Cell Differentiation %K Coculture Techniques %K Drug Carriers %K Epithelial Cells %K Humans %K immunohistochemistry %K Intestinal Mucosa %K Microscopy, Confocal %K Microscopy, Electron, Scanning %K Microscopy, Electron, Transmission %K Nanoparticles %K Nystatin %K Peyer's Patches %K Pinocytosis %K Polystyrenes %K Reproducibility of Results %K Temperature %X

An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.

%B Eur J Pharm Sci %V 30 %P 380-91 %8 2007 Apr %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/17291730?dopt=Abstract %R 10.1016/j.ejps.2006.12.006 %0 Journal Article %J Microbiology %D 2006 %T AggA is required for aggregation and increased biofilm formation of a hyper-aggregating mutant of Shewanella oneidensis MR-1. %A De Windt, Wim %A Gao, Haichun %A Krömer, Wolfgang %A Van Damme, Petra %A Dick, Jan %A Jan Mast %A Boon, Nico %A Zhou, Jizhong %A Verstraete, Willy %K Bacterial Adhesion %K Bacterial Proteins %K Biofilms %K Gene Expression Regulation, Bacterial %K Mutation %K Oligonucleotide Array Sequence Analysis %K Proteomics %K Shewanella %X

Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type.

%B Microbiology %V 152 %P 721-9 %8 2006 Mar %G eng %N Pt 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/16514152?dopt=Abstract %R 10.1099/mic.0.28204-0 %0 Journal Article %J Antonie Van Leeuwenhoek %D 2006 %T Biological control of the size and reactivity of catalytic Pd(0) produced by Shewanella oneidensis. %A De Windt, Wim %A Boon, Nico %A Van den Bulcke, Jan %A Rubberecht, Leen %A Prata, Filipa %A Jan Mast %A Hennebel, Tom %A Verstraete, Willy %K Biodegradation, Environmental %K Biomass %K Catalysis %K Crystallization %K Environmental Pollutants %K Hydrophobic and Hydrophilic Interactions %K Microbial Viability %K Nanoparticles %K Palladium %K Polychlorinated Biphenyls %K Shewanella %X

The interaction between Shewanella oneidensis MR-1 and the soluble metal Pd(II) during the reductive precipitation of Pd(0) determined the size and properties of the precipitated Pd(0) nanoparticles. Assessment of cell viability indicated that the bioreduction of Pd(II) was a detoxification mechanism depending on the Pd(II) concentration and on the presence and properties of the electron donor. The addition of H(2) in the headspace allowed S. oneidensis to resist the toxic effects of Pd(II). Interestingly, 25 mM formate was a less effective electron donor for bioreductive detoxification of Pd(II), since there was a 2 log reduction of culturable cells and a 20% decrease of viable cells within 60 min, followed by a slow recovery. When the ratio of Pd:cell dry weight (CDW) was below 5:2 at a concentration of 50 mg l(-1) Pd(II), most of the cells remained viable. These viable cells precipitated Pd(0) crystals over a relatively larger bacterial surface area and had a particle area that was up to 100 times smaller when compared to Pd(0) crystals formed on non-viable biomass (Pd:CDW ratio of 5:2). The relatively large and densely covering Pd(0) crystals on non-viable biomass exhibited high catalytic reactivity towards hydrophobic molecules such as polychlorinated biphenyls, while the smaller and more dispersed nanocrystals on a viable bacterial carrier exhibited high catalytic reactivity towards the reductive degradation of the anionic pollutant perchlorate.

%B Antonie Van Leeuwenhoek %V 90 %8 2006 Nov %G eng %N 4 %R 10.1007/s10482-006-9088-4 %0 Journal Article %J Microbes Infect %D 2006 %T Felid herpesvirus 1 glycoprotein G is a structural protein that mediates the binding of chemokines on the viral envelope. %A Costes, Bérénice %A Thirion, Muriel %A Dewals, Benjamin %A Jan Mast %A Ackermann, Mathias %A Markine-Goriaynoff, Nicolas %A Gillet, Laurent %A Vanderplasschen, Alain %K Animals %K Cats %K Cell Line %K Chemokines %K Chromosomes, Artificial, Bacterial %K Genome, Viral %K Glycoproteins %K Microscopy, Immunoelectron %K Protein Binding %K Recombination, Genetic %K Varicellovirus %K Viral Envelope Proteins %K Viral Structural Proteins %K Virion %X

Glycoprotein G (gG) orthologues have been described in several alphaherpesviruses. gG is expressed both as a membrane-anchored form on infected cells and as a secreted form. Recently, we reported that both forms of gG encoded by alphaherpesviruses infecting large herbivores and by Felid herpesvirus 1 (FeHV-1) bind with high affinity to a broad range of CXC, CC and C-chemokines. Based on the viral species, gG has been reported either as a structural or a non-structural protein. To date, the incorporation of FeHV-1 gG into virions has never been tested, nor the property of alphaherpesvirus structural gG to bind chemokines on the virion surface. In the present study, to address these questions, various FeHV-1 gG recombinant strains were produced using an original technique based on an infectious FeHV-1 BAC clone and restriction endonuclease mediated recombination. Using the recombinants produced, we were able to determine that FeHV-1 gG is a structural protein that acts as a chemokine-binding protein on the virion surface. In the light of these results, putative roles of gG in alphaherpesvirus infections are discussed, and an evolutionary scenario is proposed to explain the structural versus non-structural property of gG amongst alphaherpesviruses.

%B Microbes Infect %V 8 %P 2657-67 %8 2006 Sep %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/16962359?dopt=Abstract %R 10.1016/j.micinf.2006.07.014 %0 Journal Article %J J Bacteriol %D 2006 %T Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. %A Pieter-Jan Ceyssens %A Lavigne, Rob %A Wesley Mattheus %A Chibeu, Andrew %A Hertveldt, Kirsten %A Jan Mast %A Robben, Johan %A Volckaert, Guido %K Dna %K Electron %K Gene Order %K Mass Spectrometry %K Microscopy %K Molecular Sequence Data %K Nucleic Acid %K Podoviridae %K Pseudomonas aeruginosa %K Pseudomonas Phages %K Repetitive Sequences %K Sequence Analysis %K Sequence Homology %K Terminal Repeat Sequences %K Transmission %K Viral %K Viral Genes %K Viral Genome %K Viral Proteins %X

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.

