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The increasing use of multiwalled carbon nanotubes (MWCNTs) could increase the risk of allergic lung disease in occupational or consumer settings. We previously reported that MWCNTs exacerbated allergic lung disease in mice induced by extract from house dust mites (HDM), a common cause of asthma in humans. Because MWCNTs avidly bind biomolecules to form protein coronas that can modify immunotoxicity, we hypothesized that exacerbation of allergic lung disease in mice caused by coexposure to MWCNTs and HDM extract was due to the formation of an allergen corona. In a first set of experiments, male and female C57BL/6J mice were coexposed to MWCNTs and HDM extract over 3 weeks compared to MWCNTs or HDM extract alone. In a second set of experiments, mice were exposed to pristine MWCNTs or MWCNTs with an HDM allergen corona (HDM-MWCNTs). HDM-MWCNTs were formed by incubating MWCNTs with HDM extract, where ∼7% of proteins adsorbed to MWCNTs, including Der p 1 and Der p 2. At necropsy, bronchoalveolar lavage fluid was collected from lungs to assess lactate dehydrogenase, total protein and inflammatory cells, while lung tissue was used for histopathology, qPCR, and Western blotting. Compared to MWCNTs or HDM extract alone, coexposure to MWCNTs and HDM extract or exposure to HDM-MWCNTs increased pathological outcomes associated with allergic lung disease (eosinophilia, fibrosis, mucous cell metaplasia), increased mRNAs associated with fibrosis ( Col1A1 , Arg1 ) and enhanced STAT6 phosphorylation in lung tissue. These findings indicated that exacerbation of HDM-induced allergic lung disease by MWCNTs is due to an allergen corona.

Multi-walled carbons nanotubes (MWCNTs) are used in materials for the construction, automotive, and aerospace industries. Workers and consumers are exposed to these materials via inhalation. Existing recommended exposure limits are based on MWCNT exposures that do not take into account more realistic co-exposures. Our goal was to understand how a common allergen, house dust mites, interacts with pristine MWCNTs and lung fluid proteins. We used gel electrophoresis, western blotting, and proteomics to characterize the composition of the allergen corona formed from house dust mite extract on the surface of MWCNTs. We found that the corona is dominated by der p 2, a protein associated with human allergic responses to house dust mites. Der p 2 remains adsorbed on the surface of the MWCNTs following subsequent exposures to lung fluid proteins. The high concentration of der p 2, localized on surface of MWCNTs, has important implications for house dust mite-induced allergies and asthma. This research provides a detailed characterization of the complex house dust mite-lung fluid protein coronas for future cellular and in vivo studies. These studies will help to address the molecular and biochemical mechanisms underlying the exacerbation of allergic lung disease by nanomaterials., Competing Interests: Ethics Declarations Conflicts of interest The authors declare that they have no conflicts of interest.

Background: Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been reported to exert strong pro-inflammatory and pro-fibrotic adjuvant effects in mouse models of allergic lung disease. However, the molecular mechanisms through which MWCNTs exacerbate allergen-induced lung disease remain to be elucidated. We hypothesized that protease-activated receptor 2 (PAR2), a G-protein coupled receptor previously implicated in the pathogenesis of various diseases including pulmonary fibrosis and asthma, may play an important role in the exacerbation of house dust mite (HDM) allergen-induced lung disease by MWCNTs., Methods: Wildtype (WT) male C57BL6 mice and Par2 KO mice were exposed to vehicle, MWCNTs, HDM extract, or both via oropharyngeal aspiration 6 times over a period of 3 weeks and were sacrificed 3-days after the final exposure (day 22). Bronchoalveolar lavage fluid (BALF) was harvested to measure changes in inflammatory cells, total protein, and lactate dehydrogenase (LDH). Lung protein and RNA were assayed for pro-inflammatory or profibrotic mediators, and formalin-fixed lung sections were evaluated for histopathology., Results: In both WT and Par2 KO mice, co-exposure to MWCNTs synergistically increased lung inflammation assessed by histopathology, and increased BALF cellularity, primarily eosinophils, as well as BALF total protein and LDH in the presence of relatively low doses of HDM extract that alone produced little, if any, lung inflammation. In addition, both WT and par2 KO mice displayed a similar increase in lung Cc1-11 mRNA, which encodes the eosinophil chemokine CCL-11, after co-exposure to MWCNTs and HDM extract. However, Par2 KO mice displayed significantly less airway fibrosis as determined by quantitative morphometry compared to WT mice after co-exposure to MWCNTs and HDM extract. Accordingly, at both protein and mRNA levels, the pro-fibrotic mediator arginase 1 (ARG-1), was downregulated in Par2 KO mice exposed to MWCNTs and HDM. In contrast, phosphorylation of the pro-inflammatory transcription factor NF-κB and the pro-inflammatory cytokine CXCL-1 was increased in Par2 KO mice exposed to MWCNTs and HDM., Conclusions: Our study indicates that PAR2 mediates airway fibrosis but not eosinophilic lung inflammation induced by co-exposure to MWCNTs and HDM allergens., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Semiconducting transparent metal oxides are critical high mobility materials for flexible optoelectronic devices such as displays. We introduce the continuous liquid metal printing (CLMP) technique to enable rapid roll-to-roll compatible deposition of semiconducting two-dimensional (2D) metal oxide heterostructures. We leverage CLMP to deposit 10 cm 2 -scale nanosheets of InO x and GaO x in seconds at a low process temperature ( T < 200 °C) in air, fabricating heterojunction thin film transistors with 100× greater I on / I off , 4× steeper subthreshold slope, and a 50% increase in mobility over pure InO x channels. Detailed nanoscale characterization of the heterointerface by X-ray photoelectron spectroscopy, UV-vis, and Kelvin probe elucidates the origins of enhanced electronic transport in these 2D heterojunctions. This combination of CLMP with the electrostatic control induced by the heterostructure architecture leads to high performance (μ lin up to 22.6 cm 2 /(V s)) while reducing the process time for metal oxide transistors by greater than 100× compared with sol-gels and vacuum deposition methods.

