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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.

Physiologically-based kinetic (PBK) modeling is a valuable tool to understand the kinetics of nanoparticles (NPs) in vivo. However, estimating PBK parameters remains challenging and commonly requires animal studies. To develop predictive models to estimate PBK parameter values based on NP characteristics, a database containing PBK parameter values and corresponding NP characteristics is needed. As a first step toward this objective, this study estimates PBK parameters for gold NPs (AuNPs) and provides a comparison of two different NPs. Two animal experiments are conducted in which varying doses of AuNPs attached with polyethylene glycol (PEG) are administered intravenously to rats. The resulting Au concentrations are used to estimate PBK model parameters. The parameters are compared with PBK parameters previously estimated for poly(alkyl cyanoacrylate) NPs loaded with cabazitaxel and for LipImage 815. This study shows that a small initial database of PBK parameters collected for three NPs is already sufficient to formulate new hypotheses on NP characteristics that may be predictive of PBK parameter values. Further research should focus on developing a larger database and on developing quantitative models to predict PBK parameter values.

Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha®, and non-resorbable filler Bio-Alcamid®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value < 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1β and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.

The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to ENM through inflammatory reactions. The NLRP3 inflammasome responds to a wide range of ENM, and its activation is associated with various inflammatory diseases. Recently, anisotropic ENM have become of increasing interest, but knowledge of their effects on the immune system is still limited. The objective of the study was to compare the effects of gold ENM of different shapes on NLRP3 inflammasome activation and related signalling pathways. Differentiated THP-1 cells (wildtype, ASC-or NLRP3-deficient), were exposed to PEGylated gold nanorods, nanostars, and nanospheres, and, thus, also different surface chemistries, to assess NLRP3 inflammasome activation. Next, the exposed cells were subjected to gene expression analysis. Nanorods, but not nanostars or nanospheres, showed NLRP3 inflammasome activation. ASC-or NLRP3-deficient cells did not show this effect. Gene Set Enrichment Analysis revealed that gold nanorod-induced NLRP3 inflammasome activation was accompanied by downregulated sterol/cholesterol biosynthesis, oxidative phosphorylation, and purinergic receptor signalling. At the level of individual genes, downregulation of Paraoxonase-2, a protein that controls oxidative stress, was most notable. In conclusion, the shape and surface chemistry of gold nanoparticles determine NLRP3 inflammasome activation. Future studies should include particle uptake and intracellular localization., This research was funded by the EU FP7 project FutureNanoNeeds (GA N° 604602), RIVM, and VITO.

Nanoparticles including nanomedicines are known to be recognised by and interact with the immune system. As these interactions may result in adverse effects, for safety evaluation, the presence of such interactions needs to be investigated. Nanomedicines in particular should not unintendedly interact with the immune system, since patient’s exposure is not minimised as in the case of ‘environmental’ nanoparticles, and repeated exposure may be required. NLRP3 inflammasome activation and dendritic cell (DC) maturation are two types of immune mechanisms known to be affected by nanoparticles including nanomedicines. NLRP3 inflammasome activation results in production of the pro-inflammatory cytokines IL-1β and IL-18, as well as a specific type of cell death, pyroptosis. Moreover, chronic NLRP3 inflammasome activation has been related to several chronic diseases. Upon maturation, DC activate primary T cells; interference with this process may result in inappropriate activation and skewing of the adaptive immune response. Here, we evaluated the effect of two nanomedicines, representing nanostructured lipid carriers and polymers, on these two assays. Moreover, with a view to possible future standardisation and regulatory application, these assays were subject to an inter-laboratory comparison study using common SOPs. One laboratory performed three independent NLRP3 inflammasome activation experiments, while the other performed a single experiment. Two laboratories each performed three independent DC maturation experiments. While the nanostructured lipid carrier only showed marginal effects, the polymers showed major cytotoxicity. No evidence for inflammasome activation or DC maturation was demonstrated. Intra- and inter-laboratory comparison showed clearly reproducible results. Graphical abstract

Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project “Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)” identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine. Graphical abstract

The application of nanomaterials in medicine has led to novel pharmaceuticals and medical devices that have demonstrated a strong potential for increasing the efficacy/performance and safety of therapeutic and diagnostic procedures to address a wide range of diseases. However, the successful translation of these technologies from their inception (proof-of-concept) to clinical practice has been challenged by substantial gaps in the scientific and technical capacity of R&D companies, especially SMEs, to keep up with the ever-evolving regulatory expectations in the emerging area of nanomedicine. To address these challenges, the EU Horizon 2020 project REFINE has developed a Decision Support System (DSS) to support developers of nanotechnology-enabled health products in bringing their products to the clinic. The REFINE DSS has been developed to support experts, innovators, and regulators in the implementation of intelligent testing strategies (ITS) for efficient preclinical assessment of nanotechnology-enabled health products. The DSS applies logical rules provided by REFINE experts which generate prioritized lists of assays to be performed (i.e. ITSs) for physicochemical characterisation and for immunotoxicological endpoints. The DSS has been tested against several case studies and was validated by internal project experts as well as external ones. Graphical abstract

Significant advances have been made in the development of Adverse Outcome Pathways (AOPs) over the last decade, mainly focused on the toxicity mechanisms of chemicals. These AOPs, although relevant to manufactured nanomaterials (MNs), do not currently capture the reported roles of size-associated properties of MNs on toxicity. Moreover, some AOs of relevance to airborne exposures to MNs such as lung inflammation and fibrosis shown in animal studies may not be targeted in routine regulatory decision making. The primary objective of the present study was to establish an approach to advance the development of AOPs of relevance to MNs using existing, publicly available, nanotoxicology literature. A systematic methodology was created for curating, organizing and applying the available literature for identifying key events (KEs). Using a case study approach, the study applied the available literature to build the biological plausibility for ‘tissue injury’, a KE of regulatory relevance to MNs. The results of the analysis reveal the various endpoints, assays and specific biological markers used for assessing and reporting tissue injury. The study elaborates on the limitations and opportunities of the current nanotoxicology literature and provides recommendations for the future reporting of nanotoxicology results that will expedite not only the development of AOPs for MNs but also aid in application of existing data for decision making.

Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection. The studied nanoformulations were nanostructured lipid carriers incorporating the fluorescent dye IR780-oleyl, and polymeric nanoparticles containing the anticancer agent cabazitaxel. The biodistribution was approximated by quantification of the cargo in blood and relevant organs. Several clear and systematic differences in biodistribution were observed, with the most pronounced being a much higher (more than 50-fold) measured concentration ratio between cabazitaxel in all organs vs. blood, as compared to IR780-oleyl. Normalized dose linearity largely showed opposite trends between the two compounds after injection. Cabazitaxel showed a higher brain accumulation than IR780-oleyl with increasing dose injected. Interestingly, cabazitaxel showed a notable and prolonged accumulation in lung tissue compared to other organs. The latter observations could warrant further studies towards a possible therapeutic indication within lung and conceivably brain cancer for nanoformulations of this highly antineoplastic compound, for which off-target toxicity is currently dose-limiting in the clinic. Graphical abstract

Aim: Nanomaterials and nanomedicinal products tend to interfere with various commonly used assays, including regulatory required endotoxin detection methods for medicines. We developed a method to quantify endotoxin levels that is compatible with nanomaterials and nanomedicinal products. Materials & methods: The method is based on measuring endotoxin indirectly via 3-hydroxylated fatty acids of lipid-A, using Ultra High Performance Liquid Chromatography coupled with mass spectrometry. The outcome was related to results of the commonly used Limulus Amebocyte Lysate method. Results: The ultra high performance liquid chromatography coupled with mass spectrometry method has clear advantages compared with other endotoxin determination assays; particularly the absence of nanospecific interference. Conclusion: The method is sensitive, straightforward and accurate in determining and quantifying endotoxin in nanomedicinal product samples.

Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing., Highlights • Calu-3+MDM model shows the epithelial monolayer integrity under prolonged ALI culture. • Calu-3+MDM model has an increased sensitivity in inflammatory responses to LPS aerosol. • Combination of Calu-3+MDM model is a preferred option for long-term ALI exposure to inhaled aerosols.

For batch release of legacy vaccines such as DTaP, in vivo potency release assays are required. We quantified the variability of in vivo potency release assays for four DTaP (Diphtheria, Tetanus, acellular Pertussis) products of different manufacturers. With their large CV (Coefficients of Variance) ranging from 16% to 132%, these in vivo assays are of limited value to ensure their potency is consistent and similar to the clinical batches used for the marketing authorisation. Our data show that, although individual potency test results show high variability, the DTaP batches are manufactured with great consistency, because repeated potency testing yields similar averages for the different batches. The economic impact of variability of in vivo tests is significant since it may result in the need for greater amount of antigen than may be required or for repeating a test. For monitoring the consistency of potency, in vitro assays are superior to in vivo assays. Animal-free potency determination is common practice for newly developed vaccines under modern GMP quality systems. However, replacement of in vivo potency tests for legacy vaccines like DTaP is challenging and would require a 'reverse characterisation' strategy in which the antigens are further characterised at the level of drug substance and drug product to identify critical quality attributes (CQA) that can be tested with in vitro assays. Based on these an updated set of release tests without animal tests can be proposed. Our data can serve as benchmark for the innovative methods.

The toxicity of silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) has been associated with their dissolution or ability to release metal ions while the toxicity of cerium dioxide (CeO2) NPs has been related to their ability to induce or reduce oxidative stress dependent on their surface redox state. To examine the underlying biochemical mechanisms, multiple omics technologies were applied to characterise the responses at the molecular level in cells exposed to various metal-based particles and their corresponding metal ions. Human lung epithelial carcinoma cells (A549) were exposed to various Ag, ZnO, and CeO2 NPs, Ag and ZnO micro-sized particles (MPs), Ag ions (Ag+) and zinc ions (Zn2+) over a 24 h time course. Molecular responses at exposure levels that caused ∼20% cytotoxicity were characterised by direct infusion mass spectrometry lipidomics and polar metabolomics and by RNAseq transcriptomics. All Ag, Zn and ZnO exposures resulted in significant metabolic and transcriptional responses and the great majority of these molecular changes were common to both ionic and NP exposures and characteristic of metal ion exposure. The low toxicity CeO2 NPs elicited few molecular changes, showing slight evidence of oxidative stress for only one of the four CeO2 NPs tested. The multiple omics analyses highlight the main pathways implicated in metal ion-mediated effects. These results can be used to establish adverse outcome pathways as well as strategies to group nanomaterials for risk assessment.

Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO2, Mn2O3, CuO, ZnO, Co3O4 and WO3; 27–108 μg ml−1) using human primary 3-dimensional airway epithelium (MucilAir™) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO3 and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin-6 and -8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA-DR, CD80, CD83 and CD86 after a 48 hour exposure. Only Mn2O3 consistently upregulated DC maturation markers. Furthermore, by addition of medium from metal oxide NP-exposed 3-dimensional airway cultures to metal oxide NP-exposed DC cultures, the interplay between lung epithelium and DCs was studied. Such an interplay was again only observed for Mn2O3 and in one of five DC donors. Our results show that, even when using dosages that represent very high in vivo exposure levels, up to 27 hours of constant human airway exposure, metal oxide NPs cause minimal proinflammatory effects and that epithelial cells not necessarily interfere with DC maturation upon metal oxide NP exposure. The present approach exemplifies a relevant translation towards human safety assessment. Copyright © 2017 John Wiley & Sons, Ltd.