%B J Bacteriol %V 188 %8 2006 Oct %G eng %N 19 %R 10.1128/JB.00831-06 %0 Journal Article %J Int J Syst Evol Microbiol %D 2006 %T Helicobacter cynogastricus sp. nov., isolated from the canine gastric mucosa. %A Van den Bulck, K %A Decostere, A %A Baele, M %A Vandamme, P %A Jan Mast %A Ducatelle, R %A Haesebrouck, F %K Animals %K Bacterial Proteins %K Catalase %K Cluster Analysis %K DNA, Bacterial %K DNA, Ribosomal %K Dog Diseases %K Dogs %K Electrophoresis, Polyacrylamide Gel %K Flagella %K Gastric Mucosa %K Genes, rRNA %K Helicobacter %K Helicobacter Infections %K Molecular Sequence Data %K movement %K Oxidoreductases %K Phylogeny %K Proteome %K RNA, Bacterial %K RNA, Ribosomal, 16S %K Sequence Analysis, DNA %K Sequence Homology, Nucleic Acid %K Temperature %K urease %X

A Gram-negative, microaerophilic helical rod, isolated from the gastric mucosa of a dog and designated strain JKM4(T), was subjected to a polyphasic taxonomic study. The tightly coiled organism, measuring 10-18 mum long and up to 1 mum wide, was motile by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril running along the external side of the helix. Strain JKM4(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 30 and 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed > 97 % similarity to Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, three species previously isolated from the canine gastric mucosa. Protein profiling of strain JKM4(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin and from Helicobacter canis. Additionally, the urease gene sequence of strain JKM4(T) was different from those of urease genes of H. felis, H. bizzozeronii, H. salomonis and "Candidatus Helicobacter heilmannii". It is thus proposed that strain JKM4(T) (=LMG 23188(T)) represents a novel species within this genus, Helicobacter cynogastricus sp. nov.

%B Int J Syst Evol Microbiol %V 56 %P 1559-64 %8 2006 Jul %G eng %N Pt 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/16825630?dopt=Abstract %R 10.1099/ijs.0.63860-0 %0 Journal Article %J Vet Rec %D 2006 %T Inclusion body disease in snakes: a review and description of three cases in boa constrictors in Belgium. %A Vancraeynest, D %A Pasmans, F %A Martel, A %A Chiers, K %A Meulemans, G %A Jan Mast %A Zwart, P %A Ducatelle, R %K Animals %K Arachnid Vectors %K Belgium %K Boidae %K Diagnosis, Differential %K Inclusion Bodies, Viral %K Microscopy, Electron, Transmission %K Mites %K Myeloid Cells %K prevalence %K Prognosis %K Retroviridae Infections %K Snakes %X

Inclusion body disease, a fatal disorder in Boidae, is reviewed, and three cases in boa constrictors, the first reported cases in Belgium, are described. The snakes showed nervous signs, and numerous eosinophilic intracytoplasmic inclusions, which are considered to be characteristic of the disease, were found in the liver and pancreas. The disease is suspected to be caused by a retrovirus, but transmission electron microscopic examinations of several tissues from one of the snakes did not reveal particles with a typical retroviral morphology.

%B Vet Rec %V 158 %P 757-60 %8 2006 Jun 03 %G eng %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/16751310?dopt=Abstract %0 Journal Article %J Vaccine %D 2006 %T Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response. %A Jan Mast %A Nanbru, Cécile %A Decaesstecker, Mireille %A Bénédicte Lambrecht %A Couvreur, Bernard %A Meulemans, Guy %A Thierry van den Berg %K Animals %K Antibodies, Viral %K Chick Embryo %K Chickens %K Epitopes %K Genes, Viral %K Hemagglutination Inhibition Tests %K HN Protein %K Immunoglobulin G %K Immunoglobulin M %K Molecular Sequence Data %K Mutation %K Newcastle Disease %K Newcastle disease virus %K Sequence Analysis, DNA %K Specific Pathogen-Free Organisms %K Vaccines, Attenuated %K Viral Fusion Proteins %K Viral Vaccines %X

To reduce the embryonic pathogenicity of Newcastle disease virus (NDV), escape mutants of the La Sota strain were produced with selected monoclonal antibodies. Immunoselection resulted in the elimination of an epitope by single amino acid substitution (F and HN molecule) or in a conformational change (HN molecule). The embryonic pathogenicity of these escape mutants was reduced and their dose was optimised for in ovo vaccination. Because antibody responses and protection of in ovo vaccinated chicks were similar to controls vaccinated at hatch with the La Sota strain, immunoselection appears a valuable technique to produce attenuated NDV strains, which are candidate in ovo vaccines.

%B Vaccine %V 24 %P 1756-65 %8 2006 Mar 10 %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/16343701?dopt=Abstract %R 10.1016/j.vaccine.2005.10.020 %0 Journal Article %J Int J Syst Evol Microbiol %D 2005 %T Arcobacter cibarius sp. nov., isolated from broiler carcasses. %A Houf, Kurt %A On, Stephen L W %A Coenye, Tom %A Jan Mast %A Van Hoof, Jan %A Vandamme, Peter %K Animals %K Arcobacter %K Base Composition %K Chickens %K DNA, Bacterial %K DNA, Ribosomal %K Gram-Negative Bacterial Infections %K Molecular Sequence Data %K Nucleic Acid Hybridization %K Phenotype %K Poultry Diseases %K RNA, Ribosomal, 16S %K Sequence Analysis, DNA %X

Twenty Gram-negative, rod-shaped, slightly curved, non-spore-forming bacteria that gave a negative result in Arcobacter species-specific PCR tests but that yielded an amplicon in an Arcobacter genus-specific PCR test were isolated from 13 unrelated broiler carcasses. Numerical analysis of the profiles obtained by SDS-PAGE of whole-cell proteins clustered all isolates in a single group distinct from the other Arcobacter species. DNA-DNA hybridization among four representative strains exhibited DNA binding values above 91 %. DNA-DNA hybridization with reference strains of the current four Arcobacter species revealed binding levels below 47 %. The G+C contents ranged between 26.8 and 27.3 mol%. Pairwise comparison of 16S rRNA gene sequences revealed the mean values for similarity to the type strain of Arcobacter cryaerophilus (97.5 %), Arcobacter butzleri (96.5 %), Arcobacter skirrowii (96.0 %) and Arcobacter nitrofigilis (95.0 %). The levels of similarity to Campylobacter and Helicobacter species were below 88 and 87 %, respectively. The isolates could be distinguished from other Arcobacter species by the following biochemical tests: catalase, oxidase and urease activities; reduction of nitrate; growth at 25 and 37 degrees C under aerobic conditions; growth on 2-4 % (w/v) NaCl media; and susceptibility to cephalothin. These data demonstrate that the 20 isolates represent a single novel Arcobacter species, for which the name Arcobacter cibarius sp. nov. is proposed, with LMG 21996(T) (=CCUG 48482(T)) as the type strain.

