A local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-related genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is individual genetic factors. In a previous study, we identified a coding SNP in the hephaestin gene (HEPH) that protects against developing asbestos-related thoracic cancer. Heph is a ferroxidase that promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights into the molecular basis of its protective activity. We showed that HephD568H forms a complex with Fpn1 and possesses full ferroxidase activity. Although HephD568H is more efficiently recruited to the plasma membrane, it is impaired in binding iron-deficient Tfn, whose interaction with wild-type (WT) ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph is expressed in the human lung by pericytes and fibroblasts, and lung pericytes were shown to respond to iron demand by upregulating the iron exporter pair. These results extend the paradigm of local iron regulation discovered at the blood–brain barrier to the pulmonary vasculature. Furthermore, they establish a mechanistic link between changes in iron sensing and the risk of developing asbestos-related malignancies.

Monkeypox virus (Mpox) has been recognized for causing distinct skin lesions and is primarily transmitted through skin and sexual contact. To date, the transmissibility and pathogenesis of the Mpox virus in distal human lung has never been completely explored. Here the transmission pathways and Mpox tropism on patient-derived air-liquid epithelium (ALE) model fabricated using isolated primary human alveolar epithelial cells (hAECs) were investigated. hAECs were cultured and exposed to the Mpox virus clade IIb isolated from patient. DNA, proteins, and the tropism were elucidated using polymerase chain reaction (PCR), Western blot and high-content fluorescent imaging. Transmission electron microscopy (TEM) was employed to systematically observe the cellular distribution of viral particles. Viral titers were determined by TCID50 assay. Innate immune response and inflammatory mediators were measured using Milliplex® multiplex and ELISA analysis. Pathology at alveolar barrier integrity was determined using transepithelial electrical resistance (TEER) analysis. The study included mock-infected cells as control. Mpox virus significantly infected 42.82% of total hAEC populations. The prominent observed pathology included a significant reduction in TEER values, loss of tight junction protein, presence of tunneling nanotubes (TNTs) and syncytium morphology. Four stages of Mpox biogenesis were clearly observed without significant activation of IL-6, MIP1alpha, TNF-α, and Galectin-9, although IL-1β were subtly promoted. The developed patient-derived ALE is a versatile model for Mpox virus clade IIb infection reflecting respiratory transmission competence of the Mpox. Postinfection lung pathogenesis demonstrated alveolar barrier damage without significant inflammation, raising concerns about possible immune evasion by the virus.

Hemorrhage is the leading cause of trauma death, and innovation in hemostatic technology is important. The strongly hydrophobic carbon nanofiber (CNF) coating has previously been shown to have excellent hemostatic properties. However, the understanding of how CNF coating guides the coagulation cascade and the biosafety of CNF as hemostatic agents has yet to be explored. Here, our thrombin generation assay investigation showed that CNF induced fast blood coagulation via factor (F) XII activation of the intrinsic pathway. We further performed studies of a rat vein injury and demonstrated that the CNF gauze enabled a substantial reduction of blood loss compared to both the plain gauze and kaolin-imbued gauze (QuikClot). Analysis of blood samples from the model revealed no acute toxicity from the CNF gauze, with no detectable CNF deposition in any organ, suggesting that the immobilization of CNF on our gauze prevented the infiltration of CNF into the bloodstream. Direct injection of CNF into the rat vein was also investigated and found not to elicit overt acute toxicity or affect animal survival or behavior. Finally, toxicity assays with primary keratinocytes revealed minimal toxicity responses to CNF. Our studies thus supported the safety and efficacy of the CNF hemostatic gauze, highlighting its potential as a promising approach in the field of hemostatic control.

Abstract Tyre wear particles (TWPs) are a predominant component of particulate air pollution from road transport and emissions may increase in future due to increasing use of heavier cars. We hypothesized that inhaled respirable TWPs would have adverse effects on human lung alveolar epithelium, which would differ between alveolar type 1 (AT1; gas exchanging) and type 2 (AT2; secretory) cells. Two real-time road-generated samples were each collected into Milli-Q water using custom-made apparatus whilst driving a car for 2h: S1) high, behind the tyre (“pure” TWP) and S2) low, sideways to the tyre (mixed TWP/brake/road wear). Alveolar epithelial type 1-like (AT1L) and type 2-like (AT2L) cell lines were exposed for 24h to each TWP sample at 62.5-250µg/ml. The response differed between AT1L and AT2L cells. AT1L cell viability/metabolism and cytotoxity (MTT/LDH assays) were unaffected by either TWP sample, whereas AT2L cell metabolic activity increased (MTT measures metabolic activity) at all concentrations of TWP, ~20%-30% (p<0.01). S1 induced IL-6 mediator release (~5-fold; p<0.0001) and IL-8 mediator release (~2.2-fold; p<0.01) by AT1L cells, whereas S2 did not. S1 and S2 induced IL-6 (S1, ~10-fold, p<0.0001: S2, ~4-fold, p<0.0001) and IL-8 (S1, ~4-fold, p<0.0001: S2, ~2-fold, p<0.0001) release by AT2L cells, thus mediator release was significantly greater for S1 and greater than for AT1L cells. These data indicate that AT2L cells were more sensitive to TWP than AT1L cells; the “pure” TWP were significantly more bioreactive than the mixed TWP/brake/road wear particles.

Abstract Plastic has unique properties with multiple applications. Microsized plastic fragments (0.1−1000µm; secondary microplastics) are generated throughout the lifespan of plastic materials; primary microplastics are manufactured. Microplastics are present in PM2.5. With increased use of electric cars and reduction in tail pipe emissions, the amount and proportion of microplastics in airborne PM2.5 could increase. We know little about the effects of microplastic inhalation on human health. Hypothesis: Inhaled plastic microparticles will have adverse effects on the respiratory epithelium, both independently and in combination with PM2.5. Two atmospheric plastics, polypropylene (PP) and polyamide (PA), were generated and characterized prior to study. They had a similar size distribution (1-3µm), polydispersity (PA: 0.9002; PP: 0.9037) and charge (PA: -9.66; PP: -5.79) in culture medium. Human alveolar epithelial type 1-like cells (TT1) were exposed for 24h to 1.25-80µg/ml of PP, PA, and PM2.5; viability fell after PA exposure to 10µg/ml (~10%), reaching ~30% at 80µg/ml (p<0.0001); only 40 and 80µg/ml of PP caused a 15 and 20% drop in viability, respectively (p<0.01). PM2.5 had no effect on viability. Although plastics had no effect on IL-6 or IL-8 release, PM2.5 stimulated release at 20-80µg/ml, reaching 10-fold (p<0.001) and 2.5-fold (p<0.05) for IL6 and IL8 respectively. Exposure to plastic/PM2.5 ratios from 0.0001:1 to 1:1 had no consistent effect. TEM suggests internalization of PA, PP and PM2.5 by TT1 cells. Thus, microplastics exhibit different responses (increased cell death) to PM2.5 (increased mediator production). The inconsistent response to particle mixtures is likely dependent upon the proportion of microplastics.

Research suggests that cleaning product exposure has adverse effects on respiratory health (Svanes, Ø. et al. AJRCCM 2018;197:1157-1163). This could be attributed to the activation of airway sensory nerves, which cause reflex coughing, and bronchospasm with associated shortness of breath. We hypothesised that alkylphenols, which are commonly used as non-ionic surfactants in cleaning products (Suen, J.L. et al. KJMS 2012;28:43-48), may induce adverse respiratory effects following exposure. The prevalent alkylphenol, 4-tert-octylphenol (4-t-OP), was investigated in vitro using vagus nerve and airway smooth muscle tissue from guinea pigs and in vivo using electrophysiology recordings of single afferent nerve fibres. 4-t-OP caused depolarisation of vagus nerves in vitro and single nerve firing in vivo, suggesting it can activate airway sensory nerves. 4-t-OP also increased tracheal pressure in vivo and airway smooth muscle contraction in vitro, indicating it can induce bronchospasm. We then pharmacologically investigated ion channels known to play a role in airway reflexes and determined that antagonists for TRPM3 (isosakuranetin, 5µM) and TRPV4 (GSK2193874, 10µM) inhibited 4-t-OP (10µM)-induced nerve depolarisation by 56% and 44% respectively. The same antagonists inhibited 4-t-OP-induced smooth muscle contraction by 39% and 30%. This suggests that 4-t-OP acts via two distinct pathways involving TRPM3 and TRPV4. Our data shows that 4-t-OP interacts with airway sensory nerves and airway smooth muscle via activation of TRPM3 and TRPV4, suggesting a novel mechanism by which alkylphenols may contribute to respiratory symptoms following cleaning product exposure.