The discipline of immunotoxicology had its origins in the early 1970s, following the recognition of altered immune function and increased sensitivity to infections and cancers after exposure to environmental chemicals and therapeutic drugs. Reduced resistance to infectious disease was a well-documented consequence of primary and acquired immunodeficiencies, but a novel finding following xenobiotic exposure. The awareness of the consequences of altered immune function was likely heightened by the HIV epidemic, leading some to inappropriately characterize xenobiotic-induced immunosuppression as “chemical AIDS”, although it is now clear that mild to moderate suppression is the most likely outcome of inadvertent exposure. The human health implications of studies in which chemical exposure reduced resistance to infection, drove an early focus on immunosuppression within the toxicology community. Allergic hypersensitivity was well known to clinicians and symptoms were readily apparent, and therefore was not the initial focus of the developing toxicology subspecialty of immunotoxicology. The first review in the field of immunotoxicology was published by Vos in 1977, and, as research expanded during the years that followed, many of the assays, methodologies and approaches that are currently used to identify potential immunotoxicants were developed. Over the years, advances in our understanding of basic immunology have made it clear that allergy, immunosuppression and, in some cases, autoimmunity, are a matter of polarization of the immune response by immunotoxicants, rather than independent outcomes of chemical exposure.

Various nanomedicinal products (NMPs) have been reported to induce an adverse immune response, which may be related to their tendency to accumulate in or target cells of the immune system. Therefore, before their market authorization, NMPs should be thoroughly evaluated for their immunotoxic potential. Nonclinical regulatory immunotoxicity testing of nonbiological medicinal products, including NMPs, is currently performed by following the guideline S8 “Immunotoxicity Studies for Human Pharmaceuticals” of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). However, this guideline does not cover all the immunotoxicity endpoints reported for NMPs in the literature, such as complement activation related pseudo allergy, hypersensitivity and immunosuppression. In addition, ICH‐S8 does not provide any nanospecific testing considerations, which is important given their tendency to interfere with many commonly used toxicity assays. We therefore propose a nonclinical regulatory immunotoxicity assessment strategy, which considers the immunotoxicity endpoints currently missing in the ICH‐S8. We also list the known pitfalls related to the testing of NMPs and how to tackle them. Next to defining the relevant physicochemical and pharmacokinetic properties of the NMP and its intended use, the proposed strategy includes an in vitro assay battery addressing various relevant immunotoxicity endpoints. A weight of evidence evaluation of this information can be used to shape the type and design of further in vivo investigations. The final outcome of the immunotoxicity assessment can be included in the overall risk assessment of the NMP and provide alerts for relevant endpoints to address during clinical investigation. This article is categorized under:Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in NanomedicineToxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials, Proposed strategy for immunotoxicity assessment of nanomedicinal products.

Organs-on-chip (OC) have gained much interest as animal-free toxicity testing methods due to their closer resemblance to human tissues and longer culture viability than conventional in vitro methods. The current paper discusses where and how OCs may take a role in the transition to a more predictive, animal-free safety assessment for regulatory purposes. From a preliminary analysis of a repeated dose toxicity database, ten organs of priority for OC development for regu­latory use have been identified. For a number of these organs (lung, skin, liver, kidney, heart, and intestine), OCs are already at rather advanced stages of development, such that involvement of regulators becomes of value in the optimi­zation towards fitness-for-purpose of these methods. For organs such as testis, spleen, brain, and stomach, OCs are much more premature, if existing at all. Therefore, developmental work on OCs for these latter organs is expected to stay in the academic arena for the coming time. A number of technical recommendations and some challenges to reaching final implementation are discussed. We recommend that the development of OCs goes forward together with the development of adverse outcome pathways (AOP) and that they are combined with other methods into integrated testing strategies. Overall, opportunities exist, but much still needs to be done. In our view, regular interactions in multi-stakeholder work­shops on the application of animal-free innovations such as OCs will be beneficial.

Nanomaterials (NMs) are attractive for biomedical and pharmaceutical applications because of their unique physicochemical and biological properties. A major application area of NMs is drug delivery. Many nanomedicinal products (NMPs) currently on the market or in clinical trials are most often based on liposomal products or polymer conjugates. NMPs can be designed to target specific tissues, eg, tumors. In virtually all cases, NMPs will eventually reach the immune system. It has been shown that most NMs end up in organs of the mononuclear phagocytic system, notably liver and spleen. Adverse immune effects, including allergy, hypersensitivity, and immunosuppression, have been reported after NMP administration. Interactions of NMPs with the immune system may therefore constitute important side effects. Currently, no regulatory documents are specifically dedicated to evaluate the immunotoxicity of NMs or NMPs. Their immunotoxicity assessment is performed based on existing guidelines for conventional substances or medicinal products. Due to the unique properties of NMPs when compared with conventional medicinal products, it is uncertain whether the currently prescribed set of tests provides sufficient information for an adequate evaluation of potential immunotoxicity of NMPs. The aim of this study was therefore, to compare the current regulatory immunotoxicity testing requirements with the accumulating knowledge on immunotoxic effects of NMPs in order to identify potential gaps in the safety assessment. This comparison showed that immunotoxic effects, such as complement activation-related pseudoallergy, myelosuppression, inflammasome activation, and hypersensitivity, are not readily detected by using current testing guidelines. Immunotoxicity of NMPs would be more accurately evaluated by an expanded testing strategy that is equipped to stratify applicable testing for the various types of NMPs.

The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO 2 NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO 2 NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO 2 NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO 2 NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo. Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO 2 NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO 2 NP, OVA plus rutile TiO 2 NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated. All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile

We examined the association between statin use and the risk of rheumatoid arthritis (RA), with special focus on describing the patterns of risks of RA during statin exposure in a large population-based cohort in the United Kingdom. In the Clinical Practice Research Datalink, patients aged ≥40 years with at least one prescription of statins (1995–2009) were selected, and matched by age (+/-5 years), sex, practice and date of first prescription of statins to non-users. The follow-up period of statin use was divided into periods of current, recent and past exposure, with patients moving between these three exposure categories over time. Time-dependent Cox models were used to derive hazard ratios (HRs) of RA, adjusted for disease history and previous drug use. The study population included 1,023,240 patients, of whom 511,620 were statin users. No associations were found between RA and current (HRadj,1.06;99%CI:0.88–1.27) or past statin users (HRadj,1.18;99%CI:0.88–1.57). However, in patients who currently used statins, hazard rates were increased shortly after the first prescription of statins and then gradually decreased to baseline level. The risk of developing RA was increased in recent statin users, as compared to non-users (HRadj,1.39;99% CI:1.01–1.90). The risk of RA is substantially increased in the first year after the start of statins and then diminishes to baseline level. These findings may suggest that statins might accelerate disease onset in patients susceptible to develop RA, but in other patients, statins are probably safe and well tolerated, even after prolonged use. Alternatively, we cannot rule out that confounding by cardiovascular risk factors and ascertainment bias may have influenced the findings.

Objectives To examine the association between the use of statins and the risk of systemic lupus erythematosus (SLE) with focus on describing the patterns of risks over time.Setting A population-based cohort study using the UK Clinical Practice Research Datalink.Participants All patients aged 40 years or older who had at least one prescription of statins during the period 1995-2009 were selected and matched by age, sex, practice and date of first prescription to non-users. The follow-up period of statin users was divided into periods of current, recent and past exposure, with patients moving among these three exposure categories over time. Current statin users were also stratified into 1 year of use.Main outcome measures Time-dependent Cox models were used to calculate HRs of SLE, adjusted for disease history and previous drug exposure.Results We included 1 039 694 patients, of whom 519 847 were statin users. Current statin users did not have an increased risk of developing SLE among patients aged >= 40 years (HRadjusted 0.75, 95% CI 0.53 to 1.07). Current statin users who continued the therapy for >1 year had a 38% lower risk of developing SLE (HRadjusted 0.62, 95% CI 0.42 to 0.93). When more specific definitions for SLE were used, this latter finding, however, was not observed.Conclusions Our findings showed no effect of statins on the risk of developing SLE among patients aged >= 40 years. Further research is needed to study the long-term effects of statins on SLE.

Pertussis vaccines are routinely administered to infants to protect them from whooping cough. Still, an adequate safety test for pertussis toxin (PT), one of the main antigens in these vaccines, is not available. The histamine sensitization test is currently the only assay accepted by regulatory authorities to test for the absence of active PT in vaccines. This is however, a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e., ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PT. The in vitro safety test for PT that we developed is based on the clinical effects of PT in humans. For this, human cell lines were chosen based on the cell types involved in the clinical effects of PT. These cell lines were exposed to PT and analyzed by microarray. In this review, we discuss the clinical effects of PT and the mechanisms that underlie them. The approach taken may provide as an example for other situations in which an in vitro assay based on clinical effects in humans is required.

In the current report, a new strategy for risk assessment of nanomaterials is described, which builds upon previous project outcomes and is developed within the FP7 NANoREG project. NANoREG has the aim to develop, for the long term, new testing strategies adapted to a high number of nanomaterials where many factors can affect their environmental and health impact. In the proposed risk assessment strategy, approaches for (Quantitative) Structure Activity Relationships ((Q)SARs), grouping and read across are integrated and expanded to guide the user how to prioritise those nanomaterial applications that may lead to high risks for human health. info:eu-repo/semantics/publishedVersion

Background: Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) have anti-inflammatory and immuno-modulating properties which are effective in the treatment of cardiovascular disease and in suppressing the symptoms of ongoing autoimmunity. On the other hand, these anti-inflammatory and immuno-modulating effects may facilitate the development of autoimmunity potentially resulting in autoimmune diseases such as rheumatic diseases. Objectives: To determine whether ACE inhibitors and ARBs use was associated with an increased risk of incident rheumatoid arthritis (RA). Methods: A matched case-control study was conducted among antihypertensive patients using the Netherlands Information Network of General Practice (LINH) database from 2001 through 2006. Cases were patients over 40 years of age with a first-time diagnosis of RA. Each case was matched to up to five controls for age, sex, and index date which was selected one year before the first diagnosis of RA. ACE inhibitors and ARBs exposure was considered to be any prescription issued in the period before index date. Logistic regression analysis was used to estimate odds ratios (ORs) and their 95% confidence intervals (CI). Results: Our study included 211 cases and 667 matched controls. After controlling for potential confounders, users of ACE inhibitors or ARBs had adjusted ORs of 1.10 (95% CI: 0.77–1.57) and 1.06 (0.74–1.53), respectively. The adjusted ORs of incident RA among current and past users of ACE inhibitors were 1.14 (0.73–1.77) and 0.60 (0.27–1.30). Current and past use of ARBs had adjusted ORs of 1.38 (0.79–2.41) and 0.27 (0.05–1.51), respectively. Conclusions: Use of ACE inhibitors or ARBs is not associated with an increased risk of developing RA. (aut. ref.)