We previously reported that delivery of nickel nanoparticles (NiNPs) and bacterial lipopolysaccharide (LPS) into the lungs of mice synergistically increased IL-6 production and inflammation, and male mice were more susceptible than female mice. The primary goal of this study was to utilize an in vitro human lung epithelial cell model (BEAS-2B) to investigate the intracellular signaling mechanisms that mediate IL-6 production by LPS and NiNPs. We also investigated the effect of sex hormones on NiNP and LPS-induced IL-6 production in vitro. LPS and NiNPs synergistically induced IL-6 mRNA and protein in BEAS-2B cells. TPCA-1, a dual inhibitor of IKK-2 and STAT3, blocked the synergistic increase in IL-6 caused by LPS and NiNPs, abolished STAT3 activation, and reduced C/EBPβ. Conversely, SC144, an inhibitor of the gp130 component of the IL-6 receptor, enhanced IL-6 production induced by LPS and NiNPs. Treatment of BEAS-2B cells with sex hormones (17β-estradiol, progesterone, or testosterone) or the anti-oxidant NAC, had no effect on IL-6 induction by LPS and NiNPs. These data suggest that LPS and NiNPs induce IL-6 via STAT3 and C/EBPβ in BEAS-2B cells. While BEAS-2B cells are a suitable model to study mechanisms of IL-6 production, they do not appear to be suitable for studying the effect of sex hormones., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Per- and polyfluoroalkyl substances (PFAS) have been associated with respiratory diseases in humans, yet the mechanisms through which PFAS cause susceptibility to inhaled agents is unknown. Herein, we investigated the effects of ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), an emerging PFAS, on the pulmonary immune response of mice to carbon black nanoparticles (CBNP). We hypothesized that pulmonary exposure to GenX would increase susceptibility to CBNP through suppression of innate immunity., Methods: Male C57BL/6 mice were exposed to vehicle, 4 mg/kg CBNP, 10 mg/kg GenX, or CBNP and GenX by oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) was collected at 1 and 14 days postexposure for cytokines and total protein. Lung tissue was harvested for histopathology, immunohistochemistry (Ki67 and phosphorylated (p)-STAT3), western blotting (p-STAT3 and p-NF-κB), and qRT-PCR for cytokine mRNAs., Results: CBNP increased CXCL-1 and neutrophils in BALF at both time points evaluated. However, GenX/CBNP co-exposure reduced CBNP-induced CXCL-1 and neutrophils in BALF. Moreover, CXCL-1, CXCL-2 and IL-1β mRNAs were increased by CBNP in lung tissue but reduced by GenX. Western blotting showed that CBNP induced p-NF-κB in lung tissue, while the GenX/CBNP co-exposed group displayed decreased p-NF-κB. Furthermore, mice exposed to GenX or GenX/CBNP displayed increased numbers of BALF macrophages undergoing mitosis and increased Ki67 immunostaining. This was correlated with increased p-STAT3 by western blotting and immunohistochemistry in lung tissue from mice co-exposed to GenX/CBNP., Conclusions: Pulmonary exposure to GenX suppressed CBNP-induced innate immune response in the lungs of mice yet promoted the proliferation of macrophages and lung epithelial cells.

Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mesothelioma is a cancer of the lung pleura primarily associated with inhalation of asbestos fibers. Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials that pose a potential risk for mesothelioma due to properties that are similar to asbestos. Inhaled MWCNTs migrate to the pleura in rodents and some types cause mesothelioma. Like asbestos, there is a diversity of MWCNT types. We investigated the neoplastic potential of tangled (tMWCNT) versus rigid (rMWCNT) after chronic exposure using serial passages of rat mesothelial cells in vitro. Normal rat mesothelial (NRM2) cells were exposed to tMWCNTs or rMWCNTs for 45 weeks over 85 passages to determine if exposure resulted in transformation to a neoplastic phenotype. Rat mesothelioma (ME1) cells were used as a positive control. Osteopontin (OPN) mRNA was assayed as a biomarker of transformation by real time quantitative polymerase chain reaction (qPCR) and transformation was determined by a cell invasion assay. Exposure to rMWCNTs, but not tMWCNTs, resulted in transformation of NRM2 cells into an invasive phenotype that was similar to ME1 cells. Moreover, exposure of NRM2 cells to rMWCNTs increased OPN mRNA that correlated with cellular transformation. These data suggest that OPN is a potential biomarker that should be further investigated to screen the carcinogenicity of MWCNTs in vitro., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

There is increasing evidence that inhaled multi-walled carbon nanotubes (MWCNTs) can have harmful effects on the respiratory system. Rodent studies suggest that individuals with asthma may be susceptible to the adverse pulmonary effects of MWCNTs. Asthma is an allergic lung disease characterized by a T H 2 immune response that results in chronic airway disease characterized by eosinophilic lung inflammation, airway mucous cell metaplasia, and airway fibrosis. Signal transducer and activator of transcription 6 (STAT6) is a transcription factor with multiple roles in T H 2 type inflammation. Herein we sought to examine the role of STAT6 in the exacerbation of house dust mite (HDM) allergen-induced allergic airway disease by MWCNTs. Male wild type (WT) and STAT6 knockout ( Stat6 KO) mice were dosed via intranasal aspiration on days 0, 2, 4, 14, 16 and 18 with either vehicle, HDM extract, MWCNTs, or a combination of HDM and MWCNTs. Necropsy was performed on day 21 to collect bronchoalveolar lavage fluid (BALF), serum and lung tissue. MWCNTs exacerbated HDM-induced allergic endpoints, including eosinophilic lung inflammation, mucous cell metaplasia, and serum IgE levels. HDM-induced eosinophilic lung inflammation, mucous cell metaplasia, and serum IgE and exacerbation of these endpoints by MWCNTs were ablated in Stat6 KO mice. In addition, airway fibrosis was significantly increased by the combination of HDM and MWCNTs in WT mice but not in Stat6 KO mice. These findings provide new mechanistic insight by demonstrating a requirement for STAT6 in MWCNT-induced exacerbation of allergic respiratory disease., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or relationships that could have appeared to influence the work reported in this paper.