Tyre wear particles (TWPs) produced due to friction between tyres and road surface, and release of complex organic and inorganic material into the environment, may have adverse effects on the lung. The respiratory health effects of TWPs are unknown. The aim was to assess the bioreactivity of TWPs (collected into water in real-time; RT) on human lung alveolar epithelium using novel human alveolar epithelial type 1-like (AT1L) and type 2-like (AT2L) cell lines. A custom-made apparatus was used to collect RT generated samples; RT1, directly behind and above the tyre (peak diameters, 100nm and 750nm; DLS) and RT2, side-on and lower (peak diameters, 365 and 5590nm; DLS), during 2h driving a car. AT1L and AT2L were exposed to 50-250µg/ml of each sample for 24 h. Neither sample affected AT1L cell viability, metabolism or cytotoxicity. In contrast, the metabolic activity (MTT) of AT2L increased by 1.2-1.3-fold (p<0.01) with increasing concentrations of TWP. Although AT1L mediator release was significantly increased following RT1 exposure - CXCL-8 (2.2-fold; p<0.01) and IL-6 (5-fold; p<0.0001) - RT2 had no effect. The release of IL6 and CXCL-8 by AT2L was substantially induced after RT1 (up to approx. 10- and 4-fold, p<0.0001) and RT2 (up to approx. 4- and 2-fold, p<0.0001), respectively. These results indicate AT2L cells were more sensitive to TWP than AT1L cells. Relatively pure, smaller TWP collected directly behind the tyre were remarkably more bioreactive than the larger, mixed TWP/brake/road wear particles collected nearer the road surface. These data suggest that inhalation of respirable airborne TWP may have undesirable effects in the deep lung.

Nanoencapsulation of pesticide atrazine improves efficacy. However, this study discovered adverse bioreactivity in alveolar type 1 epithelial cells (a major recipient of inhaled nano-sized particles), following exposure to nanoencapsulated atrazine.

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM + cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

Lung cancer (LC) is one of the most deadly cancers worldwide, with very low survival rates, mainly due to poor management, which has barely changed in recent years. Nanomedicines, especially gold nanomaterials, with their unique and size-dependent properties offer a potential solution to many challenges in the field. The versatility afforded by the shape, size, charge and surface chemistry of gold nanostructures allows them to be adapted for many applications in the diagnosis, treatment and imaging of LC. In this review, a survey of the most recent advances in the field is presented with an emphasis on the optical properties of gold nanoscale materials and their use in cancer management. Gold nanoparticle toxicology has also been a focus of interest for many years but the studies have also sometimes arrived at contradictory conclusions. To enable extrapolation and facilitate the development of medicines based on gold nanomaterials, it must be assumed that each design will have its own unique characteristics that require evaluation before translation to the clinic. Advances in the understanding and recognition of the molecular signatures of LC have aided the development of personalised medicines. Tailoring the treatment to each case should, ideally increase the survival outcomes as well as reduce medical costs. This review seeks to present the potential of gold nanomaterials in LC management and to provide a unified view, which will be of interest to those in the field as well as researchers considering entering this highly important area of research.

Tuberculosis (TB) is caused by a bacterial infection that affects a number of human organs, primarily the lungs, but also the liver, spleen, and spine, causing key symptoms of fever, fatigue, and persistent cough, and if not treated properly, can be fatal. Every year, 10 million individuals become ill with active TB resulting with a mortality approximating 1.5 million. Current treatment guidelines recommend oral administration of a combination of first-line anti-TB drugs for at least 6 months. While efficacious under optimum conditions, ‘Directly Observed Therapy Short-course’ (DOTS) is not without problems. The long treatment time and poor pharmacokinetics, alongside drug side effects lead to poor patient compliance and has accelerated the emergence of multi-drug resistant (MDR) organisms. All this, combined with the limited number of newly discovered TB drugs to treat MDR-TB and shorten standard therapy time, has highlighted the need for new targeted drug delivery systems. In this respect, there has been recent focus on micro- and nano-particle technologies to prepare organic or/and metal particles loaded with TB drugs to enhance their efficacy by targeted delivery via the inhaled route. In this review, we provide a brief overview of the current epidemiology of TB, and risk factors for progression of latent stage tuberculosis (LTBI) to the active TB. We identify current TB treatment regimens, newly discovered TB drugs, and identify studies that have used micro- or nano-particles technologies to design a reliable inhalation drug delivery system to treat TB more effectively.