Respiratory sensitization provides a case study for a new approach to chemical safety evaluation, as the prevalence of respiratory sensitization has increased considerably over the last decades, but animal and/or human experimental/predictive models are not currently available. Therefore, the goal of a working group was to design a road map to develop an ASAT approach for respiratory sensitisers. This approach should aim at (i) creating a database on respiratory functional biology and toxicology, (ii) applying data analyses to understand the multi-dimensional sensitization response, and how this predisposes to respiratory inflammation and irritation, and (iii) building a systems model out of these analyses, adding pharmacokinetic-pharmacodynamic modeling to predict respiratory responses to low levels of sensitisers. To this end, the best way forward would be to follow an integrated testing approach. Experimental research should be targeted to (i) QSAR-type approaches to relate potential as a respiratory sensitizer to its chemical structure, (ii) in vitro models and (iii) in vitro-in vivo extrapolation/validation. © 2011 Elsevier Ltd.

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is, due to its endotoxic activity, responsible for the relatively high reactogenicity of whole-cell vaccines. In addition, lipopolysaccharide has strong immune stimulating properties, which makes it, potentially, an interesting vaccine component. In a previous study, we have shown that expression of two lipopolysaccharide-modifying enzymes, i.e., PagP and PagL, modulates the endotoxic activity of the Gram-negative bacterium Bordetella pertussis, the causative agent of whooping cough. To assess the consequences of PagP and PagL expression on the efficacy and reactogenicity of whole-cell pertussis vaccines, we have immunised mice and challenged them intranasally with wild-type B. pertussis. Vaccine efficacy, B. pertussis-specific antibody responses, and cytokine profiles were evaluated. The results show that expression of PagL, but not of PagP, significantly increases vaccine efficacy without altering vaccine reactogenicity. Therefore, PagL-expressing B. pertussis strains may form a basis for the development of a new and safer whole-cell pertussis vaccine, as higher vaccine efficacies may allow a reduced vaccine dosage. These data show, for the first time, that lipopolysaccharide composition is an important determinant for the efficacy of whole-cell pertussis vaccines.

Host genetics determines the course of Bordetella pertussis infection in mice. Previously, we found four loci, Tlr4 and three novel loci, designated Bps 1–3, that are involved in the control of B. pertussis infection. The purpose of the present study was to identify candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. We, therefore, studied the course of mRNA expression in the lungs after B. pertussis infection. Of the 22,000 genes investigated, 1,841 were significantly differentially expressed with 1,182 genes upregulated and 659 genes downregulated. Upregulated genes were involved in immune-related processes, such as the acute-phase response, antigen presentation, cytokine production, inflammation, and apoptosis, while downregulated genes were mainly involved in nonimmune processes, such as development and muscle contraction. Pathway analysis revealed the involvement of granulocyte function, toll-like receptor signaling pathway, and apoptosis. Nine of the differentially expressed genes were located in Bps-1, 13 were located in Bps-2, and 62 were located in Bps-3. We conclude that B. pertussis infection induces a wide and complex response, which appears to be partly specific for B. pertussis and partly nonspecific. We envisage that these data will be helpful in identifying polymorphic genes that affect the susceptibility and course of B. pertussis infection in humans. Electronic supplementary material The online version of this article (doi:10.1007/s00251-007-0227-5) contains supplementary material, which is available to authorized users. Raw and normalized data of the experiment can be accessed at the online database ArrayExpress http://www.ebi.ac.uk/arrayexpress/

Aim: A horizon scan of nanomedicinal product on the market or undergoing clinical investigation by analyzing the current nanomedicinal landscape. Materials & Methods: The horizon scan includes a search of literature, clinical trial registries and the internet. Results: This horizon scan yielded 175 nanomedicinal products. Most products were intended for cancer treatment, followed by infectious diseases. Polymer conjugates, liposomes and protein nanoparticles were the most used structures for nanomedicinal products. Conclusions: This paper provides an overview of nanomedicinal products on the market or undergoing clinical investigation, their application areas and specific properties.

This is the report of a workshop organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods that are of importance to the biosciences and which replace, reduce or refine the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organization of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (Anonymous, 1994). The workshop on "The use of in vitro systems for evaluating Immunotoxicity" was held at ECVAM (Ispra), Italy, on 24th-26th November 2003. The participants represented academia, national organizations, international regulatory bodies and industry. The aim of the workshop was to review the state-of-the-art in the field of in vitro immunotoxicology, and to develop strategies towards the replacement of in vivo testing. At the end of this report are listed the recommendations that should be considered for prevalidation and validation of relevant and reliable procedures, that could replace the use of animals in chemical and cosmetics toxicity testing.