Background: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs)., Methods and Results: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples., Conclusion: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

Background: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000)., Methods: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs., Results: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy., Conclusions: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Engineered nanomaterials (ENMs) are products of the emerging nanotechnology industry and many different types of ENMs have been shown to cause chronic inflammation in the lungs of rodents after inhalation exposure, suggesting a risk to human health. Due to the increasing demand and use of ENMs in a variety of products, a careful evaluation of the risks to human health is urgently needed. An assessment of the immunotoxicity of ENMs should consider susceptibility factors including sex, pre-existing diseases, deficiency of specific genes encoding proteins involved in the innate or adaptive immune response, and co-exposures to other chemicals. This review will address evidence from experimental animal models that highlights some important issues of susceptibility to chronic lung inflammation and systemic immune dysfunction after pulmonary exposure to ENMs.

Nickel nanoparticles (NiNPs) are increasingly used in nanotechnology applications, yet information on sex differences in NiNP-induced lung disease is lacking. The goal of this study was to explore mechanisms of susceptibility between male and female mice after acute or subchronic pulmonary exposure to NiNPs. For acute exposure, male and female mice received a single dose of NiNPs with or without LPS by oropharyngeal aspiration and were necropsied 24 h later. For subchronic exposure, mice received NiNPs with or without LPS six times over 3 weeks prior to necropsy. After acute exposure to NiNPs and LPS, male mice had elevated cytokines (CXCL1 and IL-6) and more neutrophils in bronchoalveolar lavage fluid (BALF), along with greater STAT3 phosphorylation in lung tissue. After subchronic exposure to NiNPs and LPS, male mice exhibited increased monocytes in BALF. Moreover, subchronic exposure of male mice to NiNP only induced higher CXCL1 and CCL2 in BALF along with increased alveolar infiltrates and CCL2 in lung tissue. STAT1 in lung tissue was induced by subchronic exposure to NiNPs in females but not males. Males had a greater induction of IL-6 mRNA in liver after acute exposure to NiNPs and LPS, and greater CCL2 mRNA in liver after subchronic NiNP exposure. These data indicate that susceptibility of males to acute lung inflammation involves enhanced neutrophilia with increased CXCL1 and IL-6/STAT3 signaling, whereas susceptibility to subchronic lung inflammation involves enhanced monocytic infiltration with increased CXCL1 and CCL2. STAT transcription factors appear to play a role in these sex differences. This study demonstrates sex differences in the lung inflammatory response of mice to NiNPs that has implications for human disease.

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m 3 of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 µm) composed of MWCNTs (average diameter 16 nm; average length 2.4 µm; 0.52% Ni). Mice then received 25 µg of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53 +/- ) mice has not been investigated. We hypothesized that p53 +/- mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53 +/+ ) or p53 +/- mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53 +/+ or p53 +/- mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53 +/- and p53 +/+ mice. A constitutively larger area of CD45R + /CD3 + lymphoid tissue was observed in p53 +/- mice compared to p53 +/+ mice. Importantly, p53 +/- mice had larger granulomas induced by rMWCNTs as compared to p53 +/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

Carbon nanotubes (CNTs) are engineered nanomaterials (ENMs) with numerous beneficial applications. However, they could pose a risk to human health from occupational or consumer exposures. Rodent models demonstrate that exposure to CNTs via inhalation, instillation, or aspiration results in pulmonary fibrosis. The severity of the fibrogenic response is determined by various physicochemical properties of the nanomaterial such as residual metal catalyst content, rigidity, length, aggregation status, or surface charge. CNTs are also increasingly functionalized post-synthesis with organic or inorganic agents to modify or enhance surface properties. The mechanisms of CNT-induced fibrosis involve oxidative stress, innate immune responses of macrophages, cytokine and growth factor production, epithelial cell injury and death, expansion of the pulmonary myofibroblast population, and consequent extracellular matrix accumulation. A comprehensive understanding of how physicochemical properties affect the fibrogenic potential of various types of CNTs should be considered in combination with genetic variability and gain or loss of function of specific genes encoding secreted cytokines, enzymes, or intracellular cell signaling molecules. Here, we cover the current state of the literature on mechanisms of CNT-exposed pulmonary fibrosis in rodent models with a focus on physicochemical characteristics as principal drivers of the mechanisms leading to pulmonary fibrosis. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials., (© 2017 Wiley Periodicals, Inc.)

Purpose of Review: The explosive growth of the nanotechnology industry has necessitated the examination of engineered nanomaterials (ENMs) for their toxicity. The unique properties that make ENMs useful also make them a health risk, and individuals with pre-existing diseases such as asthma are likely more susceptible. This review summarizes the current literature on the ability of ENMs to both exacerbate and directly cause asthma., Recent Findings: Recent studies highlight the ability of metal nanoparticles (NPs) and carbon nanotubes (CNTs) to not only exacerbate pre-existing asthma in animal models but also initiate allergic airway disease directly. CNTs alone are shown to cause airway mucus production, elevated serum IgE levels, and increased T H 2 cytokine levels, all key indicators of asthma. The ability of ENMs to modulate the immune response in asthma varies depending on their physicochemical properties and exposure timing. CNTs consistently exacerbate asthma, as do Ni and TiO 2 NPs, whereas some NPs like Au attenuate asthma. Evidence is strong that ENMs can contribute to allergic airway disease; however, more work is required to determine their mechanisms, and more epidemiological studies are needed to validate results from animal models.

Background: Pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs) is influenced by physicochemical characteristics and genetic susceptibility. We hypothesized that contrasting rigidities of tangled (t) versus rod-like (r) MWCNTs would result in differing immunologic or fibrogenic responses in mice and that these responses would be exaggerated in transgenic mice lacking the signal transducer and activator of transcription-1 (STAT1), a susceptible mouse model of pulmonary fibrosis., Methods: Male wild type (Stat1 +/+ ) and STAT1-deficient (Stat1 -/- ) mice were exposed to 4 mg/kg tMWCNTs, rMWCNTs, or vehicle alone via oropharyngeal aspiration and evaluated for inflammation at one and 21 days post-exposure via histopathology, differential cell counts, and cytokine levels in bronchoalveolar lavage fluid (BALF). Granuloma formation, mucous cell metaplasia, and airway fibrosis were evaluated by quantitative morphometry. Airway epithelial cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Cytokine protein levels in BALF and serum IgE levels were measured by ELISA. Lung protein Smad2/3 levels and activation were measured by Western blot. Lung mRNAs were measured by PCR., Results: There was a 7-fold difference in rigidity between tMWCNTs and rMWCNTs as determined by static bending ratio. Both MWCNT types resulted in acute inflammation (neutrophils in BALF) after one-day post-exposure, yet only rMWCNTs resulted in chronic inflammation at 21 days as indicated by neutrophil influx and larger granulomas. Both MWCNTs induced BrdU uptake in airway epithelial cells, with the greatest proliferative response observed in rMWCNT-exposed mice after one-day. Only rMWCNTs induced mucous cell metaplasia, but this index was not different between genotypes. Stat1 -/- mice had higher levels of baseline serum IgE than Stat1 +/+ mice. Greater airway fibrosis was observed with rMWCNTs compared to tMWCNTs, and exaggerated airway fibrosis was seen in the Stat1 -/- mouse lungs with rMWCNTs but not tMWCNTs. Increased fibrosis correlated with elevated levels of TGF-β1 protein levels in the BALF of Stat1 -/- mice exposed to rMWCNTs and increased lung Smad2/3 phosphorylation., Conclusions: Rigidity plays a key role in the toxicity of MWCNTs and results in increased inflammatory, immunologic, and fibrogenic effects in the lung. STAT1 is an important protective factor in the fibroproliferative response to rMWCNTs, regulating both induced TGF-β1 production and Smad2/3 phosphorylation status. Therefore, both rigidity and genetic susceptibility should be major considerations for risk assessment of MWCNTs.

Carbon nanotubes (CNTs), a prototypical engineered nanomaterial, have been increasingly manufactured for a variety of novel applications over the past two decades. However, since CNTs possess fiber-like shape and cause pulmonary fibrosis in rodents, there is concern that mass production of CNTs will lead to occupational exposure and associated pulmonary diseases. The aim of this study was to use contemporary proteomics to investigate the mechanisms of cellular response in E10 mouse alveolar epithelial cells in vitro after exposure to multi-walled CNTs (MWCNTs) that were functionalized by atomic layer deposition (ALD). ALD is a method used to generate highly uniform and conformal nanoscale thin-film coatings of metals to enhance novel conductive properties of CNTs. We hypothesized that specific types of metal oxide coatings applied to the surface of MWCNTs by ALD would determine distinct proteomic profiles in mouse alveolar epithelial cells in vitro that could be used to predict oxidative stress and pulmonary inflammation. Uncoated (U)-MWCNTs were functionalized by ALD with zinc oxide (ZnO) to yield Z-MWCNTs or aluminum oxide (Al 2 O 3 ) to yield A-MWCNTs. Significant differential protein expression was found in the following critical pathways: mTOR/eIF4/p70S6K signaling and Nrf-2 mediated oxidative stress response increased following exposure to Z-MWCNTs, interleukin-1 signaling increased following U-MWCNT exposure, and inhibition of angiogenesis by thrombospondin-1, oxidative phosphorylation, and mitochondrial dysfunction increased following A-MWCNT exposure. This study demonstrates that specific types of metal oxide thin film coatings applied by ALD produce distinct cellular and biochemical responses related to lung inflammation and fibrosis compared to uncoated MWCNT exposure in vitro.

Commercialization of multiwalled carbon nanotubes (MWCNT)-based applications has been hampered by concerns regarding their lung toxicity potential. Hyaluronic acid (HA) is a ubiquitously found polysaccharide, which is anti-inflammatory in its native high molecular weight form. HA-functionalized smart MWCNTs have shown promise as tumor-targeting drug delivery agents and can enhance bone repair and regeneration. However, it is unclear whether HA functionalization could reduce the pulmonary toxicity potential of MWCNTs. Using in vivo and in vitro approaches, we investigated the effectiveness of MWCNT functionalization with HA in increasing nanotube biocompatibility and reducing lung inflammatory and fibrotic effects. We utilized three-dimensional cultures of differentiated primary human bronchial epithelia to translate findings from rodent assays to humans. We found that HA functionalization increased stability and dispersion of MWCNTs and reduced postexposure lung inflammation, fibrosis, and mucus cell metaplasia compared with nonfunctionalized MWCNTs. Cocultures of fully differentiated bronchial epithelial cells (cultivated at air-liquid interface) and human lung fibroblasts (submerged) displayed significant reduction in injury, oxidative stress, as well as pro-inflammatory gene and protein expression after exposure to HA-functionalized MWCNTs compared with MWCNTs alone. In contrast, neither type of nanotubes stimulated cytokine production in primary human alveolar macrophages. In aggregate, our results demonstrate the effectiveness of HA functionalization as a safer design approach to eliminate MWCNT-induced lung injury and suggest that HA functionalization works by reducing MWCNT-induced epithelial injury.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Background: Atomic layer deposition (ALD) is a method for applying conformal nanoscale coatings on three-dimensional structures. We hypothesized that surface functionalization of multi-walled carbon nanotubes (MWCNTs) with polycrystalline ZnO by ALD would alter pro-inflammatory cytokine expression by human monocytes in vitro and modulate the lung and systemic immune response following oropharyngeal aspiration in mice., Methods: Pristine (U-MWCNTs) were coated with alternating doses of diethyl zinc and water over increasing ALD cycles (10 to 100 ALD cycles) to yield conformal ZnO-coated MWCNTs (Z-MWCNTs). Human THP-1 monocytic cells were exposed to U-MWCNTs or Z-MWCNTs in vitro and cytokine mRNAs measured by Taqman real-time RT-PCR. Male C57BL6 mice were exposed to U- or Z-MWCNTs by oropharyngeal aspiration (OPA) and lung inflammation evaluated at one day post-exposure by histopathology, cytokine expression and differential counting of cells in bronchoalveolar lavage fluid (BALF) cells. Lung fibrosis was evaluated at 28 days. Cytokine mRNAs (IL-6, IL-1β, CXCL10, TNF-α) in lung, heart, spleen, and liver were quantified at one and 28 days. DNA synthesis in lung tissue was measured by bromodeoxyuridine (BrdU) uptake., Results: ALD resulted in a conformal coating of MWCNTs with ZnO that increased proportionally to the number of coating cycles. Z-MWCNTs released Zn(+2) ions in media and increased IL-6, IL-1β, CXCL10, and TNF-α mRNAs in THP-1 cells in vitro. Mice exposed to Z-MWCNTs by OPA had exaggerated lung inflammation and a 3-fold increase in monocytes and neutrophils in BALF compared to U-MWCNTs. Z-MWCNTs, but not U-MWCNTs, induced IL-6 and CXCL10 mRNA and protein in the lungs of mice and increased IL-6 mRNA in heart and liver. U-MWCNTs but not Z-MWCNTs stimulated airway epithelial DNA synthesis in vivo. Lung fibrosis at 28 days was not significantly different between mice treated with U-MWCNT or Z-MWCNT., Conclusions: Pulmonary exposure to ZnO-coated MWCNTs produces a systemic acute phase response that involves the release of Zn(+2), lung epithelial growth arrest, and increased IL-6. ALD functionalization with ZnO generates MWCNTs that possess increased risk for human exposure.

