Development of a lung-liver in vitro coculture model for inhalation-like toxicity assessment.

Animal models are considered prime study models for inhalation-like toxicity assessment. However, in light of animal experimentation reduction (3Rs), we developed and investigated an alternative in vitro method to study systemic-like responses to inhalation-like exposures. A coculture platform was established to emulate inter-organ crosstalks between a pulmonary barrier, which constitutes the route of entry of inhaled compounds, and the liver, which plays a major role in xenobiotic metabolism. Both compartments (Calu-3 insert and HepG2/C3A biochip) were jointly cultured in a dynamically-stimulated environment for 72 h. The present model was characterized using acetaminophen (APAP), a well-documented hepatotoxicant, to visibly assess the passage and circulation of a xenobiotic through the device. Based on viability and functionality parameters the coculture model showed that the bronchial barrier and the liver biochip can successfully be maintained viable and function in a dynamic coculture setting for 3 days. In a stress-induced environment, present results reported that the coculture model emulated active and functional in vitro crosstalk that seemingly was responsive to xenobiotic exposure doses. The hepatic and bronchial cellular responses to xenobiotic exposure were modified in the coculture setting as they displayed earlier and stronger detoxification processes, highlighting active and functional organ crosstalk between both compartments.

Nose-only inhalations of high-dose alumina nanoparticles/hydrogen chloride gas mixtures induce strong pulmonary pro-inflammatory response: a pilot study.

OBJECTIVE: Solid composite propellants combustion, in aerospace and defense fields, can lead to complex aerosols emission containing high concentrations of alumina nanoparticles (Al2O3 NPs) and hydrogen chloride gas (HClg). Exposure to these mixtures by inhalation is thus possible but literature data toward their pulmonary toxicity are missing. To specify hazards resulting from these combustion aerosols, a pilot study was implemented. MATERIALS AND METHODS: Male Wistar rats were nose-only exposed to Al2O3 NPs (primary size 13 nm, 10 g/L suspension leading to 20.0-22.1 mg/m3 aerosol) and/or to HClg aerosols (5 ppm target concentration) following two exposure scenarios (single exposures (SE) or repeated exposures (RE)). Bronchoalveolar lavage fluids (BALF) content and lungs histopathology were analyzed 24 h after exposures. RESULTS: Repeated co-exposures increased total proteins and LDH concentrations in BALF indicating alveolar-capillary barrier permeabilization and cytolysis. Early pulmonary inflammation was induced after RE to Al2O3 NPs ± HClg resulting in PMN, TNF-α, IL-1ß, and GRO/KC increases in BALF. Both exposure scenarios resulted in pulmonary histopathological lesions (vascular congestions, bronchial pre-exfoliations, vascular and interalveolar septum edemas). Lung oxidative damages were observed in situ following SE. CONCLUSION: Observed biological effects are dependent on both aerosol content and exposure scenario. Results showed an important pro-inflammatory effect of Al2O3 NPs/HClg mixtures on the lungs of rat 24 h after exposure. This pilot study raises concerns toward potential long-term pulmonary toxicity of combustion aerosols and highlights the importance for further studies to be led in order to define dose limitations and exposure thresholds for risk management at the work place.

Improving Quality in Nanoparticle-Induced Cytotoxicity Testing by a Tiered Inter-Laboratory Comparison Study.

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.

Predicting the in vivo pulmonary toxicity induced by acute exposure to poorly soluble nanomaterials by using advanced in vitro methods.