Hexachlorobenzene (HCB) is a persistent environmental pollutant with toxic effects in man and rat. Reported adverse effects are hepatic porphyria, neurotoxicity, and adverse effects on the reproductive and immune system. To obtain more insight into HCB-induced mechanisms of toxicity, we studied gene expression levels using DNA microarrays. For 4 weeks, Brown Norway rats were fed a diet supplemented with 0, 150, or 450 mg HCB/kg. Spleen, mesenteric lymph nodes (MLN), thymus, blood, liver, and kidney were collected and analyzed using the Affymetrix rat RGU-34A GeneChip microarray. Most significant (p < 0.001) changes, compared to the control group, occurred in spleen, followed by liver, kidney, blood, and MLN, but only a few genes were affected in thymus. This was to be expected, as the thymus is not a target organ of HCB. Transcriptome profiles confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria, and the reproductive system. In line with previous histopathological findings were increased transcript levels of markers for granulocytes and macrophages. New findings include the upregulation of genes encoding proinflammatory cytokines, antioxidants, acute phase proteins, mast cell markers, complements, chemokines, and cell adhesion molecules. Generally, gene expression data provide evidence that HCB induces a systemic inflammatory response, accompanied by oxidative stress and an acute phase response. In conclusion, this study confirms previously observed (immuno)toxicological effects of HCB but also reveals several new and mechanistically relevant gene products. Thus, transcriptome profiles can be used as markers for several of the processes that occur after HCB exposure.

Dendritic cells (DC) are pivotal in the induction of adaptive immune responses because they can activate naive T-cells. Moreover, they steer these adaptive immune responses by integrating various stimuli, such as from different pathogen associated molecular patterns and the cytokine milieu. Immature DC are very well capable of ingesting protein antigens, whereas mature DC are efficient presenters of peptides to naive T cells. Human DC can be readily cultured from peripheral blood mononuclear cells, which are isolated from human blood. There is a strong need to monitor in a high-throughput fashion the immunogenicity of candidate vaccines during the process of vaccine development. Furthermore, regulators require efficacy and safety testing for batch release. For some vaccines, these tests require animal testing, causing pain and discomfort, which cannot be contested because it would interfere with the test results. With the aims of promoting vaccine development and reducing the number of animals for batch release testing, we propose to use more broadly human DC for vaccine immunogenicity testing. In this commentary, this proposition is illustrated by several examples in which the maturation of human DC was successfully used to test for vaccine and adjuvant immunogenicity.

Titanium dioxide white pigment consists of particles of various sizes, from which a fraction is in the nano range (

Background: Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5 - 200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica.Methods: Rats were orally exposed to 100, 1000 or 2500 mg/kg bw/day of SAS, or to 100, 500 or 1000 mg/kg bw/day of NM-202 (a representative nanostructured silica for OECD testing) for 28 days, or to the highest dose of SAS or NM-202 for 84 days.Results: SAS and NM-202 were extensively characterized as pristine materials, but also in the feed matrix and gut content of the animals, and after in vitro digestion. The latter indicated that the intestinal content of the mid/high-dose groups had stronger gel-like properties than the low-dose groups, implying low gelation and high bioaccessibility of silica in the human intestine at realistic consumer exposure levels. Exposure to SAS or NM-202 did not result in clearly elevated tissue silica levels after 28-days of exposure. However, after 84-days of exposure to SAS, but not to NM-202, silica accumulated in the spleen. Biochemical and immunological markers in blood and isolated cells did not indicate toxicity, but histopathological analysis, showed an increased incidence of liver fibrosis after 84-days of exposure, which only reached significance in the NM-202 treated animals. This observation was accompanied by a moderate, but significant increase in the expression of fibrosis-related genes in liver samples. Conclusions: although only few adverse effects were observed, additional studies are warranted to further evaluate the biological relevance of observed fibrosis in liver and possible accumulation of silica in the spleen in the NM-202 and SAS exposed animals respectively. In these studies, dose-effect relations should be studied at lower dosages, more representative of the current exposure of consumers, since only the highest dosages were used for the present 84-day exposure study. © 2014 van der Zande et al.; licensee BioMed Central Ltd.

Exposure to ultraviolet light, especially UVB wavelengths, can impair immune responses in animals and humans. It is remarkable that this immunomodulation is not restricted to the exposed skin but is also found at other sites, i.e. systemic (distant) immunosuppression. A frequently proposed hypothesis is that UVB exposure inhibits, specifically, T helper 1 (Th1)-mediated immune responses. The major reason for this is that contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH), both Th1-mediated immune responses, are very sensitive to UVB. For this reason these models are frequently used for photoimmunology studies. In the present study, the effects of UVB exposure were investigated in classical models for Th1-mediated immunity, i.e. CHS models in which picrylchloride or oxazolone were used as low-molecular-weight chemical antigens. In these models, CHS responsiveness and cytokines were measured, the latter by both reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The CHS responses to both contact sensitizers (picrylchloride and oxazolone) were suppressed significantly by pre-exposure to repeated suberythemal UVB exposure. Interferon-gamma (IFN-gamma), interleukin (IL)-12 and IL-4, but not IL-10, were detectable in spleen and draining lymph nodes of sensitized BALB/c mice. Repeated UVB exposure prior to sensitization at a distant locus inhibited both IFN-gamma and IL-12 but not IL-4. In BALB/c mice sensitized with ovalbumin (OVA) in the absence of complete Freund's adjuvant, a model for Th2-mediated immunity, OVA-specific serum IgE and cytokine profiles in the spleen were analysed. Sensitization did lead to a significant increase in OVA-specific IgE serum titres. Pre-exposure to UVB resulted in a decreased OVA-specific IgE serum titre. Both RT-PCR and ELISA showed increased levels of IFN-gamma, IL-4 and IL-10 in the spleens of OVA-sensitized mice. The production of IFN-gamma and IL-4 was not affected by UVB pre-exposure. In contrast, the production of IL-10 was significantly increased. This was probably caused by an up-regulation of Th2 cells. It is remarkable that IFN-gamma is significantly suppressed by UVB in Th1-mediated immune reactions but not in Th2-mediated immune reactions where it even appears to increase. IL-10, which is up-regulated by UVB pre-exposure and produced by, among others, Th2 cells, may represent a shift from Th1- to Th2-mediated immune mechanisms. However, IL-10 can also inhibit Th2 responses, which might be the reason for a decreased IgE titre in the Th2 model. From the results of this study it is concluded that UVB exposure prior to sensitization/immunization not only inhibits Th1-mediated but also Th2-mediated immune responses.