%B Int J Syst Evol Microbiol %V 55 %P 713-7 %8 2005 Mar %G eng %N Pt 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/15774649?dopt=Abstract %R 10.1099/ijs.0.63103-0 %0 Journal Article %J J Gen Virol %D 2005 %T Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31. %A Misinzo, G %A Meerts, P %A Bublot, M %A Jan Mast %A Weingartl, H M %A Nauwynck, H J %K Animals %K Cell Line %K Cell Membrane %K Circoviridae Infections %K Circovirus %K Clathrin %K Endocytosis %K Hydrogen-Ion Concentration %K Microscopy, Confocal %K Microscopy, Fluorescence %K Monocytes %K Recombination, Genetic %K Swine %K Virion %K Wasting Syndrome %X

Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47+/-5.0 % of cells. Internalization was continuous, with 70.5+/-9.7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75.5+/-7.0 % reduction) and latrunculin B (71.0+/-3.0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50.2+/-6.3 % reduction), hypertonic medium (56.4+/-5.7 % reduction), cytosol acidification (59.1+/-7.1 % reduction) and amantadine (52.6+/-6.7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47.0+/-7.9 % reduction) and chloroquine diphosphate (49.0+/-5.6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.

%B J Gen Virol %V 86 %P 2057-68 %8 2005 Jul %G eng %N Pt 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/15958685?dopt=Abstract %R 10.1099/vir.0.80652-0 %0 Journal Article %J Vet Pathol %D 2005 %T Ultrastructural changes of the tracheal epithelium after vaccination of day-old chickens with the La Sota strain of Newcastle disease virus. %A Jan Mast %A Nanbru, C %A Thierry van den Berg %A Meulemans, G %K Animals %K Antibodies, Viral %K Chickens %K Goblet Cells %K Immunoglobulin G %K Immunoglobulin M %K Newcastle Disease %K Newcastle disease virus %K Poultry Diseases %K Respiratory Mucosa %K Specific Pathogen-Free Organisms %K Trachea %K Vaccination %K Viral Vaccines %X

The progression of tracheal lesions induced by vaccination of day-old specific pathogen-free chicks with the La Sota strain of Newcastle disease virus (NDV) was examined by relating surface changes as observed by scanning electron microscopy with subcellular changes seen by transmission electron microscopy. NDV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. Activation of goblet cells peaked within 4 days postvaccination. Afterward, the activation levels gradually decreased. At the level of the ciliated cells, a marked increase in the proportion of nonciliated to ciliated cells and later an almost complete deciliation of the tracheal surface were observed because a simple squamous to cuboidal epithelium replaced the original pseudostratified epithelium. Fifteen days postvaccination, all epithelial damage was restored. Because the observed vaccination-induced lesions are detrimental to epithelial integrity and function as a barrier against invading microorganisms, they might explain at the ultrastructural level the secondary complications of vaccination with the La Sota strain against NDV.

%B Vet Pathol %V 42 %P 559-65 %8 2005 Sep %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/16145202?dopt=Abstract %R 10.1354/vp.42-5-559 %0 Journal Article %J Vet Microbiol %D 2005 %T Virulence-associated traits in avian Escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks. %A Dominique Vandekerchove %A Vandemaele, F %A Adriaensen, C %A Zaleska, M %A Hernalsteens, J-P %A De Baets, L %A Butaye, P %A Van Immerseel, F %A P Wattiau %A Laevens, H %A Jan Mast %A Goddeeris, B %A Pasmans, F %K Adhesins, Escherichia coli %K Animals %K Chickens %K Colicins %K Escherichia coli %K Escherichia coli Infections %K Female %K Gene Expression %K Genotype %K Iron %K Phenotype %K Poultry Diseases %K virulence %X

Colibacillosis appears to be of increasing importance in layer flocks. The aim of this study was to determine characteristics of avian pathogenic Escherichia coli associated with the occurrence of colibacillosis outbreaks at flock level. Forty E. coli strains originating from layers from healthy flocks ('control isolates'), consisting of 25 caecal and 15 extra-intestinal isolates, were compared with 40 strains isolated from layers originating from colibacillosis-affected flocks ('outbreak isolates'), consisting of 20 caecal and 20 extra-intestinal isolates. The examined characteristics were adhesins, invasivity in T84 cell culture, serum resistance, iron uptake, colicin production, and toxinogenicity. The following traits were significantly more often detected in the outbreak isolates than in the control isolates: tsh, iss, iucA, iutA, irp2, fyuA, iroC, cvaC, colicin and colicin V production. A comparison of the extra-intestinal outbreak isolates and the caecal control isolates yielded the same results as when the caecal isolates, extra-intestinal isolates and total number of isolates of the outbreak and the control group were compared. When comparing the caecal and extra-intestinal isolates within the control and within the outbreak group, no significant differences were detected. The O78 and O2 groups showed significant differences with other O-types and NT strains for prevalence of most of the same characteristics. The combination of type 1 fimbriae, tsh, serum resistance, iss, traT, iucA, fyuA, iroC and colicin or colicin V production was significantly more often present in extra-intestinal outbreak isolates than in extra-intestinal control isolates. Only the combination of serum resistance, fyuA and colicin production was present in all outbreak isolates, with a significantly lower prevalence in the control isolates. None of the characteristics or combinations examined were exclusive to the outbreak isolates.

