Development and validation of a human bronchial epithelial spheroid model to study respiratory toxicity in vitro.

The use of laboratory animals in research has been extensively criticized. While most of the critique has been centered around the ethical aspect, also the economic and scientific aspects have been frequently mentioned as points of concern. As a result, the use of alternative methods has gradually become more enticing. The most used alternatives to laboratory animals are the 2D monolayer cell cultures. However, the limited translatability of these monolayer cell cultures to in vivo has led to the development of 3D cell cultures that are believed to better capture the in vivo physiology and pathology. Here we report on the development of a physiologically more relevant 3D cell model (spheroids) comprised of human bronchial epithelial (16HBE14o-) cells, for use in respiratory toxicity research. Culturing 16HBE14o-cells as hanging-drops led to the formation of stable spheroids which showed an increased expression of CLDN1 when compared to 2D monolayer cultured cells. In addition, cell-cycle analysis revealed an increased sub-G0 population and signs of G0/G1 arrest in spheroids. Afterwards, standard operating procedures (SOPs) were established, and existing protocols optimized, for compatibility with spheroids. Spheroids were successfully used to assess cytotoxicity, genotoxicity, apoptosis/necrosis, and oxidative stress after exposure to known cytotoxic or genotoxic compounds. The development of the bronchial epithelial spheroids and the establishment of SOPs can contribute to a more reliable toxicity assessment of chemicals and may aid in bridging the gap between in vivo and in vitro experiments.

Exposure to silicates and systemic autoimmune-related outcomes in rodents: a systematic review.

BACKGROUND: Autoimmunity can result from the interplay between genetic background and effects of environmental and/or occupational exposure to hazardous materials. Several compounds, including silica dust, have been linked with systemic autoimmunity and systemic autoimmune diseases, based on epidemiological evidence. For asbestos, a strong link with systemic autoimmune diseases does not yet exist, however, several studies have documented features of autoimmunity following asbestos exposure. Even so, human studies are limited in their ability to identify and examine isolated exposures, making it difficult to demonstrate causation or to assess pathogenic mechanisms. Therefore, this systematic review examines the existing animal evidence regarding autoimmunity and exposure to silicates (silica and asbestos). METHODS: PubMed and EMBASE were systematically searched for peer-reviewed studies examining systemic autoimmune disease-related outcomes after silicate exposure in rodents. Literature databases were searched up to September 2021 for studies written in English and where the full text was available. Search strings were established based on a PECO (Population, Exposure, Comparator, Outcome) format. After title, abstract, and full-text screening, thirty-four studies were identified for further analysis. Quality assessment through ToxR tool and qualitative analysis of the results was performed. RESULTS: Although there was significant heterogeneity in the included studies in terms of exposure protocol and genetic background of the rodent models used, it was noted that both genetic background and exposure to silicates [(crystalline) silica and asbestos] are highly relevant to the development of (sub-) clinical systemic autoimmune disease. CONCLUSION: Parallels were observed between the findings from the animal (this review) and human (epidemiological) studies, arguing that experimental animal models are valuable tools for examining exacerbation or development of autoimmune disease after silicate exposure. However, genetic background and synergism between exposures should be considered in future studies.

Neurotoxicity of four frequently used nanoparticles: a systematic review to reveal the missing data.

Systemic exposure to nanoparticles (NPs) adversely affects different organs, including the nervous system. We systematically extracted data from publication on PubMed and Embase database up to the year 2020, and analyzed in vitro and in vivo neurotoxicity of 4 of the most well studied NPs (silver NPs, carbon-based NPs, iron NPs and silica NPs). A relatively good correlation was observed between in vitro and in vivo effects, including genotoxicity, oxidative stress, apoptosis and pro-inflammatory effects. However, crucial knowledge gap exists in current understanding of the underlying mechanisms. Some of the critical knowledge gaps and research needs identified in relation to neurotoxicity of nanoparticles include (1) lack of physio-chemical characteristics of NPs used, (2) cellular/tissue uptake of NP, (3) NP translocation across the blood-brain barrier (BBB), (4) Effect of exposure routes.

Associations between occupational and environmental exposures and organ involvement in sarcoidosis: a retrospective case-case analysis.

BACKGROUND: Sarcoidosis most commonly affects lungs and intrathoracic lymph nodes, but any other organ can be involved. In epidemiological studies, many occupational and environmental exposures have been linked to sarcoidosis but their relationship with the disease phenotype has barely been studied. OBJECTIVE: To investigate how occupational and environmental exposures prior to diagnosis relate to organ involvement in patients with sarcoidosis METHODS: We retrospectively studied patients seen at a sarcoidosis clinic between 2017 and 2020. Patients were included if they had a clinical presentation consistent with sarcoidosis and histologically confirmed epithelioid granulomas or had Löfgren syndrome. In a case-case analysis using multivariable logistic regression we calculated odds ratios (OR) of prespecified exposure categories (based on expert ascertainment) for cases with a given organ involvement versus cases without this organ involvement. RESULTS: We included 238 sarcoidosis patients. Sarcoidosis limited to pulmonary involvement was associated with exposure to inorganic dust prior to diagnosis (OR 2.11; 95% confidence interval [CI] 1.11-4.17). Patients with liver involvement had higher odds of contact with livestock (OR 3.68; 95% CI 0.91-12.7) or having jobs with close human contact (OR 4.33; 95% CI 1.57-11.3) than patients without liver involvement. Similar associations were found for splenic involvement (livestock: OR 4.94, 95% CI 1.46-16.1; close human contact: OR 3.78; 95% CI 1.47-9.46). Cardiac sarcoidosis was associated with exposure to reactive chemicals (OR 5.08; 95% CI 1.28-19.2) or livestock (OR 9.86; 95% CI 1.95-49.0). Active smokers had more ocular sarcoidosis (OR 3.26; 95% CI 1.33-7.79). CONCLUSIONS: Our study indicates that, in sarcoidosis patients, different exposures might be related to different organ involvements-hereby providing support for the hypothesis that sarcoidosis has more than one cause, each of which may promote a different disease phenotype.

Cobalt exposure via skin alters lung immune cells and enhances pulmonary responses to cobalt in mice.

Cobalt has been associated with allergic contact dermatitis and occupational asthma. However, the link between skin exposure and lung responses to cobalt is currently unknown. We investigated the effect of prior dermal sensitization to cobalt on pulmonary physiological and immunological responses after subsequent challenge with cobalt via the airways. BALB/c mice received epicutaneous applications (25 µL/ear) with 5% CoCl2*6H2O (Co) or the vehicle (Veh) dimethyl sulfoxide (DMSO) twice; they then received oropharyngeal challenges with 0.05% CoCl2*6H2O or saline five times, thereby obtaining four groups: Veh/Veh, Co/Veh, Veh/Co, and Co/Co. To detect early respiratory responses noninvasively, we performed sequential in vivo microcomputed tomography (µCT). One day after the last challenge, we assessed airway hyperreactivity (AHR) to methacholine, inflammation in bronchoalveolar lavage (BAL), innate lymphoid cells (ILCs) and dendritic cells (DCs) in the lungs, and serum IgE. Compared with the Veh/Veh group, the Co/Co group showed increased µCT-derived lung response, increased AHR to methacholine, mixed neutrophilic and eosinophilic inflammation, elevated monocyte chemoattractant protein-1 (MCP-1), and elevated keratinocyte chemoattractant (KC) in BAL. Flow cytometry in the Co/Co group demonstrated increased DC, type 1 and type 2 conventional DC (cDC1/cDC2), monocyte-derived DC, increased ILC group 2, and natural cytotoxicity receptor-ILC group 3. The Veh/Co group showed only increased AHR to methacholine and elevated MCP-1 in BAL, whereas the Co/Veh group showed increased cDC1 and ILC2 in lung. We conclude that dermal sensitization to cobalt may increase the susceptibility of the lungs to inhaling cobalt. Mechanistically, this enhanced susceptibility involves changes in pulmonary DCs and ILCs.

Carbon Nanotube- and Asbestos-Induced DNA and RNA Methylation Changes in Bronchial Epithelial Cells.

Carbon nanotubes (CNTs) are nanoscale tube-shaped carbon materials used in many industrial areas. Their fiber shape has caused concerns about their toxicity given their structural similarity with asbestos. The aim here was to elucidate the effect of CNTs and asbestos exposure on global DNA and RNA methylation and the methylation of genes associated with cell cycle, inflammation, and DNA damage processes in human lung cells. Human bronchial epithelial cells (16HBE14o-) were exposed for 24 h to 25 and 100 µg/mL CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) and 2.5 µg/mL asbestos (chrysotile, amosite, and crocidolite). Global DNA and RNA (hydroxy)methylation to cytosines was measured by a validated liquid chromatography tandem-mass spectrometry method. Global RNA methylation to adenines was measured by a colorimetric ELISA-like assay. Gene-specific DNA methylation status at certain cytosine-phosphate-guanine (CpG) sites of cyclin-dependent kinase inhibitor 1A ( CDKN1A), serine/threonine kinase ( ATM), and TNF receptor-associated factor 2 ( TRAF2) were analyzed by using bisulfite pyrosequencing technology. Only MWCNT-exposed cells showed significant global DNA hypomethylation of cytosine and global RNA hypomethylation of adenosine. SWCNT, MWCNT, and amosite exposure decreased DNA methylation of CDKN1A. ATM methylation was affected by chrysotile, SWCNT, and MWCNT. However, SWCNT exposure led to DNA hypermethylation of TRAF2. These findings contribute to further understanding of the effect of CNTs on different carcinogenic pathways.

Epigenetic effects of carbon nanotubes in human monocytic cells.

Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine-phosphate-guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

Neuro-immune interactions in chemical-induced airway hyperreactivity.

Asthma may be induced by chemical sensitisers, via mechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivo model, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient Kit(Wsh) (/Wsh) mice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

Toluene diisocyanate and methylene diphenyl diisocyanate: asthmatic response and cross-reactivity in a mouse model.

Both 2,4-toluene diisocyanate (TDI) and 4,4-methylene diphenyl diisocyanate (MDI) can cause occupational asthma. In this study, we optimized our mouse model of chemical-induced asthma in the C57Bl/6 mice strain using the model agent TDI. Furthermore, we validated MDI in this mouse model and investigated whether cross-reactivity between TDI and MDI is present. On days 1 and 8, C57Bl/6 mice were dermally treated (20 µl/ear) with 3 % MDI, 2 % TDI or the vehicle acetone olive oil (AOO) (3:2). On day 15, they received a single oropharyngeal challenge with 0.04 % MDI, 0.01 % TDI or the vehicle AOO (4:1). One day later, airway hyperreactivity (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL) were assessed. Furthermore, total serum IgE levels, lymphocyte subpopulations in auricular lymph nodes and cytokine levels in supernatants of lymphocytes were measured. Both dermal sensitization with TDI or MDI resulted in increased total serum IgE levels along with T and B cell proliferation in the auricular lymph nodes. The auricular lymphocytes showed an increased release of both Th2 and Th1 cytokines. Mice sensitized and challenged with either TDI or MDI showed AHR, along with a predominant neutrophil lung inflammation. Mice sensitized with MDI and challenged with TDI or the other way around showed no AHR, nor BAL inflammation. Both TDI and MDI are able to induce an asthma-like response in this mouse model. However, cross-reactivity between both diisocyanates remained absent.

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice.

BACKGROUND: Pulmonary exposure to nanoparticles (NPs) may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis. However, only few studies have investigated hemostatic effects after pulmonary exposure. METHODS: We used Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1(-/-)) mice which have a disturbed circadian rhythm and procoagulant phenotype, to study the pulmonary and hemostatic toxicity of multi-walled carbon nanotubes (MWCNTs) and zinc oxide (ZnO) NPs after subacute pulmonary exposure. Bmal1(-/-) and wild-type (Bmal1(+/+)) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128 µg MWCNTs or 32 or 64 µg ZnO NPs. RESULTS: MWCNTs caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F)VII. In contrast, the ZnO NPs seemed to suppress the inflammatory (decreased neutrophils in Bmal1(-/-) mice) and oxidative response (increased total glutathione in Bmal1(-/-) mice), but were also procoagulant with a significant increase of FVIII. The procoagulant effects, as well as the significant correlations between the pulmonary endpoints (inflammation and oxidative stress) and hemostasis parameters were more pronounced in Bmal1(-/-) mice than in Bmal1(+/+) mice. CONCLUSIONS: The Bmal1(-/-) mouse is a sensitive animal model to study the procoagulant effects of engineered NPs. The MWCNTs and ZnO NPs showed different pulmonary toxicity but both NPs induced procoagulant effects, suggesting different mechanisms of affecting hemostasis. However, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects.

Pulmonary inflammation in mice with collagen-induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN-γ.

Following immunization with collagen II (CII) in complete Freund's adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen-induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild-type DBA/1 mice. In IFN-γ receptor-deficient (IFN-γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN-γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT-PCR on affected tissue. Treatment with a TNF-α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN-γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.