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

The 1980s mark the starting point of nanotechnology: the capacity to synthesise, manipulate and visualise matter at the nanometre scale. New powers to reach the nanoscale brought us the unprecedented possibility to directly target at the scale of biomolecular interactions, and the motivation to create smart nanostructures that could circumvent the hurdles hindering the success of traditional pharmacological approaches. Forty years on, the progressive integration of bio- and nanotechnologies is starting to produce a transformation of the way we detect, treat and monitor diseases and unresolved medical problems [ 1]. While much of the work remains in research laboratories, the first nano-based treatments, vaccines, drugs, and diagnostic devices, are now receiving approval for commercialisation and clinical use. In this special issue we review recent advances of nanomedical approaches to combat antibiotic resistance, treatment and detection of cancers, targeting neurodegerative diseases, and applications as diverse as dentistry and the treatment of tuberculosis. We also examine the use of advanced smart nanostructured materials in areas such as regenerative medicine, and the controlled release of drugs and treatments. The latter is currently poised to bring ground-breaking changes in immunotherapy: the advent of ‘vaccine implants’ that continuously control and improve immune responses over time. With the increasingly likely prospect of ending the COVID 19 pandemic with the aid of a nanomedicine-based vaccine (both Moderna and BioNTech/Pfizer vaccines are based on lipid nanoparticle formulations), we are witnessing the coming of age of nanomedicine. This makes it more important than ever to concentrate on safety: in parallel to pursuing the benefits of nanomedine, we must strengthen the continuous focus on nanotoxicology and safety regulation of nanomedicines that can deliver the medical revolution that is within our grasp.

Studies using Langmuir-Wilhelmy balance (LWB) systems have demonstrated that engineered nanoparticles (NPs) can disrupt the surface tension behaviour of model lung surfactants. However, the majority of such studies used approaches that do not accurately mimic in vivo deposition at the air-liquid interface of inhaled particles using instead NP suspensions. The present study investigated for the first time the effect of the mode of delivery of NPs to the interface on the compression surface pressure (II) - mean molecular area (Mma) isotherms of surfactant monolayers, using an LWB. A novel system to deposit a well characterised aerosol of cerium oxide nanoparticles (CeO2 NPs) on a dipalmitoylphosphatidylcholine (DPPC) monolayer was developed. The effects of depositing CeO2 NPs from an aerosol were compared with two alternative approaches used in the literature: CeO2 NPs suspended in chloroform and deposited on a DPPC monolayer and mixtures of CeO2 NPs and DPPC in chloroform deposited directly on the subphase. Experiments were undertaken using a physiologically relevant subphase and temperature, 37 degrees C, with additional experiments at 21 degrees C. In each case Langmuir-Blodgett films were imaged using scanning electron microscopy and the distribution of cerium mapped using time of flight secondary ion mass spectrometry. The results clearly demonstrate that mode of delivery has a differential effect on surface tension behaviour and that caution should be exercised in the use of solvent deposition techniques because of effects on NP agglomeration state and the pattern of distribution of NP agglomerates, which may not accurately reflect the behaviour in vivo of inhaled particles. The study findings therefore suggest that aerosol deposition techniques are to be preferred, however, there are issues that need to be investigated further, including the effect of control air exposures, and also a need to explore the effects of cycling and the use of more complete lung surfactant models, before such a recommendation can be confirmed.

Airway and alveolar epithelial cells play a key role in health and disease. This review provides an introduction into the principles of epithelial cell culture, emerging developments and foreseeable future applications.http://bit.ly/33NxojQ

Cerium dioxide nanoparticles (CeO2NPs) have been used as diesel fuel-borne catalysts for improved efficiency and pollutant emissions. Concerns that such material may influence diesel exhaust particle (DEP) effects within the lung upon inhalation, prompted us to examine particle responses in mice in the presence and absence of the common allergen house dust mite (HDM). Repeated intranasal instillation of combined HDM and DEP increased airway mucin, eosinophils, lymphocytes, IL-5, IL-13, IL-17A and plasma IgE, which were further increased with CeO2NPs co-exposure. A single co-exposure of CeO2NPs and DEP after repeated HDM exposure increased macrophage and IL-17A levels above DEP induced levels. CeO2NPs exposure in the absence of HDM also resulted in increased levels of plasma IgE and airway mucin staining, changes not observed with repeated DEP exposure alone. These observations indicate that CeO2NPs can modify exhaust particulate and allergen induced inflammatory events in the lung with the potential to influence conditions such as allergic airway disease.