This review summarizes the literature on mammalian toxicity of ZnO nanoparticles (NPs) published between 2009 and 2011. The toxic effects of ZnO NPs are due to the compound's solubility. Whether the increased intracellular [Zn(2+)] is due to the NPs being taken up by cells or to NP dissolution in medium is still unclear. In vivo airway exposure poses an important hazard. Inhalation or instillation of the NPs results in lung inflammation and systemic toxicity. Reactive oxygen species (ROS) generation likely plays an important role in the inflammatory response. The NPs do not, or only to a minimal extent, cross the skin; this also holds for sunburned skin. Intraperitoneal administration induces neurological effects. The NPs show systemic distribution; target organs are liver, spleen, lung, and kidney and, in some cases, the heart. In vitro exposure of BEAS-2B bronchial epithelial cells and A549 alveolar adenocarcinoma cells results in cytotoxicity, increased oxidative stress, increased intracellular [Ca(2+)], decreased mitochondrial membrane potential, and interleukin (IL)-8 production. Decreased contractility of airway smooth muscle cells poses an additional hazard. In contrast to the results for BEAS-2B and A549 cells, in RKO colon carcinoma cells ZnO NPs and not Zn(2+) induce cytotoxicity and mitochondrial dysfunction. Short-term exposure of skin cells results in apoptosis but not in an inflammatory response, while long-term exposure leads to increased ROS generation, decreased mitochondrial activity, and formation of tubular intercellular structures. Macrophages, monocytes, and dendritic cells are affected; exposure results in cytotoxicity, oxidative stress, intracellular Ca(2+) flux, decreased mitochondrial membrane potential, and production of IL-1β and chemokine CXCL9. The NPs are phagocytosed by macrophages and dissolved in lysosomes. In vitro the Comet assay and the cytokinesis-blocked micronucleus assay show genotoxicity, whereas the Ames test does not. This is, however, not confirmed by in vivo genotoxicity assays. Protein binding results in increased stability.

It has been suggested that statins can both stimulate and suppress the immune system, and thereby, may influence autoimmune diseases. Therefore, we studied effects of statins on innate and adaptive immunity, and self-tolerance by measuring serological levels of C-reactive protein (CRP), neopterin, immunoglobulin E (IgE) antibodies and the presence of autoantibodies (antinuclear antibodies (ANA) and IgM rheumatoid factor (RF)) in the general population. We conducted a nested case-control study within the population-based Doetinchem cohort. Data from health questionnaires, serological measurements and information on medication from linkage to pharmacy-dispensing records were available. We selected 332 statin users (cases) and 331 non-users (controls), matched by age, sex, date of serum collection, history of cardiovascular diseases, diabetes mellitus type II and stroke. Multivariate regression analyses were performed to estimate effect of statins on the immune system. The median level of CRP in statin users (1.28 mg/L, interquartile range (IQR): 0.59-2.79) was lower than in non-users (1.62 mg/L, IQR: 0.79-3.35), which after adjustment was estimated to be a 28% lower level. We observed an inverse association between duration of statin use and CRP levels. Elevated levels of IgE (>100 IU/mL) were more prevalent in statin users compared to non-users. A trend towards increased levels of IgE antibodies in statin users was observed, whereas no associations were found between statin use and levels of neopterin or the presence of autoantibodies. In this general population sub-sample, we observed an anti-inflammatory effect of statin use and a trend towards an increase of IgE levels, an surrogate marker for Th (helper) 2 responses without a decrease in neopterin levels, a surrogate marker for Th1 response and/or self-tolerance. We postulate that the observed decreased inflammatory response during statin therapy may be important but is insufficient to induce loss of self-tolerance.

We thank Mathieu and colleagues for their interest in our recent publication [1] and for reporting their very interesting experiments showing that intraperitoneal but not subcutaneous simvastatin inhibits arthritis progression in the mouse collagen-induced arthritis (CIA) model [2]. These results are in line with those of Palmer and colleagues, who showed that intraperitoneal but not oral simvastatin administration inhibited pre-existing CIA [3]. According to Mathieu and colleagues, in our study we found that 'oral atorvastatin and pravastatin had no effect on the arthritis score after CIA induction' [2]. This statement is not correct, however. In our study, the number of arthritic animals increased from seven out of 12 in the nonstatin-treated controls to 12 out of 12 animals given oral atorvastatin and pravastatin, both after CIA induction (Figure 3A in [1]). Moreover, the summed arthritis score increased from 21 in the nonstatin-treated controls to 37 for oral pravastatin and 49 for oral atorvastatin, both after CIA induction (Figure 3B in [1]). Since our aim was to investigate the possible effects of statin intake upon arthritis development and severity, we chose to administer statins orally-the common route of administration for patients receiving statins. The findings from Mathieu and colleagues [2] underscore that statins have an immunomodulatory activity. Altogether, one may conclude that the outcome of statins on immune responses may depend on the type of statin, the dose, and the route of administration. Whereas we acknowledge that statins may have beneficial immunological effects, statins may also play a role in the induction of autoimmunity, as we have also shown in a general population-based study [4].