Sampling strategies and analytical techniques for assessment of airborne micro and nano plastics.

The atmosphere is pervasively polluted by microplastics and nano plastics (M/NPs) released into indoor and outdoor areas. However, various methodologies and their limitations along with non-standardization make the comparison of information concerning their prevalence difficult. Such diversity in techniques greatly limits the interpretation of results. Herein, We extracted data from publications on PubMed and Embase database up to the year 2022 regarding sampling strategies, identification methods, and reporting data for M/NPs quantification. In this review, 5 major areas for measuring airborne M/NPs have been identified including pre-sampling/ sampling/ post-sampling/ analysis/ and contamination avoidance. There are many challenges specific to each of those sections that need to be resolved through further method development and harmonization. This review mainly focuses on the different methods for collecting atmospheric M/NPs and also the analytical tools which have been used for their identification. While passive sampling is the most user-friendly method, the most precise and reproducible approach for collecting plastic particles is an active method which is directly followed by visual counting as the most common physical analysis technique. Polymers collected using visual sorting are most frequently identified by spectroscopy (FTIR; Raman). However, destructive analytical techniques (thermal degradation) also provide precise chemical information. In all cases, the methods were screened for advantages, limitations, and fieldwork abilities. This review outlines and critiques knowledge gaps, and recommendations to support standardized and comparable future research.

Impact of a Polymer-Based Nanoparticle with Formoterol Drug as Nanocarrier System In Vitro and in an Experimental Asthmatic Model.

The implementation of nanotechnology in pulmonary delivery systems might result in better and more specific therapy. Therefore, a nano-sized drug carrier should be toxicologically inert and not induce adverse effects. We aimed to investigate the responses of a polymer nano drug carrier, a lysine poly-hydroxyethyl methacrylate nanoparticle (NP) [Lys-p(HEMA)], loaded with formoterol, both in vitro and in vivo in an ovalbumin (OVA) asthma model. The successfully synthesized nanodrug formulation showed an expectedly steady in vitro release profile. There was no sign of in vitro toxicity, and the 16HBE and THP-1 cell lines remained vital after exposure to the nanocarrier, both loaded and unloaded. In an experimental asthma model (Balb/c mice) of ovalbumin sensitization and challenge, the nanocarrier loaded and unloaded with formoterol was tested in a preventive strategy and compared to treatment with the drug in a normal formulation. The airway hyperresponsiveness (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL), both cellular and biochemical, were assessed. The application of formoterol as a regular drug and the unloaded and formoterol-loaded NP in OVA-sensitized mice followed by a saline challenge was not different from the control group. Yet, both the NP formulation and the normal drug application led to a more deteriorated lung function and increased lung inflammation in the OVA-sensitized and -challenged mice, showing that the use of the p(HEMA) nanocarrier loaded with formoterol needs more extensive testing before it can be applied in clinical settings.

Proteome changes in auricular lymph nodes and serum after dermal sensitization to toluene diisocyanate in mice.

Some reactive chemicals, such as diisocyanates, are capable of initiating an allergic response, which can lead to occupational asthma after a latency period. Clinical symptoms such as cough, wheezing, and dyspnea occur only late, making it difficult to intervene at an early stage. So far, most studies using proteomics in lung research have focused on comparisons of healthy versus diseased subjects. Here, using 2D-DIGE, we explored proteome changes in the local draining lymph nodes and serum of mice dermally sensitized once or twice with toluene-2,4-diisocyanate (TDI) before asthma is induced. In the lymph nodes, we found 38 and 58 differentially expressed proteins after one and two treatments, respectively, between TDI-treated and vehicle-treated mice. In serum, seven and 16 differentially expressed proteins were detected after one and two treatments, respectively. We identified 80-85% of the differentially expressed proteins by MS. Among them, lymphocyte-specific protein-1, coronin 1a, and hemopexin were verified by Western blotting or ELISA in an independent group of mice. This study revealed alterations in the proteomes early during sensitization in a mouse model before the onset of chemical-induced asthma. If validated in humans, these changes could lead to earlier diagnosis of TDI-exposed workers.

Nano-titanium dioxide modulates the dermal sensitization potency of DNCB.