%B Vet Microbiol %V 108 %P 75-87 %8 2005 Jun 15 %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/15917135?dopt=Abstract %R 10.1016/j.vetmic.2005.02.009 %0 Journal Article %J Avian Dis %D 2003 %T Association of Streptococcus gallolyticus strains of high and low virulence with the intestinal tract of pigeons. %A Kimpe, A %A Decostere, A %A Hermans, K %A Jan Mast %A Haesebrouck, E %K Animals %K Bacterial Adhesion %K Bird Diseases %K Columbidae %K immunohistochemistry %K Intestines %K Microscopy, Electron, Scanning %K Streptococcal Infections %K Streptococcus %K virulence %X

We investigated the ability of a high virulence (STR 357) and a low virulence (STR 598) strain of Streptococcus gallolyticus to attach to the intestinal tract of pigeons. For that purpose, first of all, two groups of six pigeons were anesthetized and ligatures were placed at the beginning of duodenum, jejunum, ileum, and colon. The obtained intestinal loops of the birds of the first and second group were injected with S. gallolyticus strains STR 357 and STR 598, respectively. At 15, 30, and 60 min postinoculation, two pigeons of each group were euthanatized and the various intestinal loops were sampled for histologic, immunohistochemical, and electron microscopic examination. Both the high and low virulence strains were able to adhere to the intestinal mucosa. Indeed, all samples dearly showed numerous coccal-shaped bacteria that stained positively with S. gallolyticus antiserum and were lining up against the intestinal epithelium. Likewise, on electron microscopic examination, cocci were seen in the mucus covering the intestinal epithelium. Second, the association of S. gallyticus strains of differing virulence with the intestinal tissue was determined quantitatively. Experiments were performed as described above. The number of S. gallolyticus bacteria that adhered to the intestinal epithelium was determined by plating out 10-fold serial dilutions of the segments. No significant differences in the number of adhered bacteria were found between the strains of high and low virulence.

%B Avian Dis %V 47 %P 559-65 %8 2003 Jul-Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/14562882?dopt=Abstract %R 10.1637/6081 %0 Journal Article %J Dev Comp Immunol %D 2002 %T Dynamics of immune cell infiltration in the caecal lamina propria of chickens after neonatal infection with a Salmonella enteritidis strain. %A Van Immerseel, Filip %A De Buck, Jeroen %A De Smet, Isabel %A Jan Mast %A Haesebrouck, Freddy %A Ducatelle, Richard %K Animals %K Animals, Newborn %K B-Lymphocytes %K Cecum %K Chickens %K Granulocytes %K Immunoglobulin A %K Leukocytes %K Liver %K Macrophages %K Poultry Diseases %K Salmonella enteritidis %K Salmonella Infections, Animal %K Spleen %K T-Lymphocytes %X

Dynamics of leucocyte infiltration and bacterial invasion in the caecal wall were studied after oral infection of 2-day-old chicks with Salmonella enterica ser. Enteritidis. Bacteria invaded the lamina propria of the caecal wall from 12h post-challenge onwards. Bacteriological examination of internal organs (liver, spleen) showed a peak in Salmonella bacteria at 3days post-infection, after which the number of bacteria decreased. Immunohistochemistry revealed macrophages and T-lymphocytes invading the caecal propria mucosae from 24h after challenge onwards, while B-lymphocytes came somewhat later, subsequently organising into follicular aggregates. An early increase in granulocytes was partly masked by the response to natural flora. While the B-lymphocyte and granulocyte populations were maintained, T-lymphocyte and macrophage populations were already reducing by 10days post-challenge. The infiltration of macrophages and T-lymphocytes in the caecal wall, followed by B-lymphocytes, is the result of an inflammatory response, caused by invading bacteria at this site. The structural maturation of the mucosa-associated lymphoid tissues is antigen driven, since B-cells organise in a follicular pattern.

%B Dev Comp Immunol %V 26 %P 355-64 %8 2002 May %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11888650?dopt=Abstract %0 Journal Article %J Vaccine %D 2002 %T The effect of vaccination with a Salmonella enteritidis aroA mutant on early cellular responses in caecal lamina propria of newly-hatched chickens. %A Van Immerseel, Filip %A De Buck, Jeroen %A De Smet, Isabel %A Jan Mast %A Haesebrouck, Freddy %A Ducatelle, Richard %K 3-Phosphoshikimate 1-Carboxyvinyltransferase %K Alkyl and Aryl Transferases %K Animals %K Animals, Newborn %K Antibodies, Bacterial %K B-Lymphocytes %K Cecum %K Chickens %K Genes, Bacterial %K Granulocytes %K Immunoglobulin A %K Macrophages %K Mutation %K Salmonella enteritidis %K Salmonella Infections %K Salmonella Vaccines %K T-Lymphocytes %X

When newly hatched chicks are inoculated with a Salmonella strain, they induce a rapid onset of resistance to intestinal colonization by other Salmonella strains. The exact mechanism of this early colonization-inhibition is not known. To study host-related contributions to this phenomenon, the kinetics of immune cell infiltration in the caecal wall was analyzed during the first 10 days after vaccination of newly hatched chickens with a Salmonella enterica serovar Enteritidis aroA mutant, and infection 1 day later with a virulent S. enterica serovar Enteritidis strain. These data were correlated with bacterial colonization and clearance of the Salmonella Enteritidis challenge strain. Bacteriological data showed that vaccinated animals had a much lower number of challenge bacteria in their organs and caecal contents the first days post-challenge, relative to unvaccinated animals. Immunohistochemical analysis of the caecal lamina propria revealed that heterophils started infiltrating the caecal lamina propria from 12 h post-vaccination. Macrophages and T-lymphocytes started infiltrating from 20 h and B-lymphocytes from 24 h post-vaccination. These data imply that immune cells already colonized the caecal wall at the time of challenge in vaccinated animals. The presence and activity of these cells in the caecal wall shortly after administration of a Salmonella Enteritidis aroA mutant might contribute to the inhibition of colonization of a virulent Salmonella strain, subsequently administered.

%B Vaccine %V 20 %P 3034-41 %8 2002 Jul 26 %G eng %N 23-24 %1 http://www.ncbi.nlm.nih.gov/pubmed/12126917?dopt=Abstract %0 Journal Article %J Br J Nutr %D 2000 %T Dietary L-carnitine supplementation increases antigen-specific immunoglobulin G production in broiler chickens. %A Jan Mast %A Buyse, J %A Goddeeris, B M %K Animals %K Antibody Specificity %K Carnitine %K Chickens %K Dietary Supplements %K Immunoglobulin G %K sex factors %K Temperature %K Weight gain %X

The usefulness of supplementary dietary L-carnitine as an immunomodulator to increase antigen-specific antibody levels was analysed in 2-6-week-old broilers. The chickens received commercial feeds either unsupplemented (starter feed 17.8 mg carnitine/kg, finisher diet 22.9 mg carnitine/kg) or supplemented with L-carnitine (100 mg carnitine/kg added to feed). At 14 d of age, both groups were distributed in equal numbers and sex ratios over two environmentally controlled chambers where temperature (28 degrees) was either reduced immediately to 20 degrees, or gradually to 22 degrees at 36 d of age. Antigen-specific immunoglobulin (Ig)M, IgG, IgA and total Ig responses were measured following two immunizations with bovine serum albumin (BSA). The typical BSA-specific IgM responses followed by IgG responses to the primary immunization were boosted by the secondary immunization. The kinetics of these responses were not altered by L-carnitine treatment. However, BSA-specific total Ig and IgG, but not IgM, responses were significantly increased by dietary L-carnitine supplementation, after both the primary and the secondary immunization. No significant influence of the sex of the chicks or the imposed environmental temperature on Ig responses was found. Temperature treatment and sex, but not L-carnitine supplementation, did significantly influence body-weight gain: cockerels were heavier than females and this became most evident in the second half of the rearing period. Further, lowering the temperature increased body weight. In conclusion, dietary L-carnitine supplementation appeared to be beneficial in enhancing specific humoral responses on vaccination.

%B Br J Nutr %V 83 %P 161-6 %8 2000 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/10743495?dopt=Abstract %0 Journal Article %J J Reprod Fertil %D 2000 %T Effect of TNF-alpha on LH and IGF-I modulated chicken granulosa cell proliferation and progesterone production during follicular development. %A Onagbesan, O M %A Jan Mast %A Goddeeris, B %A Decuypere, E %K Analysis of Variance %K Animals %K Cell Division %K Cell Line, Transformed %K Cells, Cultured %K Chickens %K Culture Media, Conditioned %K Dose-Response Relationship, Drug %K Female %K Granulosa Cells %K Insulin-Like Growth Factor I %K Luteinizing Hormone %K Macrophages %K Ovarian Follicle %K Progesterone %K Recombinant Proteins %K Stimulation, Chemical %K Tumor Necrosis Factor-alpha %X

This study demonstrates the effects of recombinant human tumour necrosis factor a (rhTNF-alpha) and conditioned medium of the HD11-transformed chicken macrophage cell line on cultured chicken granulosa cells. Effects were studied on basal, IGF-I- and LH-stimulated progesterone production and cell proliferation. Recombinant human TNF-alpha stimulated basal progesterone production in a dose-dependent manner in the granulosa cells of the largest follicle but had no effect on cells from the third largest follicle. TNF-alpha stimulated and sometimes inhibited progesterone production stimulated by IGF-I and LH alone or in combination depending on the size of the follicle and the concentration of LH or IGF-I applied. However, the inhibitory effect of TNF-alpha was significantly more pronounced in cells from the third largest follicle when high concentrations of IGF-I, LH or a combination of both were applied. TNF-alpha had no effect on basal cell proliferation in both the largest and the third largest follicles, but regulated responses to IGF-I and a combination IGF-I and LH in the cells of the third largest follicle but not those of the largest follicle. The data indicate that the normal hierarchy of follicles is maintained in the chicken ovary through the regulation of the activity of IGF-I and its interaction with LH. Conditioned medium of LPS-activated HD11 macrophages mimicked the effects of TNF-alpha and its interaction with IGF-I and LH on progesterone production and cell proliferation. The observation that the HD11-conditioned medium contained TNF-alpha indicates that TNF-alpha produced by macrophages found in chicken follicles modulates granulosa cell growth and differentiation.

%B J Reprod Fertil %V 120 %8 2000 Nov %G eng %N 2 %R 10.1530/jrf.0.1200433 %0 Journal Article %J Br Poult Sci %D 2000 %T Enhanced specific antibody response to bovine serum albumin in pigeons due to L-carnitine supplementation. %A Janssens, G P %A Jan Mast %A Goddeeris, B M %A Cox, E %A Hesta, M %A De Wilde, R O %K Animals %K Body weight %K Carnitine %K Columbidae %K Enzyme-Linked Immunosorbent Assay %K Female %K Immunoglobulin G %K Immunoglobulin M %K Linear Models %K Random Allocation %K Serum Albumin, Bovine %X

1. Thirty adult female pigeons (Columba livia domestica) were randomly divided into 3 equal groups; the 1st and 2nd groups were immunised with bovine serum albumin (BSA) at 0 and 20 d, the 2nd group also received 1 g L-carnitine per litre of drinking water from -5 to 25 d post-immunisation (dpi) and the 3rd group, a control group, received neither treatment. 2. Body weights and serum samples were taken at 0, 5, 10, 15, 20, 25, 30 and 35 dpi. 3. Both BSA-specific IgG and IgM responses were enhanced by about 10% by L-carnitine supplementation. 4. L-carnitine supplemented pigeons showed a higher water consumption. Body weight loss during the onset of the immune response showed a slight tendency to be counteracted by L-carnitine supplementation. 5. The impact of L-carnitine on resistance and resilience to an immunological challenge is discussed.

%B Br Poult Sci %V 41 %P 448-53 %8 2000 Sep %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11128385?dopt=Abstract %R 10.1080/713654972 %0 Journal Article %J Vet Immunol Immunopathol %D 1999 %T Development of immunocompetence of broiler chickens. %A Jan Mast %A Goddeeris, B M %K Aging %K Animals %K Antigens, CD57 %K Chick Embryo %K Chickens %K Immunocompetence %K Immunoglobulin G %K Immunoglobulin M %K Serum Albumin, Bovine %X

Subpopulations of T-cells, B-cells, macrophages and ellipsoid-associated reticular cells (EARC) could be demonstrated by immunohistochemical staining early in the development of chicken spleen. However, the typical structures of the spleen, such as the peri-arteriolar lymphoid sheath (PALS) and the ellipsoids with their surrounding ring of macrophages, were only formed around embryonic day (ED) 20. These structures and especially the B-cell compartment, i.e., the peri-ellipsoid lymphoid sheath (PELS) gradually matured during the first week posthatch. Therefore, we analysed at what age broiler chickens could generate a humoral response against the thymus-dependent antigen bovine serum albumin (BSA). Chickens were immunised in ovo (ED16 and ED18) and at 1, 7 and 12 days of age and subsequent BSA-specific immunoglobulin (Ig) M and IgG responses were measured up to 10 days postimmunisation (DPI). No major differences were observed in the relative growth rates, while hatchability was only slightly reduced. Only in chicks immunised on 12 days of age, IgM and IgG responses were high with a normal kinetic pattern. In chicks immunised on 7 days of age, responses were just detectable, but they were absent in chicks immunised in ovo and on the day of hatching (Day 1). In a subsequent experiment, 1-, 7- and 12-day-old chicks were BSA-immunised and Ig responses were measured for a longer period up to the age of 28 days. The IgG response of chicks immunised at 1 day of age was lower and occurred later (from 28 DPI) than the response of chicks immunised at 7 and 14 days of age (from 14 DPI). It was not increased by a booster immunisation on 29 days of age, in contrast to the response of chicks immunised at 7 and 14 days of age. These findings indicate that vaccination at 1 day of age does not activate the B-cell response resulting in antibody production and support the idea that the immune function of the late embryonic and neonatal chickens is not entirely developed due to the incomplete structural organisation of their secondary immune organs.

%B Vet Immunol Immunopathol %V 70 %8 1999 Sep 20 %G eng %N 3-4 %R https://doi.org/not-available %0 Journal Article %J Am J Vet Res %D 1999 %T Effects of inhalation of dust and endotoxin on respiratory tracts of pigs. %A Urbain, B %A Jan Mast %A Beerens, D %A N'Guyen, T Q %A Goddeeris, B %A Ansay, M %A Gustin, P %K Albumins %K Animal Feed %K Animals %K Antibodies, Monoclonal %K Bronchoalveolar Lavage %K Bronchoalveolar Lavage Fluid %K Dust %K Endotoxins %K Escherichia coli %K Flow Cytometry %K Fluorescent Antibody Technique %K Inhalation Exposure %K L-Lactate Dehydrogenase %K Leukocyte Count %K Lung %K Nasal Lavage Fluid %K Radioimmunoassay %K Random Allocation %K Respiratory Tract Diseases %K Swine %K Swine Diseases %X

OBJECTIVE: To assess the effects of inhalation of feed flour dust and dustborne endotoxin on respiratory tracts of pigs.

ANIMALS: 29 healthy Belgian Landrace pigs.

PROCEDURE: Pigs housed in an environmental chamber were exposed for 6 days to feed flour dust (1 to 15 mg/m3) and dustborne endotoxins (50 to 2,500 ng/m3). Effects were evaluated by measuring albumin concentration, lactate dehydrogenase (LDH) activity, cell composition of nasal lavage (NL) and bronchoalveolar lavage (BAL) fluids and blood, and percentages of CD4+ and CD8+ T lymphocytes in blood and lavage fluids. Dustborne endotoxin was obtained by mixing endotoxins from Escherichia coli (serotype O127:B8) with feed flour before spraying the flour in the environmental chamber.

RESULTS: Exposure did not affect cell composition of NL fluid or blood. Total cell counts of BAL fluids were increased in all groups exposed to dust. Macrophage counts were increased in pigs exposed to inhalable dust concentrations as low as 4.4 mg/m3, and lymphocyte counts were increased in groups exposed to high dust concentrations. Percentages of CD4+ and CD8+ T lymphocytes in blood and lavage fluids were unchanged. In all dust-exposed groups, albumin content of BAL fluid was increased, whereas LDH activity was unaffected. Macrophage and lymphocyte infiltration and edema in the bronchi were identified by light microscopy. Effects attributable to E. coli endotoxin exposure were not identified.

CONCLUSIONS: Inhalation of feed flour dust did not affect nasal mucosa but did induce bronchial airway inflammation. Dustborne endotoxins did not have effects attributable to endotoxin alone.

%B Am J Vet Res %V 60 %P 1055-60 %8 1999 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/10490071?dopt=Abstract %0 Journal Article %J Cell Tissue Res %D 1998 %T CD57, a marker for B-cell activation and splenic ellipsoid-associated reticular cells of the chicken. %A Jan Mast %A Goddeeris, B M %K Animals %K Antigens, CD57 %K B-Lymphocytes %K Biomarkers %K Capillaries %K Chickens %K Flow Cytometry %K immunohistochemistry %K Lymphocyte Activation %K Plasma Cells %K Spleen %K T-Lymphocytes %X

We have demonstrated that the ellipsoid-associated reticular cells of chicken spleen express CD57, a marker for B-cell activation. These cells are characterised by their spindle-shaped morphology, tissue distribution and the absence of certain leucocyte-specific markers. They are phagocytotic and possess high endogenous non-specific esterase activity. Previous reports failed to detect CD57 expression on ellipsoid-associated reticular cells, probably because the tissue sections were differently treated before immunohistochemistry. CD57 is also expressed by a small number of T-cells in the spleen and the caecal tonsils. This number is highly variable between individual chickens depending on the activation state of the immune system. Moreover, CD57 is expressed by bursal lymphocytes (90% or more) but not by B-cells of the peripheral blood. More interestingly, we have been able to discriminate and quantify three B-cell populations of the secondary lymphoid organs, i.e. resting B-cells, germinal centre B-cells and plasma cells, based on their expression levels of CD57 and Bu-1 (a pan B-cell marker). Thus, CD57 should be considered as a B-cell activation marker, rather than as a marker for bursal B-cells; it is also a valuable marker for the immunohistochemical study of ellipsoid-associated reticular cells of chicken spleen.

%B Cell Tissue Res %V 291 %8 1998 Jan %G eng %N 1 %R not available %0 Journal Article %J Vet Immunol Immunopathol %D 1998 %T Characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01. %A Jan Mast %A Goddeeris, B M %A Peeters, K %A Vandesande, F %A Berghman, L R %K Animals %K Antibodies, Monoclonal %K Antibody Specificity %K Chick Embryo %K Chickens %K Dendritic Cells %K Female %K Flow Cytometry %K immunohistochemistry %K Macrophages %K Mammals %K Monocytes %K Species Specificity %K Tissue Distribution %X

The distribution, function and ontogeny of the mononuclear phagocyte system of the chicken were characterised using the monoclonal antibody (MAb) KUL01. KUL01 specifically recognises chicken monocytes, macrophages and interdigitating cells, as well as activated microglia cells. Its tissue distribution allowed to discriminate KUL01 from all earlier described MAb, reactive with mononuclear phagocytes. The specificity of KUL01 for mononuclear phagocytes was further confirmed in functional assays: KUL01-positive macrophages in spleen and liver actively took up colloidal carbon, while monocytes and spleen and gut macrophages contained non-specific esterase and acid phosphatase activities characteristic for antigen-processing. Further, it was demonstrated that KUL01-reactive peripheral blood monocytes express MHC-II, but not CD4. In all tissues investigated, the same morphological subtypes of macrophages were detected in chicken at similar localisations as in mammals, indicating a high degree of conservation between the mononuclear phagocyte system of the chicken and of mammals.

%B Vet Immunol Immunopathol %V 61 %8 1998 Feb 27 %G eng %N 2-4 %R https://doi.org/not-available %0 Journal Article %J Infect Immun %D 1998 %T High-level expression of Chlamydia psittaci major outer membrane protein in COS cells and in skeletal muscles of turkeys. %A Vanrompay, D %A Cox, E %A Jan Mast %A Goddeeris, B %A Volckaert, G %K Amino Acid Sequence %K Animals %K Antibodies, Monoclonal %K Antigens, Bacterial %K Bacterial Outer Membrane Proteins %K Base Sequence %K Chlamydophila psittaci %K COS Cells %K Gene Expression %K Membrane Proteins %K Molecular Sequence Data %K Muscle, Skeletal %K Plasmids %K Transfection %K Turkeys %X

The omp1 genes encoding the major outer membrane proteins (MOMPs) of avian Chlamydia psittaci serovar A and D strains were cloned and sequenced. The nucleotide sequences of the avian C. psittaci serovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avian C. psittaci serovar A strain 6BC, 84.6 and 99.8% identical to that of the avian C. psittaci serovar D strain NJ1, 79.1 and 81.1% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of the Chlamydia trachomatis L2 strain, and 57.5 and 60.4% identical to that of the Chlamydia pneumoniae IOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.

%B Infect Immun %V 66 %8 1998 Nov %G eng %N 11 %R https://doi.org/not-available %0 Journal Article %J Domest Anim Endocrinol %D 1998 %T In ovo treatment with an aromatase inhibitor masculinizes postnatal hormone levels, abdominal fat pad content, and GH pulsatility in broiler chickens. %A Dewil, E %A Buyse, J %A Veldhuis, J D %A Jan Mast %A De Coster, R %A Decuypere, E %K Adipose Tissue %K Analysis of Variance %K Animals %K Aromatase Inhibitors %K Body Composition %K Body weight %K Chick Embryo %K Chickens %K Dose-Response Relationship, Drug %K Enzyme Inhibitors %K Estradiol %K Female %K Growth Hormone %K Injections %K Male %K Random Allocation %K Sex Characteristics %K Testosterone %K thyroxine %K Time Factors %K triazoles %K Triiodothyronine %X

Vorozole, a selective aromatase inhibitor, was administered in ovo to test the specific embryonic role of estrogen in conferring the sex distinction in GH release and body phenotype in broilers. On Day 6 of incubation, eggs were injected with saline or with different concentrations of vorozole. Postnatal blood samples were analyzed for T3, T4, GH, estradiol (E2), and testosterone (T). At the age of 4 wk, control and vorozole-treated birds were cannulated, and serial blood samples were withdrawn every 10 min for 5 hr, wherein GH pulsatility characteristics were determined using deconvolution analysis. The proportional abdominal fat pad weight was reduced significantly in the treated groups, especially in female birds. The vorozole treatment increased plasma T3, E2, T, and GH concentrations, and decreased T4. The frequency of the GH pulses was lower and the interval between the bursts (min) was higher in the vorozole-treated group, as were the mass secreted per burst (ng/ml), the amplitude (ng/ml/min) and the production rate (ng/ml/5 hr). In conclusion, early in ovo treatment with a potent aromatase inhibitor is able to increase the mean serum T3 and GH concentration and masculinize the GH pulse pattern, resulting in an economically favorable decrease in abdominal fat pad content in male and female broilers at slaughter age.

%B Domest Anim Endocrinol %V 15 %P 115-27 %8 1998 Mar %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/9532425?dopt=Abstract %0 Journal Article %J Vet Immunol Immunopathol %D 1998 %T Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens. %A Desmidt, M %A Ducatelle, R %A Jan Mast %A Goddeeris, B M %A Kaspers, B %A Haesebrouck, F %K Animals %K Antibodies, Bacterial %K Bursa of Fabricius %K Chickens %K Colony Count, Microbial %K Feces %K Immunoglobulin Isotypes %K Liver %K Poultry Diseases %K Salmonella enteritidis %K Salmonella Infections, Animal %K Salmonella Phages %K Spleen %K Time Factors %X

The role of avian humoral immunity in the clearance of S. enteritidis was evaluated through bursectomy. After oral inoculation of bursectomized and sham-treated chickens with S. enteritidis, faecal excretion of S. enteritidis was examined. Organs were collected weekly until six weeks post-inoculation (pi) for bacteriological enumeration. Antibody isotypes in serum and bile were quantified by ELISA. Faecal excretion of S. enteritidis was significantly lower in controls from 13 days pi. Numbers of S. enteritidis in caeca from controls were significantly decreased from three weeks pi. Numbers of S. enteritidis were significantly decreased at two weeks pi in the spleen and the liver and at six weeks pi in the liver. Antibodies to S. enteritidis peaked at two weeks pi in controls and were absent in bursectomized chickens. These findings indicate that elimination of S. enteritidis partly depends on humoral immunity. The intestinal humoral response appeared more effective than the systemic humoral response for elimination of S. enteritidis.