Objective: To assess whether there is an association between statin use and the occurrence of polymyalgia rheumatic (PMR) in the spontaneous reporting database of the World Health Organisation (WHO). Methods: We conducted a case/non-case study based on individual case safety reports (ICSR) in the WHO global ICSR database (VigiBase). Case reports containing the adverse event term polymyalgia rheumatica (WHOART or MedDRA Preferred Term) were defined as cases. Non-cases were all case reports containing other adverse event terms. Each case was matched to five non-cases by age, gender, and time of reporting. Case reports regarding a statin as suspected or concomitant drug were identified using the Anatomical Therapeutic Chemical (ATC) classification. Multivariate logistic regression was used to calculate reporting odds ratios (RORs) with 95% confidence intervals (CI). Results: We identified 327 reports of PMR as cases and 1635 reports of other ADRs as non-cases. Among cases, statins were more frequently reported as suspected agent (29.4%) compared to non-cases (2.9%). After adjustment for several covariates, statins were significantly associated with reports of PMR (ROR 14.21; 95% CI 9.89-20.85). Conclusion: The results of this study lends support to previous anecdotal case reports in the literature suggesting that the use of a statin may be associated with the occurrence of PMR. Further studies are needed to study the strength of the association in more detail and to elucidate the underlying mechanism.

Introduction: Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding. Methods: The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured. Results: Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFN gamma production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine. Conclusion: These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

Allergic contact dermatitis (ACD) is the most prevalent form of immunotoxicity in humans characterized by clinical manifestations such as red rashes, itchy skin and blisters. The disease is caused by skin sensitizers which are allergenic low-molecular weight chemicals. ACD is an important occupational disease that gives problems at different workplaces, including hair dressers, metal workers, construction workers, and cleaners. In addition, ACD can develop in the general population as well, since several consumer products contain skin sensitizers. Important skin sensitizers are metals (nickel, chromium), fragrances, hair dye ingredients and preservatives (Kimber et al., 2002a; Vandebriel & van Loveren, 2010).

Many chemicals can induce allergic contact dermatitis. Because evaluation of skin sensitizing potential by animal testing is prohibited for cosmetics, and screening of many chemicals is required within Registration, Evaluation, Authorisation and Restriction of Chemicals, urgent need exists for predictive in vitro assays to identify contact allergens. Keratinocytes (KC) are the first cells encountered when chemicals land on the skin. Therefore, KC form an important site of haptenization and their metabolism is likely to be important. Moreover, KC secrete mediators that affect processing and presentation of haptenized proteins by dendritic cells. To develop a KC-based in vitro assay to predict sensitizing potential of chemicals, in vitro exposure effects of eight contact sensitizers and six irritants on the KC cell line HaCaT were examined by gene profiling. Classifiers predictive of the class sensitizers or irritants were calculated, based on support vector machine (SVM) and random forest (RF) algorithms. Classifiers using high-ranking genes were 70% (SVM) and 62% (RF) accurate, based on three (SVM) and two to five (RF) features. Classifiers using oxidative stress pathway gene sets were 68-73% (SVM) and 69-71% (RF) accurate. Crossvalidation showed that the top-3 of most discriminating genes added up to 13 genes and included oxidative stress gene HMOX1 irrespective of the chemical left out. Moreover, HMOX1 was the most significantly regulated gene. Gene Set Enrichment Analysis showed upregulation of "Keap1 dependent" and "oxidative stress" gene lists. In conclusion, KC expression profiling can identify contact sensitizers, providing opportunities for nonanimal testing for sensitizing potential. Moreover, our data suggest that contact sensitizers induce the oxidative stress pathway in KC. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Background The gram-negative bacterium Bordetella pertussis is an important causative agent of pertussis, an infectious disease of the respiratory tract. After introduction of whole-cell vaccines (wP) in the 1950's, pertussis incidence has decreased significantly. Because wP were found to be reactogenic, in most developed countries they have been replaced by acellular vaccines (aP). We have previously shown a role for Toll-like receptor 4 (Tlr4) in pertussis-infected mice and the pertussis toxin (Ptx)-IgG response in wP-vaccinated children, raising the issue of the relative importance of Tlr4 in wP vaccination of mice. Here we analyze the effects of wP and aP vaccination and B. pertussis challenge, in Tlr4-deficient C3H/HeJ and wild-type C3H/HeOuJ mice. aP consists of Ptx, filamentous hemagglutinin (FHA), and pertactin (Prn). Results We show an important role of Tlr4 in wP and (to a lesser extent) aP vaccination, induction of Th1 and Th17 cells by wP but not aP vaccination, and induction of Th17 cells by infection, confirming data by Higgins et al. (J Immunol 2006, 177: 7980–9). Furthermore, in Tlr4-deficient mice, compared to wild-type controls (i) after vaccination only, Ptx-IgG (that was induced by aP but not wP vaccination), FHA-IgG, and Prn-IgG levels were similar, (ii) after infection (only), lung IL-1α and IL-1β expression were lower, (iii) after wP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while lung IL-1β, TNF-α, IFN-γ, IL-17, and IL-23 expression were lower, and lung pathology was absent, and (iv) after aP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while Ptx-IgG level was lower. Conclusion Tlr4 does not influence the humoral response to vaccination (without challenge), plays an important role in natural immunity, wP and aP efficacy, and induction of Th1 and Th17 responses, is critical for lung pathology and enhances pro-inflammatory cytokine production after wP vaccination and challenge, and diminishes Th2 responses after both wP and aP vaccination and challenge. wP vaccination does not induce Ptx-IgG. A role for LPS in the efficacy of wP underlines the usefulness of LPS analogs to improve bacterial subunit vaccines such as aP.