BACKGROUND: Animal models remain at that time a reference tool to predict potential pulmonary adverse effects of nanomaterials in humans. However, in a context of reduction of the number of animals used in experimentation, there is a need for reliable alternatives. In vitro models using lung cells represent relevant alternatives to assess potential nanomaterial acute toxicity by inhalation, particularly since advanced in vitro methods and models have been developed. Nevertheless, the ability of in vitro experiments to replace animal experimentation for predicting potential acute pulmonary toxicity in human still needs to be carefully assessed. The aim of the study was to evaluate the differences existing between the in vivo and the in vitro approaches for the prediction of nanomaterial toxicity and to find advanced methods to enhance in vitro predictivity. For this purpose, rats or pneumocytes in co-culture with macrophages were exposed to the same poorly soluble and poorly toxic TiO2 and CeO2 nanomaterials, by the respiratory route in vivo or using more or less advanced methodologies in vitro. After 24 h of exposure, biological responses were assessed focusing on pro-inflammatory effects and quantitative comparisons were performed between the in vivo and in vitro methods, using compatible dose metrics. RESULTS: For each dose metric used (mass/alveolar surface or mass/macrophage), we observed that the most realistic in vitro exposure method, the air-liquid interface method, was the most predictive of in vivo effects regarding biological activation levels. We also noted less differences between in vivo and in vitro results when doses were normalized by the number of macrophages rather than by the alveolar surface. Lastly, although we observed similarities in the nanomaterial ranking using in vivo and in vitro approaches, the quality of the data-set was insufficient to provide clear ranking comparisons. CONCLUSIONS: We showed that advanced methods could be used to enhance in vitro experiments ability to predict potential acute pulmonary toxicity in vivo. Moreover, we showed that the timing of the dose delivery could be controlled to enhance the predictivity. Further studies should be necessary to assess if air-liquid interface provide more reliable ranking of nanomaterials than submerged methods.

Air-Liquid Interface In Vitro Models for Respiratory Toxicology Research: Consensus Workshop and Recommendations.

In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats.

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions.

BACKGROUND: Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO2 and one nano-CeO2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. RESULTS: Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. CONCLUSIONS: We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.

Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro.

The health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier. The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis. Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection.

Titanium dioxide (TiO2) nanoparticles are used in many applications. Due to their small size, easy body penetration and toxicological adverse effects have been suspected. Numerous studies have tried to characterize TiO2 translocation after oral, dermal or respiratory exposure. In this study, we focused on TiO2 nanoparticle biodistribution, clearance and toxicological effects after intravenous injection, considering TiO2 translocation in the blood occurs. Using ICP-OES, transmission electron microscopy, and histological methods, we found TiO2 accumulation in liver, lungs and spleen. We estimated TiO2 nanoparticles' half life in the body to about 10 days. Clinical biomarkers were also quantified for 56 days to identify potential toxicological impact on lungs, blood, liver, spleen and kidneys. Results showed absence of toxicological effects after TiO2 intravenous injection at concentrations of 7.7 to 9.4 mg/kg.

Specific uptake and genotoxicity induced by polystyrene nanobeads with distinct surface chemistry on human lung epithelial cells and macrophages.

Nanoparticle surface chemistry is known to play a crucial role in interactions with cells and their related cytotoxic effects. As inhalation is a major route of exposure to nanoparticles, we studied specific uptake and damages of well-characterized fluorescent 50 nm polystyrene (PS) nanobeads harboring different functionalized surfaces (non-functionalized, carboxylated and aminated) on pulmonary epithelial cells and macrophages (Calu-3 and THP-1 cell lines respectively). Cytotoxicity of in mass dye-labeled functionalized PS nanobeads was assessed by xCELLigence system and alamarBlue viability assay. Nanobeads-cells interactions were studied by video-microscopy, flow cytometry and also confocal microscopy. Finally ROS generation was assessed by glutathione depletion dosages and genotoxicity was assessed by γ-H2Ax foci detection, which is considered as the most sensitive technique for studying DNA double strand breaks. The uptake kinetic was different for each cell line. All nanobeads were partly adsorbed and internalized, then released by Calu-3 cells, while THP-1 macrophages quickly incorporated all nanobeads which were located in the cytoplasm rather than in the nuclei. In parallel, the genotoxicity study reported that only aminated nanobeads significantly increased DNA damages in association with a strong depletion of reduced glutathione in both cell lines. We showed that for similar nanoparticle concentrations and sizes, aminated polystyrene nanobeads were more cytotoxic and genotoxic than unmodified and carboxylated ones on both cell lines. Interestingly, aminated polystyrene nanobeads induced similar cytotoxic and genotoxic effects on Calu-3 epithelial cells and THP-1 macrophages, for all levels of intracellular nanoparticles tested. Our results strongly support the primordial role of nanoparticles surface chemistry on cellular uptake and related biological effects. Moreover our data clearly show that nanoparticle internalization and observed adverse effects are not necessarily associated.

Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles.

As the lung is one of the main routes of exposure to manufactured nanoparticles, we developed an in vitro model resembling the alveolo-capillary barrier for the study of nanoparticle translocation. In order to provide a relevant and ethical in vitro model, cost effective and easy-to-implement human cell lines were used. Pulmonary epithelial cells (Calu-3 cell line) and macrophages (THP-1 differentiated cells) were cultivated on the apical side and pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a microporous polyester membrane (Transwell®). Translocation of non-functionalized (51 and 110 nm) and aminated (52 nm) fluorescent polystyrene (PS) nanobeads was studied in this system. The use of Calu-3 cells allowed high transepithelial electrical resistance (TEER) values (>1000 Ω cm2) in co-cultures with or without macrophages. After 24 h of exposure to non-cytotoxic concentrations of non-functionalized PS nanobeads, the relative TEER values (%/t0) were significantly decreased in co-cultures. Epithelial cells and macrophages were able to internalize PS nanobeads. Regarding translocation, Transwell® membranes per se limit the passage of nanoparticles between apical and basal side. However, small non-functionalized PS nanobeads (51 nm) were able to translocate as they were detected in the basal side of co-cultures. Altogether, these results show that this co-culture model present good barrier properties allowing the study of nanoparticle translocation but research effort need to be done to improve the neutrality of the porous membrane delimitating apical and basal sides of the model.

Correction: Effet of Combined Nitrogen Dioxide and Carbon Nanoparticle Exposure on Lung Function During Ovalbumin Sensitization in Brown Norway Rat.

[This corrects the article DOI: 10.1371/journal.pone.0045687.].

Cell cooperation and role of the P2X7 receptor in pulmonary inflammation induced by nanoparticles.

Macrophages and alveolar epithelial cells are the first targets of inhaled nanoparticles (NPs) reaching the alveoli. Mono- or co-cultures of lung epithelial (A549 or NCI-H441) and macrophage (THP-1) cell lines were used to study the cell cooperation and the involvement of the P2X7 cell death receptor during the inflammation caused by SiO2 and TiO2 NPs. Here we show that, secretion of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) in response to NPs exposure was higher in co-cultures than in mono-cultures. A functional P2X7 receptor was found in all the cell lines studied. Its involvement in IL-1ß secretion in co-cultures was demonstrated using a specific antagonist, the brilliant blue G. Furthermore, mono and co-cultures exhibited distinct secretion patterns of pro-inflammatory cytokines in response to NPs exposure, and we provide the first evidence that the P2X7 receptor is involved in the inflammation triggered by SiO2 and TiO2 NPs, by increasing IL-1ß secretion, and likely through the inflammasome pathway. Altogether, our data indicate that cell co-cultures used in this study represent valid models to study the inflammatory mechanisms of NPs within the alveoli.

Effect of combined nitrogen dioxide and carbon nanoparticle exposure on lung function during ovalbumin sensitization in Brown Norway rat.

The interaction of particulate and gaseous pollutants in their effects on the severity of allergic inflammation and airway responsiveness are not well understood. We assessed the effect of exposure to NO(2) in the presence or absence of repetitive treatment with carbon nanoparticle (CNP) during allergen sensitization and challenges in Brown-Norway (BN) rat, in order to assess their interactions on lung function and airway responses (AR) to allergen and methacholine (MCH), end-expiratory lung volume (EELV), bronchoalveolar lavage fluid (BALF) cellular content, serum and BALF cytokine levels and histological changes. Animals were divided into the following groups (n = 6): Control; CNP (Degussa-FW2): 13 nm, 0.5 mg/kg instilled intratracheally ×3 at 7-day intervals; OVA: ovalbumin-sensitised; OVA+CNP: both sensitized and exposed to CNP. Rats were divided into equal groups exposed either to air or to NO(2), 10 ppm, 6 h/d, 5d/wk for 4 weeks. Exposure to NO(2), significantly enhanced lung inflammation and airway reactivity, with a significantly larger effect in animals sensitized to allergen, which was related to a higher expression of TH1 and TH2-type cytokines. Conversely, exposure to NO(2) in animals undergoing repeated tracheal instillation of CNP alone, increased BALF neutrophilia and enhanced the expression of TH1 cytokines: TNF-α and IFN-γ, but did not show an additive effect on airway reactivity in comparison to NO(2) alone. The exposure to NO(2) combined with CNP treatment and allergen sensitization however, unexpectedly resulted in a significant decrease in both airway reactivity to allergen and to methacholine, and a reduction in TH2-type cytokines compared to allergen sensitization alone. EELV was significantly reduced with sensitization, CNP treatment or both. These data suggest an immunomodulatory effect of repeated tracheal instillation of CNP on the proinflammatory effects of NO(2) exposure in sensitized BN rat. Furthermore, our findings suggest that NO(2), CNP and OVA sensitization may significantly slow overall lung growth in parenchymally mature animals.

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats.

BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg⁻¹ pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 µg) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1ß (IL-1ß), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1ß and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.

Proteomic analysis of major and minor allergens from isolated pollen cytoplasmic granules.

Grass pollen is one of the most important vectors of aeroallergens. Under atmospheric conditions, pollen grains can release pollen cytoplasmic granules (PCGs). The allergens associated with these intrinsic subfractions induce, in laboratory animals as well as in asthmatic patients, allergic and inflammatory responses. The objectives of this study were to characterize the PCGs' intrinsic allergens and to compare them with those of pollen grains. The water-soluble proteins were extracted from pollen grains and their PCGs. IgE-binding proteins were analyzed and characterized through an allergomic strategy: 1- and 2-dimensional gel electrophoresis (1-DE and 2-DE), immunoblotting, using grass-pollen-sensitized patient sera, mass spectrometry (MS) analysis, and database searching. Several of the allergens listed in the IUIS nomenclature, Phl p 1, 4, 5, 6, and 12, were detected in pollen and PCG extracts, whereas Phl p 11 was found only in PCGs, and Phl p 2 as well as Phl p 13 were found only in pollen extract. Some other allergens not listed in the IUIS nomenclature were also characterized in both pollen and PCG extracts. Since the major grass pollen allergens were found in PCGs and because of their small size, these submicronic particles should be considered as very potent sensitizing and challenging respirable vectors of allergens.

In vivo biodistribution and biological impact of injected carbon nanotubes using magnetic resonance techniques.

BACKGROUND: Single-walled carbon nanotubes (SWCNT) hold promise for applications as contrast agents and target delivery carriers in the field of nanomedicine. When administered in vivo, their biodistribution and pharmacological profile needs to be fully characterized. The tissue distribution of carbon nanotubes and their potential impact on metabolism depend on their shape, coating, and metallic impurities. Because standard radiolabeled or fluorescently-labeled pharmaceuticals are not well suited for long-term in vivo follow-up of carbon nanotubes, alternative methods are required. METHODS: In this study, noninvasive in vivo magnetic resonance imaging (MRI) investigations combined with high-resolution magic angle spinning (HR-MAS), Raman spectroscopy, iron assays, and histological analysis ex vivo were proposed and applied to assess the biodistribution and biological impact of intravenously injected pristine (raw and purified) and functionalized SWCNT in a 2-week longitudinal study. Iron impurities allowed raw detection of SWCNT in vivo by susceptibility-weighted MRI. RESULTS: A transitional accumulation in the spleen and liver was observed by MRI. Raman spectroscopy, iron assays, and histological findings confirmed the MRI readouts. Moreover, no acute toxicological effect on the liver metabolic profile was observed using the HR-MAS technique, as confirmed by quantitative real-time polymerase chain reaction analysis. CONCLUSION: This study illustrates the potential of noninvasive MRI protocols for longitudinal assessment of the biodistribution of SWCNT with associated intrinsic metal impurities. The same approach can be used for any other magnetically-labeled nanoparticles.

Ability of pollen cytoplasmic granules to induce biased allergic responses in a rat model.

BACKGROUND: Grass pollen is one of the most important aeroallergens in Europe. It highly contributes to respiratory allergic diseases, mainly allergic rhinitis. In contact to water or airborne pollutants, pollen grains can release pollen cytoplasmic granules (PCGs) containing allergens. Because of their size (<5 µm), PCGs may penetrate deeper into the lungs to induce higher allergic responses, such as asthma. They have been associated with thunderstorm-related asthma. The aim of this study was to evaluate, with Brown Norway rats, the allergenic potential of isolated PCGs and to compare it with the allergenicity of whole timothy grass pollen. METHODS: Rats were sensitized (day 0) and challenged (day 21), in controlled comparative conditions, with pollen grains (0.5 mg) or PCGs (4.5 × 107 and 0.5 mg). At day 25, blood samples, bronchoalveolar lavage fluid (BALF) and bronchial lymph node were collected. IgE and IgG1 levels in sera were assessed by ELISA. Alveolar cells, protein and cytokine concentrations were quantified in BALF. T cell proliferation, in response to pollen or granules, was performed by lymph node assay. RESULTS: The results showed that proliferative responses of lymph node cells were similar in PCG- and pollen-sensitized rats. IgE and IgG1 levels were higher in pollen- than in PCG-sensitized rats. However, eosinophils, lymphocytes and pro-allergy cytokines in BALF were higher in PCG- than in pollen-sensitized rats. CONCLUSIONS: Thus, PCGs, able to deeply penetrate in the respiratory tract, induced local and strong allergic and inflammatory responses more linked with asthma- than rhinitis-related allergic symptoms.

Key role of water-insoluble allergens of pollen cytoplasmic granules in biased allergic response in a rat model.

BACKGROUND: : Grass pollen grain, an important aeroallergen, can disperse in the environment pollen cytoplasmic granules (PCGs) able to release water-soluble allergens when they are washed out by rainfall. The allergenicity of these washed PCGs is, however, preserved. OBJECTIVE: : The purpose of the study was to assess the allergenic potential of washed and unwashed PCGs, from Phleum pratense pollen grains, in the Brown Norway rat, and to study the IgE reactivity of sera of sensitized rats to water-soluble and water-insoluble extracts of PCGs and pollen grains. METHODS: : Rats were sensitized and challenged intratracheally with washed or unwashed PCGs or pollen grains. Using water-soluble and -insoluble extracts of pollen grains and/or PCGs, IgE ELISA and immunoblotting were performed with rat sera. Proliferation of bronchial lymph node cells was monitored by [H]-thymidine incorporation in a lymph node assay. Alveolar cells, proteins, and TH1 and TH2 cytokines were quantified in bronchoalveolar lavage fluid. RESULTS: : Rats sensitized with unwashed PCGs showed a predominant humoral response with high serum IgE and reactivity to water-soluble and -insoluble proteins together with low lymph node cell proliferation. Conversely, in rats sensitized to washed PCGs, cellular responses were higher with significant increases in eosinophils, lymphocytes, and TH2 cytokines observed in bronchoalveolar lavage fluid. CONCLUSION: : Allergic and inflammatory responses were induced by both grass pollen grains and their isolated washed and unwashed PCGs. However, on the basis of humoral and cellular responses, differential patterns were observed. Water-insoluble allergens seem to play a role in the centrally mediated inflammatory response, whereas water-soluble allergens may be involved in the peripheral humoral response.