Asthma is characterized by a T helper type 2 phenotype and by chronic allergen-induced airway inflammation (AAI). Environmental exposure to air pollution ultrafine particles (i.e., nanoparticles) exacerbates AAI, and a concern is possible exacerbation posed by engineered nanoparticles generated by emerging nanotechnologies. Signal transducer and activator of transcription (STAT) 1 is a transcription factor that maintains T helper type 1 cell development. However, the role of STAT1 in regulating AAI or exacerbation by nanoparticles has not been explored. In this study, mice with whole-body knockout of the Stat1 gene (Stat1(-/-)) or wild-type (WT) mice were sensitized to ovalbumin (OVA) allergen and then exposed to multiwalled carbon nanotubes (MWCNTs) by oropharygneal aspiration. In Stat1(-/-) and WT mice, OVA increased eosinophils in bronchoalveolar lavage fluid, whereas MWCNTs increased neutrophils. Interestingly, OVA sensitization prevented MWCNT-induced neutrophilia and caused only eosinophilic inflammation. Stat1(-/-) mice displayed increased IL-13 in bronchoalveolar lavage fluid at 1 day compared with WT mice after treatment with OVA or OVA and MWCNTs. At 21 days, the lungs of OVA-sensitized Stat1(-/-) mice displayed increased eosinophilia, goblet cell hyperplasia, airway fibrosis, and subepithelial apoptosis. MWCNTs further increased OVA-induced goblet cell hyperplasia, airway fibrosis, and apoptosis in Stat1(-/-) mice at 21 days. These changes corresponded to increased levels of profibrogenic mediators (transforming growth factor-β1, TNF-α, osteopontin) but decreased IL-10 in Stat1(-/-) mice. Finally, fibroblasts isolated from the lungs of Stat1(-/-) mice produced significantly more collagen mRNA and protein in response to transforming growth factor-β1 compared with WT lung fibroblasts. Our results support a protective role for STAT1 in chronic AAI and exacerbation of remodeling caused by MWCNTs.

The hormone cortisol is often believed to play a pivotal role in the effects of stress on human cognition. This meta-analysis is an attempt to determine the effects of acute cortisol administration on core executive functions. Drawing on both rodent and stress literatures, we hypothesized that acute cortisol administration would impair working memory and set-shifting but enhance inhibition. Additionally, because cortisol is thought to exert different nongenomic (rapid) and genomic (slow) effects, we further hypothesized that the effects of cortisol would differ as a function of the delay between cortisol administration and cognitive testing. Although the overall analyses were nonsignificant, after separating the rapid, nongenomic effects of cortisol from the slower, genomic effects of cortisol, the rapid effects of cortisol enhanced response inhibition, g+ = 0.113, p=.016, but impaired working memory, g+ = -0.315, p=.008, although these effects reversed over time. Contrary to our hypotheses, there was no effect of cortisol administration on set-shifting. Thus, although we did not find support for the idea that increases in cortisol influence set-shifting, we found that acute increases in cortisol exert differential effects on working memory and inhibition over time., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Multi-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation., Methods: THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses., Results: Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs., Conclusions: These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.

Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher's exact test to determine differentially regulated species. Significant proteins by Fisher's exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Multi-walled carbon nanotubes (MWCNTs) pose a possible human health risk for lung disease as a result of inhalation exposure. Mice exposed to MWCNTs develop pulmonary fibrosis. Lung macrophages engulf MWCNTs and produce pro-fibrogenic cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and osteopontin (OPN). Atomic layer deposition (ALD) is a novel process used to enhance functional properties of MWCNTs, yet the consequence of ALD-modified MWCNTs on macrophage biology and fibrosis is unknown., Methods: The purpose of this study was to determine whether ALD coating with aluminum oxide (Al2O3) would alter the fibrogenic response to MWCNTs and whether cytokine expression in human macrophage/monocytes exposed to MWCNTs in vitro would predict the severity of lung fibrosis in mice. Uncoated (U)-MWCNTs or ALD-coated (A)-MWCNTs were incubated with THP-1 macrophages or human peripheral blood mononuclear cells (PBMC) and cell supernatants assayed for cytokines by ELISA. C57BL6 mice were exposed to a single dose of A- or U-MWCNTs by oropharyngeal aspiration (4 mg/kg) followed by evaluation of histopathology, lung inflammatory cell counts, and cytokine levels at day 1 and 28 post-exposure., Results: ALD coating of MWCNTs with Al2O3 enhanced IL-1β secretion by THP-1 and PBMC in vitro, yet reduced protein levels of IL-6, TNF-α, and OPN production by THP-1 cells. Moreover, Al2O3 nanoparticles, but not carbon black NPs, increased IL-1β but decreased OPN and IL-6 in THP-1 and PBMC. Mice exposed to U-MWCNT had increased levels of all four cytokines assayed and developed pulmonary fibrosis by 28 days, whereas ALD-coating significantly reduced fibrosis and cytokine levels at the mRNA or protein level., Conclusion: These findings indicate that ALD thin film coating of MWCNTs with Al2O3 reduces fibrosis in mice and that in vitro phagocyte expression of IL-6, TNF-α, and OPN, but not IL-1β, predict MWCNT-induced fibrosis in the lungs of mice in vivo.

The nanotechnology revolution offers enormous societal and economic benefits for innovation in the fields of engineering, electronics, and medicine. Nevertheless, evidence from rodent studies show that biopersistent engineered nanomaterials (ENMs) stimulate immune, inflammatory, and fibroproliferative responses in the lung, suggesting possible risks for lung diseases or systemic immune disorders as a consequence of occupational, environmental, or consumer exposure. Due to their nanoscale dimensions and increased surface area per unit mass, ENMs have a much greater potential to reach the distal regions of the lung and generate ROS. High aspect ratio ENMs (e.g., nanotubes, nanofibers) activate inflammasomes in macrophages, triggering IL-1β release and neutrophilic infiltration into the lungs. Moreover, some ENMs alter allergen-induced eosinophilic inflammation by immunostimulation, immunosuppression, or modulating the balance between Th1, Th2, and Th17 cells, thereby influencing the nature of the inflammatory response. ENMs also migrate from the lungs across epithelial, endothelial, or mesothelial barriers to stimulate or suppress systemic immune responses.

Background: In vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes., Objectives: We investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response., Methods: Primary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 μg/mL equivalent to 0.37-6.0 μg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms., Results: MWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1β, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used., Conclusions: Taken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.

Background: Nickel nanoparticles (NiNPs) are increasingly used in a variety of industrial applications, including the manufacturing of multi-walled carbon nanotubes (MWCNTs). While occupational nickel exposure is a known cause of pulmonary alveolitis, fibrosis, and cancer, the health risks of NiNPs are not well understood, especially in susceptible individuals such as asthmatics. The T-box transcription factor Tbx21 (T-bet) maintains Th1 cell development and loss of T-bet is associated with a shift towards Th2 type allergic airway inflammation that characterizes asthma. The purpose of this study was to determine the role of T-bet in susceptibility to lung remodeling by NiNPs or MWCNTs., Methods: Wild-type (WT) and T-bet-/- mice were exposed to NiNPs or MWCNTs (4 mg/kg) by oropharyngeal aspiration (OPA). Necropsy was performed at 1 and 21 days. Bronchoalveolar lavage fluid (BALF) was collected for differential counting of inflammatory cells and for measurement of cytokines by ELISA. The left lung was collected for histopathology. The right lung was analyzed for cytokine or mucin (MUC5AC and MUC5B) mRNAs., Results: Morphometry of alcian-blue/periodic acid Schiff (AB/PAS)-stained lung tissue showed that NiNPs significantly increased mucous cell metaplasia in T-bet-/- mice at 21 days (p < 0.001) compared to WT mice, and increased MUC5AC and MUC5B mRNAs (p < 0.05). MWCNTs also increased mucous cell metaplasia in T-bet-/- mice, but to a lesser extent than NiNPs. Chronic alveolitis was also increased by NiNPs, but not MWCNTs, in T-bet-/- mice compared to WT mice at 21 days (P < 0.001). NiNPs also increased IL-13 and eosinophils (p < 0.001) in BALF from T-bet-/- mice after 1 day. Interestingly, the chemokine CCL2 in the BALF of T-bet-/- mice was increased at 1 and 21 days (p < 0.001 and p < 0.05, respectively) by NiNPs, and to a lesser extent by MWCNTs at 1 day. Treatment of T-bet-/- mice with a monoclonal anti-CCL2 antibody enhanced NiNP-induced mucous cell metaplasia and MUC5AC mRNA levels (p < 0.05), yet marginally reduced NiNP-induced alveolitis., Conclusion: These findings identify T-bet as a potentially important susceptibility factor for NiNP exposure and to a lesser extent for MWCNT exposure, and suggests that individuals with asthma are at greater risk.

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

There has been a conceptual shift in toxicological studies from describing what happens to explaining how the adverse outcome occurs, thereby enabling a deeper and improved understanding of how biomolecular and mechanistic profiling can inform hazard identification and improve risk assessment. Compared to traditional toxicology methods, which have a heavy reliance on animals, new approaches to generate toxicological data are becoming available for the safety assessment of chemicals, including high-throughput and high-content screening (HTS, HCS). With the emergence of nanotechnology, the exponential increase in the total number of engineered nanomaterials (ENMs) in research, development, and commercialization requires a robust scientific approach to screen ENM safety in humans and the environment rapidly and efficiently. Spurred by the developments in chemical testing, a promising new toxicological paradigm for ENMs is to use alternative test strategies (ATS), which reduce reliance on animal testing through the use of in vitro and in silico methods such as HTS, HCS, and computational modeling. Furthermore, this allows for the comparative analysis of large numbers of ENMs simultaneously and for hazard assessment at various stages of the product development process and overall life cycle. Using carbon nanotubes as a case study, a workshop bringing together national and international leaders from government, industry, and academia was convened at the University of California, Los Angeles, to discuss the utility of ATS for decision-making analyses of ENMs. After lively discussions, a short list of generally shared viewpoints on this topic was generated, including a general view that ATS approaches for ENMs can significantly benefit chemical safety analysis.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity., Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability., Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells., Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β., Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity., Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) [anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid "functionalized" (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment., Results: TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice., Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.

Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pleural diseases (fibrosis and mesothelioma) are a major concern for individuals exposed by inhalation to certain types of particles, metals, and fibers. Increasing attention has focused on the possibility that certain types of engineered nanoparticles (NPs), especially those containing nickel, might also pose a risk for pleural diseases. Platelet-derived growth factor (PDGF) is an important mediator of fibrosis and cancer that has been implicated in the pathogenesis of pleural diseases. In this study, we discovered that PDGF synergistically enhanced nickel NP (NiNP)-induced increases in mRNA and protein levels of the profibrogenic chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2), and the antifibrogenic IFN-inducible CXC chemokine (CXCL10) in normal rat pleural mesothelial 2 (NRM2) cells in vitro. Carbon black NPs (CBNPs), used as a negative control NP, did not cause a significant increase in CCL2 or CXCL10 in the absence or presence of PDGF. NiNPs prolonged PDGF-induced phosphorylation of the mitogen-activated protein kinase family termed extracellular signal-regulated kinases (ERK)-1 and -2 for up to 24 hours, and NiNPs also synergistically increased PDGF-induced hypoxia-inducible factor (HIF)-1α protein levels in NRM2 cells. Inhibition of ERK-1,2 phosphorylation with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, blocked the synergistic increase in CCL2, CXCL10, and HIF-1α levels induced by PDGF and NiNPs. Moreover, the antioxidant, N-acetyl-L-cysteine (NAC), significantly reduced HIF-1α, ERK-1,2 phosphorylation, and CCL2 protein levels that were synergistically increased by the combination of PDGF and NiNPs. These data indicate that NiNPs enhance the activity of PDGF in regulating chemokine production in NRM2 cells through a mechanism involving reactive oxygen species generation and prolonged activation of ERK-1,2.

Background: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2)., Methods: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively., Results: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction., Conclusion: This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

Remifentanil patient-controlled analgesia is well established in many centres and provides satisfactory pain relief for many women in labour. We describe a patient using remifentanil patient-controlled analgesia who suffered a respiratory arrest requiring a brief period of ventilation. In our institution, remifentanil patient-controlled analgesia has been offered to women in labour since 2009. Up to this point, we had not observed any critical incidents in over 130 patients using this mode of analgesia in our labour suite., (Anaesthesia © 2012 The Association of Anaesthetists of Great Britain and Ireland.)

We developed a dispersal method for multiwalled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on profibrogenic responses in tissue culture cells and in mouse lung. We demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA, and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells, and alveolar macrophages (AM) and induced more prominent TGF-β1 and IL-1β production in vitro and TGF-β1, IL-1β, and PDGF-AA production in vivo than nondispersed tubes. Moreover, there was good agreement between the profibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bioprocessed by AM and induced less prominent collagen deposition at sites of nongranulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects profibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

Nanoparticle drug-delivery systems offer the potential for improved efficacy of treatment, and yet there are also potential risks associated with these novel therapeutic strategies. An attractive property of carbon nanotubes (CNTs) is that the tube- or fiber-like structure allows for extensive functionalization and loading of cargo. However, a large body of evidence indicates that CNTs may have adverse effects if used in drug delivery as they have been shown to cause pulmonary fibrosis and exacerbate lung disease in rodents with pre-existing lung diseases. Major factors that cause these toxic effects are the high aspect ratio, durability and residual metal content that generate reactive oxygen species. Therefore, careful consideration should be given to the possibility that lung inflammation or fibrosis could be significant side effects caused by a CNT-based drug-delivery system, thereby outweighing any potential beneficial effects of therapeutic treatment. However, functionalization of CNTs to modulate aspect ratio, biodegradability and to remove residual metals could allow for safe design of CNTs for use in drug delivery in certain circumstances.

Background: Lamotrigine and quetiapine are commonly used in bipolar disorder, but there are no published systematic studies of their use in combination for treatment-resistant bipolar depression., Methods: We studied 39 trials in outpatients (15 with bipolar I disorder, 22 with bipolar II disorder, and 1 with bipolar disorder not otherwise specified; 1 patient had 2 trials) with depression resistant to quetiapine or lamotrigine who were taking a mean of 1.7 other prescription psychotropic medications. Patients were given either open-label lamotrigine or quetiapine naturalistically, for up to 12 weeks of combination therapy., Results: Lamotrigine (mean dose, 204.2 mg/d) plus quetiapine (mean dose, 188.5 mg/d) increased the euthymia rate (0.0% to 46.2%), decreased syndromal (79.5% to 30.8%) and subsyndromal (20.5% to 15.4%) depression rates, and improved Clinical Global Impression-Severity (mean change, -1.0) and Global Assessment of Functioning (mean change, +5.9) scores. Approximately one-fifth of patients discontinued therapy (20.5%) or required subsequent additional pharmacotherapy (20.5%). Only 10.3% discontinued due to adverse effects, and there was no significant change in mean body weight., Conclusions: The findings of this uncontrolled open pilot study must be viewed with caution. However, randomized, double-blind, placebo-controlled studies are warranted to confirm the possibility that combination therapy with lamotrigine and quetiapine is effective and well tolerated in patients with treatment-resistant bipolar depression.

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.

Objective: To assess quetiapine effectiveness in bipolar disorder (BD) patients in a clinical setting., Methods: We naturalistically administered open quetiapine to outpatients assessed with the Systematic Treatment Enhancement Program for BD (STEP-BD) Affective Disorders Evaluation, and monitored longitudinally with the STEP-BD Clinical Monitoring Form., Results: 96 patients (36 BD I, 50 BD II, 9 BD NOS, 1 Schizoaffective Bipolar Type, mean ± SD age 42.3 ± 13.8 years, 66.7% female) received quetiapine, combined with an average of 2.5 (in 66.7% of patients at least 2) other psychotropic medications and 0.9 non-psychotropic medications, started most often during depressive symptoms (53.1%) or euthymia (37.5%). Mean quetiapine duration and final dose were 385 days and 196 mg/day (50.0% of patients took ≤75 mg/day). Quetiapine was discontinued in 38.5% of trials, after on average 307 days, most often (in 19.8%) due to CNS adverse effects (primarily sedation). In 38.5% of trials quetiapine was continued on average 328 days with no subsequent psychotropic added. In 22.9% quetiapine was continued on average 613 days, but had subsequent psychotropic added after on average 113 days, most often for depressive symptoms. In 67 trials started at Stanford, quetiapine tended to primarily maintain euthymia and relieve depressive symptoms. In 29 trials started prior to Stanford, continuing quetiapine tended to primarily maintain euthymia and relieve mood elevation symptoms. Aside from sedation, quetiapine was generally well tolerated., Conclusions: In bipolar disorder outpatients quetiapine had a moderate (38.5%, with 385-day mean duration) discontinuation rate, and commonly did not require subsequent additional pharmacotherapy, suggesting effectiveness in a clinical setting., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Fibrotic reactions in the airways of the lung or the pulmonary interstitium are a common pathologic outcome after exposure to a wide variety of toxic agents, including metals, particles or fibers. The survival of mesenchymal cells (fibroblasts and myofibroblasts) is a key factor in determining whether a fibroproliferative response that occurs after toxic injury to the lung will ultimately resolve or progress to a pathologic state. Several polypeptide growth factors, including members of the platelet-derived growth factor (PDGF) family and the epidermal growth factor (EGF) family, are prosurvival factors that stimulate a replicative and migratory mesenchymal cell phenotype during the early stages of lung fibrogenesis. This replicative phenotype can progress to a matrix synthetic phenotype in the presence of transforming growth factor-β1 (TGF-β1). The resolution of a fibrotic response requires growth arrest and apoptosis of mesenchymal cells, whereas progressive chronic fibrosis has been associated with mesenchymal cell resistance to apoptosis. Mesenchymal cell survival or apoptosis is further influenced by cytokines secreted during Th1 inflammation (e.g., IFN-γ) or Th2 inflammation (e.g., IL-13) that modulate the expression of growth factor activity through the STAT family of transcription factors. Understanding the mechanisms that regulate the survival or death of mesenchymal cells is central to ultimately developing therapeutic strategies for lung fibrosis.

Engineered multi-walled carbon nanotubes (MWCNT) represent a possible health risk for pulmonary fibrosis due to their fiber-like shape and potential for persistence in the lung. We postulated that bacterial lipopolysaccharide (LPS), a ubiquitous agent in the environment that causes lung inflammation, would enhance fibrosis caused by MWCNT. Rats were exposed to LPS and then intratracheally instilled with MWCNT or carbon black (CB) nanoparticles 24 hours later. Pulmonary fibrosis was observed 21 days after MWCNT exposure, but not with CB. LPS alone caused no fibrosis but enhanced MWCNT-induced fibrosis. LPS plus CB did not significantly increase fibrosis. MWCNT increased platelet-derived growth factor-AA (PDGF-AA), a major mediator of fibrosis. PDGF-AA production in response to MWCNT, but not CB, was synergistically enhanced by LPS. Immunostaining showed PDGF-AA in bronchiolar epithelial cells and macrophages. Since macrophages engulfed MWCNT, were positive for PDGF-AA, and mediate fibroblast responses, experiments were performed with rat lung macrophages (NR8383 cells) and rat lung fibroblasts in vitro. LPS exposure increased PDGF-A mRNA levels in NR8383 cells and enhanced MWCNT-induced PDGF-A mRNA levels. Moreover, LPS increased MWCNT- or CB-induced PDGF receptor-alpha (PDGF-Ralpha) mRNA in fibroblasts. Our data suggest that LPS exacerbates MWCNT-induced lung fibrosis by amplifying production of PDGF-AA in macrophages and epithelial cells, and by increasing PDGF-Ralpha on pulmonary fibroblasts. Our findings also suggest that individuals with pre-existing pulmonary inflammation are at greater risk for the potential adverse effects of MWCNT.

Nanotechnology holds the promise of revolutionizing our society, bringing numerous beneficial innovations to improve structural materials, electronics, energy, medical imaging, and drug delivery, among other applications. However, nanomaterials present potential safety concerns, and there is accumulating evidence to suggest that nanoparticles may exert adverse effects on the lung and other organ systems. This article will overview the potential risks of engineered nanoparticles and nanotechnology on the respiratory system and highlight recent findings related to pulmonary and systemic effects of inhaled nanoparticles. Special emphasis will be given to carbon nanotubes and the possibility that these nanoparticles could represent an emerging risk for environmental and occupational lung disease, especially in individuals with pre-existing respiratory diseases such as asthma.

Background: Vanadium pentoxide (V2O5) exposure is a cause of occupational bronchitis and airway fibrosis. Respiratory syncytial virus (RSV) is a ubiquitous pathogen that causes airway inflammation. It is unknown whether individuals with pre-existing respiratory viral infection are susceptible to V2O5-induced bronchitis. We hypothesized that respiratory viral infection will exacerbate vanadium-induced lung fibrosis., Methods: In this study we investigated the effect of RSV pre- or post-exposure to V2O5 in male AKR mice. Mice were pre-exposed by intranasal aspiration to RSV or media vehicle prior to intranasal aspiration of V2O5 or saline vehicle at day 1 or day 7. A parallel group of mice were treated first with V2O5 or saline vehicle at day 1 and day 7 then post-exposed to RSV or media vehicle at day 8., Results: V2O5-induced airway inflammation and fibrosis were decreased by RSV pre- or post-exposure. Real time quantitative RT-PCR showed that V2O5 significantly increased lung mRNAs encoding pro-fibrogenic growth factors (TGF-beta1, CTGF, PDGF-C) and collagen (Col1A2), but also increased mRNAs encoding anti-fibrogenic type I interferons (IFN-alpha, -beta) and IFN-inducible chemokines (CXCL9 and CXCL10). RSV pre- or post-exposure caused a significantly reduced mRNAs of pro-fibrogenic growth factors and collagen, yet reduced RNA levels of anti-fibrogenic interferons and CXC chemokines., Conclusions: Collectively these data suggest that RSV infection reduces the severity of V2O5-induced fibrosis by suppressing growth factors and collagen genes. However, RSV suppression of V2O5-induced IFNs and IFN-inducible chemokines suggests that viral infection also suppresses the innate immune response that normally serves to resolve V2O5-induced fibrosis.

Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work suggests that minimizing inhalation of nanotubes during handling is prudent until further long-term assessments are conducted.