Nano(bio)Materials Do Not Affect Macrophage Phenotype-A Study Conducted by the REFINE Project.

Macrophages are well known for their involvement in the biocompatibility, as well as biodistribution, of nano(bio)materials. Although there are a number of rodent cell lines, they may not fully recapitulate primary cell responses, particularly those of human cells. Isolation of tissue-resident macrophages from humans is difficult and may result in insufficient cells with which to determine the possible interaction with nano(bio)materials. Isolation of primary human monocytes and differentiation to monocyte-derived macrophages may provide a useful tool with which to further study these interactions. To that end, we developed a standard operating procedure for this differentiation, as part of the Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE) project, and used it to measure the secretion of bioactive molecules from M1 and M2 differentiated monocytes in response to model nano(bio)materials, following an initial assessment of pyrogenic contamination, which may confound potential observations. The SOP was deployed in two partner institutions with broadly similar results. The work presented here shows the utility of this assay but highlights the relevance of donor variability in responses to nano(bio)materials. Whilst donor variability can provide some logistical challenges to the application of such assays, this variability is much closer to the heterogeneous cells that are present in vivo, compared to homogeneous non-human cell lines.

Evaluation of Adverse Effects of Resorbable Hyaluronic Acid Fillers: Determination of Macrophage Responses.

Resorbable tissue fillers for aesthetic purposes can induce severe complications including product migration, late swelling, and inflammatory reactions. The relation between product characteristics and adverse effects is not well understood. We hypothesized that the degree of cross-linking hyaluronic acid (HA) fillers was associated with the occurrence of adverse effects. Five experimental HA preparations similar to HA fillers were synthesized with an increasing degree of cross-linking. Furthermore, a series of commercial fillers (Perfectha®) was obtained that differ in degradation time based on the size of their particulate HA components. Cytotoxic responses and cytokine production by human THP-1-derived macrophages exposed to extracts of the evaluated resorbable HA fillers were absent to minimal. Gene expression analysis of the HA-exposed macrophages revealed the responses related to cell cycle control and immune reactivity. Our results could not confirm the hypothesis that the level of cross-linking in our experimental HA fillers or the particulate size of commercial HA fillers is related to the induced biological responses. However, the evaluation of cytokine induction and gene expression in macrophages after biomaterial exposure presents promising opportunities for the development of methods to identify cellular processes that may be predictive for biomaterial-induced responses in patients.

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

In vitro effects of aldehydes present in tobacco smoke on gene expression in human lung alveolar epithelial cells.

Tobacco smoke consists of thousands of harmful components. A major class of chemicals found in tobacco smoke is formed by aldehydes, in particular formaldehyde, acetaldehyde and acrolein. The present study investigates the gene expression changes in human lung alveolar epithelial cells upon exposure to formaldehyde, acrolein and acetaldehyde at sub-cytotoxic levels. We exposed A549 cells in vitro to aldehydes and non-aldehyde chemicals (nicotine, hydroquinone and 2,5-dimethylfuran) present in tobacco smoke and used microarrays to obtain a global view of the transcriptomic responses. We compared responses of the individual aldehydes with that of the non-aldehydes. We also studied the response of the aldehydes when present in a mixture at relative concentrations as present in cigarette smoke. Formaldehyde gave the strongest response; a total of 66 genes were more than 1.5-fold differentially expressed mostly involved in apoptosis and DNA damage related processes, followed by acetaldehyde (57 genes), hydroquinone (55 genes) and nicotine (8 genes). For acrolein and the mixture only one gene was upregulated involved in oxidative stress. No gene expression effect was found for exposure to 2,5-dimethylfuran. Overall, aldehyde responses are primarily indicative for genotoxicity and oxidative stress. These two toxicity mechanisms are linked to respiratory diseases such as cancer and COPD, respectively. The present findings could be important in providing further understanding of the role of aldehydes emitted from cigarette smoke in the onset of pulmonary diseases.

The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles.

Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The most pronounced effect of silver nanoparticles and the role of particle size in determining these effects, also in comparison to silver ions, are largely unknown. Effects of silver nanoparticles of different sizes (20, 80, 113 nm) were compared in in vitro assays for cytotoxicity, inflammation, genotoxicity and developmental toxicity. Silver nanoparticles induced effects in all endpoints studied, but effects on cellular metabolic activity and membrane damage were most pronounced. In all toxicity endpoints studied, silver nanoparticles of 20 nm were more toxic than the larger nanoparticles. In L929 fibroblasts, but not in RAW 264.7 macrophages, 20 nm silver nanoparticles were more cytotoxic than silver ions. Collectively, these results indicate that effects of silver nanoparticles on different toxic endpoints may be the consequence of their ability to inflict cell damage. In addition, the potency of silver in the form of nanoparticles to induce cell damage compared to silver ions is cell type and size-dependent.

Lipopolysaccharide analogs improve efficacy of acellular pertussis vaccine and reduce type I hypersensitivity in mice.

Pertussis is an infectious disease of the respiratory tract that is caused by the gram-negative bacterium Bordetella pertussis. Although acellular pertussis (aP) vaccines are safe, they are not fully effective and thus require improvement. In contrast to whole-cell pertussis (wP) vaccines, aP vaccines do not contain lipopolysaccharide (LPS). Monophosphoryl lipid A (MPL) and Neisseria meningitidis LpxL2 LPS have been shown to display immune-stimulating activity while exerting little endotoxin activity. Therefore, we evaluated whether these LPS analogs could increase the efficacy of the aP vaccine. Mice were vaccinated with diphtheria-tetanus-aP vaccine with aluminum, MPL, or LpxL2 LPS adjuvant before intranasal challenge with B. pertussis. Compared to vaccination with the aluminum adjuvant, vaccination with either LPS analog resulted in lower colonization and a higher pertussis toxin-specific serum immunoglobulin G level, indicating increased efficacy. Vaccination with either LPS analog resulted in reduced lung eosinophilia, reduced eosinophil numbers in the bronchoalveolar lavage fluid, and the ex vivo production of interleukin-4 (IL-4) by bronchial lymph node cells and IL-5 by spleen cells, suggesting reduced type I hypersensitivity. Vaccination with either LPS analog increased serum IL-6 levels, although these levels remained well below the level induced by wP, suggesting that supplementation with LPS analogs may induce some reactogenicity but reactogenicity considerably less than that induced by the wP vaccine. In conclusion, these results indicate that supplementation with LPS analogs forms a promising strategy that can be used to improve aP vaccines.

Lung pathology and immediate hypersensitivity in a mouse model after vaccination with pertussis vaccines and challenge with Bordetella pertussis.

While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.

p53 heterozygosity results in an increased 2-acetylaminofluorene-induced urinary bladder but not liver tumor response in DNA repair-deficient Xpa mice.

Both nucleotide excision repair (NER) and the p53 tumor suppressor protein play crucial roles in the prevention of cells becoming cancerous. This is clearly demonstrated by the fact that NER-deficient xeroderma pigmentosum patients and Li-Fraumeni patients who carry a germ-line p53 mutation are highly tumor prone. The NER-deficient Xpa and the p53(+/-) mouse models clearly mimic their human counterparts, because they are both tumor prone as well. The aim of the study presented here was to analyze the relative contribution of these two pathways in tumor suppression and to analyze a possible link between NER and p53 activation in vivo. For this, we exposed Xpa, p53(+/-), and Xpa/p53(+/-) mice to 2-acetylaminofluorene (2-AAF). We show that 2-AAF-induced urinary bladder tumor suppression is dependent on p53 status, because p53(+/-) mice were highly tumor prone. Xpa/p53(+/-) mice were even more tumor prone, whereas no increased tumor response was found in Xpa mice. Short-term assays revealed a decreased apoptotic response in Xpa/p53(+/-) mice, pointing in vivo toward a link between NER and p53-mediated apoptosis. In contrast, liver tumor response was primarily dependent on appropriate DNA repair, because Xpa-deficient mice were liver tumor prone. p53 heterozygosity had no influence on liver tumor incidences, in line with the results obtained from the short-term 2-AAF studies revealing no altered cellular response in p53(+/-) or Xpa/p53(+/-) mice. Interestingly, however, mice completely deficient in both NER and p53 (Xpa/p53(-/-) mice) showed a dramatic increase of hepatocellular proliferation accompanied by lacZ reporter gene mutations.