Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Nanotoxicology: a perspective and discussion of whether or not in vitro testing is a valid alternative

Despite the many proposed advantages related to nanotechnology, there are increasing concerns as to the potential adverse human health and environmental effects that the production of, and subsequent exposure to nanoparticles (NPs) might pose. In regard to human health, these concerns are founded upon the plethora of knowledge gained from research relating to the effects observed following exposure to environmental air pollution. It is known that increased exposure to environmental air pollution can cause reduced respiratory health, as well as exacerbate pre-existing conditions such as cardiovascular disease and chronic obstructive pulmonary disease. Such disease states have also been associated with exposure to the NP component contained within environmental air pollution, raising concerns as to the effects of NP exposure. It is not only exposure to accidentally produced NPs however, which should be approached with caution. Over the past decades, NPs have been specifically engineered for a wide range of consumer, industrial and technological applications. Due to the inevitable exposure of NPs to humans, owing to their use in such applications, it is therefore imperative that an understanding of how NPs interact with the human body is gained. In vivo research poses a beneficial model for gaining immediate and direct knowledge of human exposure to such xenobiotics. This research outlook however, has numerous limitations. Increased research using in vitro models has therefore been performed, as these models provide an inexpensive and high-throughput alternative to in vivo research strategies. Despite such advantages, there are also various restrictions in regard to in vitro research. Therefore, the aim of this review, in addition to providing a short perspective upon the field of nanotoxicology, is to discuss (1) the advantages and disadvantages of in vitro research and (2) how in vitro research may provide essential information pertaining to the human health risks posed by NP exposure.

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Quantum dot cytotoxicity in vitro: An investigation into the cytotoxic effects of a series of different surface chemistries and their core/shell materials

Abstract The aim of this study was to assess the effects of a series of different surface coated quantum dots (QDs) (organic, carboxylated [COOH] and amino [NH(2)] polytethylene glycol [PEG]) on J774.A1 macrophage cell viability and to further determine which part of the QDs cause such toxicity. Cytotoxic examination (MTT assay and LDH release) showed organic QDs to induce significant cytotoxicity up to 48 h, even at a low particle concentration (20 nM), whilst both COOH and NH(2) (PEG) QDs caused reduced cell viability and cell membrane permeability after 24 and 48 h exposure at 80 nM. Subsequent analysis of the elements that constitute the QD core, core/shell and (organic QD) surface coating showed that the surface coating drives QD toxicity. Elemental analysis (ICP-AES) after 48 h, however, also observed a release of Cd from organic QDs. In conclusion, both the specific surface coating and core material can have a significant impact on QD toxicity.

Investigating the interaction of cellulose nanofibers derived from cotton with a sophisticated 3D human lung cell coculture

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber-cell interactions.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

A 950 yr temperature reconstruction from Duckhole Lake, southern Tasmania, Australia

A lack of quantitative high resolution paleoclimate data from the Southern Hemisphere limits the ability to examine current trends within the context of long-term natural climate variability. This study presents a temperature reconstruction for southern Tasmania based on analyses of a sediment core from Duckhole Lake (43.365°S, 146.875°E). The relationship between non-destructive whole core scanning reflectance spectroscopy measurements in the visible spectrum (380–730 nm) and the instrumental temperature record (ad 1911–2000) was used to develop a calibration-in-time reflectance spectroscopy-based temperature model. Results showed that a trough in reflectance from 650 to 700 nm, which represents chlorophyll and its derivatives, was significantly correlated to annual mean temperature. A calibration model was developed (R = 0.56, p auto < 0.05, root mean squared error of prediction (RMSEP) = 0.21°C, five-year filtered data, calibration period 1911–2000) and applied down-core to reconstruct annual mean temperatures in southern Tasmania over the last c. 950 years. This indicated that temperatures were initially cool c. ad 1050, but steadily increased until the late ad 1100s. After a brief cool period in the ad 1200s, temperatures again increased. Temperatures steadily decreased during the ad 1600s and remained relatively stable until the start of the 20th century when they rapidly decreased, before increasing from ad 1960s onwards. Comparisons with high resolution temperature records from western Tasmania, New Zealand and South America revealed some similarities, but also highlighted differences in temperature variability across the mid-latitudes of the Southern Hemisphere. These are likely due to a combination of factors including the spatial variability in climate between and within regions, and differences between records that document seasonal (i.e. warm season/late summer) versus annual temperature variability. This highlights the need for further records from the mid-latitudes of the Southern Hemisphere in order to constrain past natural spatial and seasonal/annual temperature variability in the region, and to accurately identify and attribute changes to natural variability and/or anthropogenic activities.

[Mastitis management in Swiss dairy farms with udder health problems]

The objective of this study was to describe the udder health management in Swiss dairy herds with udder health problems. One hundred dairy herds with a yield-corrected somatic cell count of 200'000 to 300'000 cells/ml during 2010 were selected. Data concerning farm structure, housing system, milking technique, milking procedures, dry-cow and mastitis management were collected during farm visits between September and December 2011. In addition, quarter milk samples were collected for bacteriological culturing from cows with a composite somatic cell count ≥ 150'000 cells/ml. The highest quarter level prevalence was 12.3 % for C. bovis. Eighty-two percent of the pipeline milking machines in tie-stalls and 88 % of the milking parlours fulfilled the criteria for the vacuum drop, and only 74 % of the pipeline milking machines met the criteria of the 10-l-water test. Eighty-five percent of the farms changed their milk liners too late. The correct order of teat preparation before cluster attachment was carried out by 37 % of the farmers only. With these results, Swiss dairy farmers and herd health veterinarians can be directed to common mistakes in mastitis management. The data will be used for future information campaigns to improve udder health in Swiss dairy farms.

Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis.

BACKGROUND Exposure to food allergens through a disrupted skin barrier has been recognized as a potential factor in the increasing prevalence of food allergy. OBJECTIVE We sought to test the immunologic mechanisms by which epicutaneous sensitization to food allergens predisposes to intestinal food allergy. METHODS Mice were epicutaneously sensitized with ovalbumin or peanut on an atopic dermatitis-like skin lesion, followed by intragastric antigen challenge. Antigen-specific serum IgE levels and T(H)2 cytokine responses were measured by ELISA. Expression of type 2 cytokines and mast cell proteases in the intestine were measured by using real-time PCR. Accumulation of basophils in the skin and mast cells in the intestine was examined by using flow cytometry. In vivo basophil depletion was achieved by using diphtheria toxin treatment of Baso-DTR mice. For cell-transfer studies, the basophil population was expanded in vivo by means of hydrodynamic tail vein injection of thymic stromal lymphopoietin (TSLP) cDNA plasmid. RESULTS Sensitization to food allergens through an atopic dermatitis-like skin lesion is associated with an expansion of TSLP-elicited basophils in the skin that promote antigen-specific T(H)2 cytokine responses, increased antigen-specific serum IgE levels, and accumulation of mast cells in the intestine, promoting the development of intestinal food allergy. Critically, disruption of TSLP responses or depletion of basophils reduced the susceptibility to intestinal food allergy, whereas transfer of TSLP-elicited basophils into intact skin promoted disease. CONCLUSION Epicutaneous sensitization on a disrupted skin barrier is associated with accumulation of TSLP-elicited basophils, which are necessary and sufficient to promote antigen-induced intestinal food allergy.

Uptake efficiency of surface modified gold nanoparticles does not correlate with functional changes and cytokine secretion in human dendritic cells in vitro.

Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG+PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties.