Search

International audience; The notorious PM2.5 (2.5 μm diameter particulate matters) that can reach human lungs, considered as responsible for most deleterious effects of air pollution, are now unmasked. Anthropogenic carbon nanotubes and other carbon nanoparticles have been unambiguously identified as the major components of PM2.5 in alveolar macrophages of Parisian children. Soot and diesel particulate matter have recognized detrimental health effects. However, information on the health effects of anthropogenic carbon nanotubes, which are the main components of PM2.5 found within human alveolar cells, is still lacking. While nanotechnologists are focused on applications with a profit, notably in the field of nano-biomedicine, the health effects of nano-particulate pollutants attract little of their attention. In an attempt to bridge the research gaps between cutting-edge nanotechnologies, environmental studies and biomedical approaches, this opinion paper attempts to inform the nanotechnology community about critical issues related to airborne anthropogenic carbon nanotubes. The question that has to be urgently addressed is whether carbon nanotubes are the main culprit or act only as nano-vectors of carcinogenic polycyclic aromatic hydrocarbons and other toxic gaseous pollutants.

Compelling evidence shows that fine particulate matters (PMs) from air pollution penetrate lower airways and are associated with adverse health effects even within concentrations below those recommended by the WHO. A paper reported a dose-dependent link between carbon content in alveolar macrophages (assessed only by optical microscopy) and the decline in lung function. However, to the best of our knowledge, PM had never been accurately characterized inside human lung cells and the most responsible components of the particulate mix are still unknown. On another hand carbon nanotubes (CNTs) from natural and anthropogenic sources might be an important component of PM in both indoor and outdoor air. We used high-resolution transmission electron microscopy and energy dispersive X-ray spectroscopy to characterize PM present in broncho-alveolar lavage-fluids (n = 64) and inside lung cells (n = 5 patients) of asthmatic children. We show that inhaled PM mostly consist of CNTs. These CNTs are present in all examined samples and they are similar to those we found in dusts and vehicle exhausts collected in Paris, as well as to those previously characterized in ambient air in the USA, in spider webs in India, and in ice core. These results strongly suggest that humans are routinely exposed to CNTs., Graphical abstract, Highlights • Fine particulate matters (PMs) were characterized in broncho-alveolar lavage fluids (BALF) from asthmatic children (n = 69). • Anthropogenic carbon nanotubes were found in all samples, indicating that humans routinely breathe such nanostructures. • Light microscopy cannot discriminate between PM and lamellar bodies, thus previous studies should be reconsidered. • Fine particulate matters (PMs) from air pollution penetrate lower airways and are associated with adverse health effects even within concentrations below those recommended by the WHO. However, the most responsible components of the particulate mix are still unknown. We show that carbon nanotubes (CNTs) are present in the airways of asthmatic Parisian children. These nanostructures are similar to those present in dusts and vehicle exhausts collected in Paris, as well as to those previously observed in ambient air in the USA, in spider webs in India, and in ice core. These results suggest that humans are routinely exposed to CNTs.

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24h EC(50) values exceeded the top concentration tested (800 microg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Innate immune defense against microbial pathogens occurs by physical barriers, by recruitment of cells such as neutrophils, NK cells, and macrophages, and by secretion of molecules with antimicrobial activity. Such molecules are produced by various epithelia including skin. The importance of antimicrobial peptides has been shown in cathelicidin-deficient mice, which have increased susceptibility to skin infection by Streptococcus. Although keratinocytes increase cathelicidin expression upon injury, their role relative to neutrophil cathelicidin and their sites of peptide storage and activation have not been elucidated. Herein, it is reported that cathelicidin predominantly resides in granules of the superficial epidermis and partially localizes in lamellar bodies as determined by immunogold electron microscopy and immunoblot of lamellar bodies isolated from mice. In cultured keratinocytes, cathelicidin displays a granular distribution and partially localizes within the Golgi apparatus. Cathelicidin processing can be observed by western blot analysis in keratinocyte extracts but not in conditioned media. Further, fluorescent bacteria colocalize with cathelicidin in granules both intracellularly and at the cell surface. These observations illustrate the immune defense potential of keratinocytes acting directly through storage and processing of antimicrobial peptides.

Direct visualization of the skin barrier formation process by cryotransmission electron microscopy of vitreous epidermal sections has been performed. The results obtained differ in many aspects from those of classical chemical fixation electron microscopy. Here we show that (i) a new “organelle or branched tubular structure” containing nonlamellar or partly lamellar material, and closely corresponding in numbers, dimensions, and localization to lamellar bodies of classical chemical fixation electron micrographs, and (ii) a new “ribosome complex-like structure”, not preserved in classical electron micrographs, are omnipresent at apparent active sites of skin barrier formation. Evidence that skin barrier formation may not take place via extensive membrane fusion involving discrete lamellar bodies, but rather by a morphologically continuous membrane folding process are: (i) the often clearly nonlamellar content of structures corresponding to lamellar bodies with concomitant visualization of multilamellar membrane structures of the intercellular space; (ii) the “multifolded” appearance of the lipid matrix of the intercellular space; and (iii) the identification of extended “intracellular” multilamellar continuous structures with an optical density profile closely corresponding to that of the lipid matrix of the intercellular space. Based on the cryo-electron microscopic data presented in this study we propose that a membrane transition from cubic-like to multilamellar may take place already inside the “tubuloreticular cisternal membrane system” of upper granular cells recently reported by Elias et al.

I review how diverse inherited and acquired abnormalities in epidermal structural and enzymatic proteins converge to produce defective permeability barrier function and antimicrobial defense in patients with atopic dermatitis (AD). Although best known are mutations in filaggrin (FLG), mutations in other member of the fused S-100 family of proteins (ie, hornerin [hrn] and filaggrin 2 [flg-2]); the cornified envelope precursor (ie, SPRR3); mattrin, which is encoded by TMEM79 and regulates the assembly of lamellar bodies; SPINK5, which encodes the serine protease inhibitor lymphoepithelial Kazal-type trypsin inhibitor type 1; and the fatty acid transporter fatty acid transport protein 4 have all been linked to AD. Yet these abnormalities often only predispose to AD; additional acquired stressors that further compromise barrier function, such as psychological stress, low ambient humidity, or high-pH surfactants, often are required to trigger disease. TH2 cytokines can also compromise barrier function by downregulating expression of multiple epidermal structural proteins, lipid synthetic enzymes, and antimicrobial peptides. All of these inherited and acquired abnormalities converge on the lamellar body secretory system, producing abnormalities in lipid composition, secretion, and/or extracellular lamellar membrane organization, as well as antimicrobial defense. Finally, I briefly review therapeutic options that address this new pathogenic paradigm. © 2014 American Academy of Allergy, Asthma & Immunology.