We determined the ability of a model nanoparticle (NP) (titanium dioxide, TiO(2)) to modulate sensitization induced by a known potent dermal sensitizer (dinitrochlorobenzene) using a variant of the local lymph node assay called lymph node proliferation assay.BALB/c mice received sub-cutaneous injections of vehicle (2.5 mM sodium citrate), TiO(2) NPs (0.004, 0.04 or 0.4 mg/ml) or pigment particles (0.04 mg/ml) both stabilized in sodium citrate buffer at the base of each ear (2x50µl), before receiving dermal applications (on both ears) of 2,4-Dinitrochlorobenzene (DNCB) (2x25µl of 0.1%) or its vehicle (acetone olive oil - AOO (4:1)) on days 0, 1 and 2. On day 5, the stimulation index (SI) was calculated as a ratio of (3)HTdR incorporation in lymphocytes from DNBC-treated mice and AOO-treated controls. In a second experiment the EC(3)-value for DNCB (0 to 0.1%) was assessed in the absence or presence of 0.04 mg/ml TiO(2). In a third experiment, the lymphocyte subpopulations and the cytokine secretion profile were analyzed after TiO(2) (0.04 mg/ml) and DNCB (0.1%) treatment. Injection of NPs in AOO-treated control mice did not have any effect on lymph node (LN) proliferation. DNCB sensitization resulted in LN proliferation, which was further increased by injection of TiO(2) NPs before DNCB sensitization. The EC(3) of DNCB, with prior injection of vehicle control was 0.041%, while injection with TiO(2) decreased the EC(3) of DNCB to 0.015%. TiO(2) NPs pre-treatment did not alter the lymphocyte subpopulations, but significantly increased the level of IL-4 and decreased IL-10 production in DNCB treated animals.In conclusion, our study demonstrates that administration of nano-TiO(2) increases the dermal sensitization potency of DNCB, by augmenting a Th(2) response, showing the immunomodulatory abilities of NPs.

Assessment of Experimental Techniques That Facilitate Human Granuloma Formation in an In Vitro System: A Systematic Review.

The process of granuloma formation is complex, and due to species differences, the validity of animal studies is somewhat questioned. Moreover, the large number of animals needed to observe the different stages of development also raises ethical questions. Therefore, researchers have explored the use of human peripheral blood mononuclear cells (PBMCs), a heterogeneous population of immune cells, in an in vitro model. This review included in vitro studies that focused on exposing PBMCs-from healthy, sensitized, or diseased individuals-to antigens derived from infectious agents-such as mycobacteria or Schistosoma spp.-or inorganic antigens-such as beryllium. The reviewed studies mainly explored how human in vitro granuloma models can contribute towards understanding the pathogenesis of granulomatous diseases, especially during the early stages of granuloma formation. The feasibility of granuloma modelling was thus largely assessed via experimental techniques including (1) granuloma scoring indices (GI), (2) cell surface markers and (3) cytokine secretion profiling. While granuloma scoring showed some similarities between studies, a large variability of culture conditions and endpoints measured have been identified. The lack of any standardization currently impedes the success of a human in vitro granuloma model.

Occupational Asthma Caused by Low-Molecular-Weight Chemicals Associated With Contact Dermatitis: A Retrospective Study.

BACKGROUND: Occupational asthma (OA) may have different etiologies, but it is not clear whether the etiologic agents influence the clinical presentation, especially the co-occurrence of skin lesions. OBJECTIVE: To determine the impact of different asthmagens on the characteristics of OA, with a focus on the occurrence of prior or concomitant skin disorders. METHODS: In a retrospective analysis of patients who visited the Occupational and Environmental Disease Clinic of a tertiary referral hospital from 2009 to 2019, we classified patients into definite, probable, or possible OA according to prespecified diagnostic guidelines. In multivariate logistic regression with sensitivity analysis, we examined the relation of high- and low-molecular-weight (HMW and LMW) agents with the clinical presentation. RESULTS: Of 209 cases of OA, 66 were caused by HMW agents and 143 by LMW agents. Patients with OA exposed to LMW agents had higher odds of having (had) allergic contact dermatitis (odds ratio, 5.45 [1.80-23.70]; P < .01), compared with patients exposed to HMW agents. Conversely, HMW agents were associated with higher odds of rhinitis symptoms (odds ratio of LMW/HMW, 0.33 [0.17-0.63]; P < .001) and high total IgE (odds ratio of LMW/HMW, 0.35 [0.17-0.70]; P < .01). Risk factors for having coexisting contact dermatitis included construction work, hairdressing, and exposure to metals or epoxy resins. CONCLUSIONS: Among patients with OA, exposure to specific LMW agents was associated with a high frequency of contact dermatitis. Different types of asthmagens within HMW or LMW agents appear to determine the phenotype and comorbidity of OA.

The EXIMIOUS project-Mapping exposure-induced immune effects: connecting the exposome and the immunome.

Immune-mediated, noncommunicable diseases-such as autoimmune and inflammatory diseases-are chronic disorders, in which the interaction between environmental exposures and the immune system plays an important role. The prevalence and societal costs of these diseases are rising in the European Union. The EXIMIOUS consortium-gathering experts in immunology, toxicology, occupational health, clinical medicine, exposure science, epidemiology, bioinformatics, and sensor development-will study eleven European study populations, covering the entire lifespan, including prenatal life. Innovative ways of characterizing and quantifying the exposome will be combined with high-dimensional immunophenotyping and -profiling platforms to map the immune effects (immunome) induced by the exposome. We will use two main approaches that "meet in the middle"-one starting from the exposome, the other starting from health effects. Novel bioinformatics tools, based on systems immunology and machine learning, will be used to integrate and analyze these large datasets to identify immune fingerprints that reflect a person's lifetime exposome or that are early predictors of disease. This will allow researchers, policymakers, and clinicians to grasp the impact of the exposome on the immune system at the level of individuals and populations.

Biodistribution and pulmonary metabolic effects of silver nanoparticles in mice following acute intratracheal instillations.

The respiratory tract is the route of entry for accidentally inhaled AgNPs, which can reach the lungs and redistribute to other main organs through systemic circulation. In the present work, we aimed to evaluate silver biodistribution and biological effects after 1 or 2 intratracheal instillations (IT) of two differently sized PVP-coated AgNPs (5 and 50 nm-3 mg/kg) and ionic silver (AgNO3-1 mg/kg bw) in mice. Furthermore, nuclear magnetic resonance (NMR) metabolomics was applied to unveil pulmonary metabolic variations. Animals exposed to 5 nm AgNP (AgNP5) showed higher levels of ionic silver in organs, especially in the lung, spleen, kidney and liver, while animals exposed to 50 nm AgNP (AgNP50) showed higher levels of silver in the blood. Animals exposed to AgNP50 excreted higher amounts of silver than those exposed to AgNP5, which is consistent with higher tissue accumulation of silver in animals exposed to the latter. Lung metabolic profiling revealed several Ag-induced alterations in metabolites involved in different pathways, such as glycolysis and tricarboxylic acid (TCA) cycle, amino acid and phospholipid metabolism, and antioxidant defense. Notably, most of the metabolic changes observed after 1 IT were absent in animals subjected to 2 IT of AgNO3, or reversed for AgNPs, suggesting adaptation mechanisms to cope with the initial insult and recover homeostasis. Graphical abstract.

Effect of Graphene and Graphene Oxide on Airway Barrier and Differential Phosphorylation of Proteins in Tight and Adherens Junction Pathways.

Via inhalation we are continuously exposed to environmental and occupational irritants which can induce adverse health effects, such as irritant-induced asthma (IIA). The airway epithelium forms the first barrier encountered by these agents. We investigated the effect of environmental and occupational irritants on the airway epithelial barrier in vitro. The airway epithelial barrier was mimicked using a coculture model, consisting of bronchial epithelial cells (16HBE) and monocytes (THP-1) seeded on the apical side of a permeable support, and human lung microvascular endothelial cells (HLMVEC) grown on the basal side. Upon exposure to graphene (G) and graphene oxide (GO) in a suspension with fetal calf serum (FCS), ammonium persulfate (AP), sodium persulfate (SP) and hypochlorite (ClO-), the transepithelial electrical resistance (TEER) and flux of fluorescent labelled dextran (FD4-flux), was determined. Exposure to graphene nanoparticles (GNPs) induced an immediate negative effect on the epithelial barrier, whereas ClO- only had a negative impact after 24 h of exposure. AP and SP did not affect the barrier properties. The tight junctions (TJ) network showed less connected zonula occludens 1 (ZO-1) and occludin staining in GNP-exposed cocultures. Functional analysis of the phosphoproteomic data indicated that proteins in the adherens junction (AJ) and TJ pathways showed an altered phosphorylation due to GNP exposure. To conclude, the negative effect of GNPs on the epithelial barrier can be explained by the slightly altered the TJ organization which could be caused by alterations in the phosphorylation level of proteins in the AJ and TJ pathway.