%B Vet Immunol Immunopathol %V 63 %P 355-67 %8 1998 Jun 12 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/9656424?dopt=Abstract %0 Journal Article %J Archiv für Geflügelkunde %D 1997 %T Different methods of bursectomy induce different effects on leukocyte distribution and reactivity %A Jan Mast %A Desmidt, Miek %A Room, Gwendolien %A Martin, Claude %A Ducatelle, Richard %A Haesebrouck, F %A Davison, TF %A Kaspers, Bernd %A Goddeeris, Bruno %K bursa %K bursectomy %K chicken %K Leukocyte concentration %B Archiv für Geflügelkunde %V 61 %8 1997 %G eng %N 5 %& 238 %R https://doi.org/not-available %0 Journal Article %J Vet Immunol Immunopathol %D 1997 %T Expression of beta 2 integrins on blood leukocytes of cows with or without bovine leukocyte adhesion deficiency. %A Cox, E %A Jan Mast %A MacHugh, N %A Schwenger, B %A Goddeeris, B M %K Animals %K Antigens, CD11 %K Antigens, CD18 %K Cattle %K Cattle Diseases %K Heterozygote %K Leukocyte-Adhesion Deficiency Syndrome %K Leukocytes %K Tetradecanoylphorbol Acetate %X

Peripheral blood leukocytes of 11 normal cows, 7 cows heterozygous and 2 heifers homozygous for bovine leukocyte adhesion deficiency (BLAD) were analysed by flow cytometry for the intensity of their beta 2 integrin expression (LFA-1(CD11a/CD18), CR3 (CD11b/CD18) and CR4 (CD11c/CD18)). BLAD-homozygotes revealed no or a very weak expression of the beta 2 integrins and had a 10-fold and 4- to 5-fold increase in absolute number of neutrophils and monocytes, respectively, whereas the absolute number of lymphocytes remained normal. The mean fluorescence intensity (MFI) of the beta 2 integrins (CD18) in heterozygous animals was 56 to 90% of this in the normal cows (MFI between 14 and 512). The difference in the expression level was most pronounced for LFA-1 on the small cluster of lymphocytes with the highest MFI for LFA-1. Repeated analysis and phorbol myristate acetate stimulation revealed that the LFA-1 expression on this high-expressing cell population of the peripheral blood allowed a ready identification of BLAD-heterozygotes by flow cytometry.

%B Vet Immunol Immunopathol %V 58 %P 249-63 %8 1997 Sep 19 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/9436269?dopt=Abstract %0 Journal Article %J Vet Microbiol %D 1995 %T Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D. %A Vanrompay, D %A Jan Mast %A Ducatelle, R %A Haesebrouck, F %A Goddeeris, B %K Aerosols %K Animals %K Cells, Cultured %K Cercopithecus aethiops %K Chlamydophila psittaci %K Female %K Fluorescent Antibody Technique, Indirect %K Immunoenzyme Techniques %K Leukocytes %K Male %K Organ Specificity %K Poultry Diseases %K Psittacosis %K Turkeys %X

At 7 days of age, 4 groups, each of twenty specific pathogen free turkeys kept in isolation units were inoculated by aerosol with the Texas Turkey strain (avian Chlamydia psittaci serovar D), strain 92/1293 (avian Chlamydia psittaci serovar D), strain 84/55 (avian Chlamydia psittaci serovar A) or strain 89/1326 (avian Chlamydia psittaci serovar B). A fifth group of 4 specific pathogen free turkeys were sham inoculated controls. At daily intervals for 10 days and then twice weekly up to 34 days post infection, one bird in each group was killed and the target tissues and cells for replication and the sequence of events of serovar A, B and D infections was examined. In these turkeys, the primary site of replication was the respiratory tract. Chlamydial replication could be detected in the respiratory tract on day 1 post inoculation (p.i.) for group A, on day 3 p.i. for group B and on day 1 to 2 p.i. for groups D1 and D2. Subsequently, there was chlamydaemia and localisation in the digestive tract, in one or more parenchymatous organs, in the pericardium and in the conjunctivae. Specific immunoperoxidase staining revealed chamydiae in these organs in epithelial cells and in monomorphonuclear cells in all infected groups. The monomorphonuclear cells were identified as macrophages by double immunofluorescence staining. Chlamydiae were present in the same tissues for serovars A and D, but could not be demonstrated in proventriculus, duodenum, pancreas, ovaries and testes for serovar B. Furthermore, the intensity of replication was similar for all serovars. However, for serovar B in comparison with the other serovars, the bacteria appeared in most tissues 1 to 6 days later and the maximal replication in these tissues occurred 3 to 4 days later.

%B Vet Microbiol %V 47 %P 245-56 %8 1995 Dec %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8748540?dopt=Abstract %0 Generic %D 0 %T Characterization of the TiO2 E171 food additive %A Frederic Brassinne %A Jan Mast %E Sandra De Vos %E Eveline Verleysen %E Pieter-Jan De Temmerman %E Ledecq, Marina %X

E171 (Titanium dioxide) is an EC approved food additive (EC 1129/2011), authorized to be used as color in foodstuffs. It is widely used for its refractive properties (shiny coating, UV protection) in the food and pharmaceutical industries. It is intended and assumed to be present in bulk form. A nanofraction may be present.Dispersion is crucial step to characterize the particle properties of food additives as it allows to separate the primary particles. To better characterize titanium dioxide food additives (E171) their dispersion method was optimized.

A dispersion methodology based on the Guiot and Spalla approach was applied. It electrosterically stabilizes the (nano)materials, dispersed by sonication, using BSA at a pH determined by zeta potential measurement. Dispersion efficiency was examined by descriptive TEM and using a combination of TEM imaging and image analysis. The latter approach allows to assess the distribution of the particle properties (size, shape, surface structure) quantitatively. For both the pristine TiO2 food additive E171 and the JRC TiO2 representative test material zeta-potential measurement allowed to identify the conditions (pH) where a stable dispersion with a minimal level of agglomeration was observed. The stability of the dispersion was confirmed by descriptive TEM: preparing dispersions of TiO2 through a pH adjustment provides a stable dispersion of single primary particles and small aggregates and agglomerates.

Under the optimized conditions, the minimal external dimension of the primary particles could be measured more precisely and accurately by a combination of EM imaging and image analysis than in metastable conditions, such that the materials could be better classified according to the EC definition of a nanomaterial.

%G eng %0 Generic %D 0 %T Examples related to the assessment of solubility/dissolution and particle size distribution %A Francisco Cubada %A Jan Mast %A Hubert Rauscher %A Stefan Weigel %G eng %0 Generic %D 0 %T Guidance on reporting the results of an EM analysis %A Jan Mast %A Eveline Verleysen %G eng %0 Report %D 0 %T Titanium analyses in food before and after ban of the food additive E171 %A Régis Nkenda %A Sophie Vand Den Neucker %A Heidi Demaegdt %A Jan Mast %A Karlien Cheyns %G eng