Gold nanoparticles (AuNPs) have emerged as promising drug delivery candidates that can be leveraged for cancer therapy. Lung cancer (LC) is a heterogeneous disease that imposes a significant burden on society, with an unmet need for new therapies. Chemotherapeutic drugs such as afatinib (Afb), which is clinically approved for the treatment of epidermal growth factor receptor positive LC, is hydrophobic and has low bioavailability leading to spread around the body, causing severe side effects. Herein, we present a novel afatinib-AuNP formulation termed Afb-AuNPs, with the aim of improving drug efficacy and biocompatibility. This was achieved by synthesis of an alkyne-bearing Afb derivative and reaction with azide-functionalized lipoic acid using copper-catalyzed click chemistry, then conjugation to AuNPs via alkylthiol-gold bond formation. The Afb-AuNPs were found to possess up to 3.7-fold increased potency when administered to LC cells in vitro and were capable of significantly inhibiting cancer cell proliferation, as assessed by MTT assay and electric cell-substrate impedance sensing, respectively. Furthermore, when exposed to Afb-AuNPs, human alveolar epithelial type I-like cells, a model of the healthy lung epithelium, maintained viability and were found to release less proinflammatory cytokines when compared to free drug, demonstrating the biocompatibility of our formulation. This study provides a new platform for the development of nontraditional AuNP conjugates which can be applied to other molecules of therapeutic or diagnostic utility, with potential to be combined with photothermal therapy in other cancers.

Mycobacterium tuberculosis ( M.tb) has the extraordinary ability to adapt to the administration of antibiotics through the development of resistance mechanisms. By rapidly exporting drugs from within the cytosol, these pathogenic bacteria diminish antibiotic potency and drive the presentation of drug-tolerant tuberculosis (TB). The membrane integrity of M.tb is pivotal in retaining these drug-resistant traits. Silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) are established antimicrobial agents that effectively compromise membrane stability, giving rise to increased bacterial permeability to antibiotics. In this work, biodegradable multimetallic microparticles (MMPs), containing Ag NPs and ZnO NPs, were developed for use in pulmonary delivery of antituberculous drugs to the endosomal system of M.tb-infected macrophages. Efficient uptake of MMPs by M.tb-infected THP1 cells was demonstrated using an in vitro macrophage infection model, with direct interaction between MMPs and M.tb visualized with the use of electron FIB-SEM tomography. The release of Ag NPs and ZnO NPs within the macrophage endosomal system increased the potency of the model antibiotic rifampicin by as much as 76%, realized through an increase in membrane disorder of intracellular M.tb. MMPs were effective at independently driving membrane destruction of extracellular bacilli located at the exterior face of THP1 macrophages. This MMP system presents as an effective drug delivery vehicle that could be used for the transport of antituberculous drugs such as rifampicin to infected alveolar macrophages, while increasing drug potency. By increasing M.tb membrane permeability, such a system may prove effectual in improving treatment of drug-susceptible TB in addition to M.tb strains considered drug-resistant.

One of the most important news occurring in axillary surgery since the last St. Gallen Conference in 2017 was the publication of confirmatory long-term follow-up data from several large multicenter phase III non-inferiority trials, which clearly showed that axillary dissection can no longer be considered standard practice in all node-positive patients. Several groups are currently investigating the most accurate method to reliably determine axillary pathologic complete response after neoadjuvant chemotherapy to omit axillary dissection in initially clinically node-positive patients. Concerning breast surgery, after publication of the broadly endorsed definition of “no ink on tumor” for negative margins, many groups have demonstrated the expected decrease in re-excision rates. More evidence is needed to evaluate the adequate margin width in the neoadjuvant setting, where an increased risk of local recurrence has been shown compared to the upfront surgery setting. Besides narrowing margins and local down-staging by neoadjuvant therapy, another potential way to increase breast conservation rates is eliminating multicentricity as a contraindication. This requires high-volume tumorectomy, which has been demonstrated to be oncologically safe in a large series of oncoplastic reduction mammoplasties. However, the beneficial impact of oncoplastic surgery on quality of life still needs to be confirmed. The Oncoplastic Breast Consortium (OPBC) is a rapidly growing global non-profit organization that is committed to identifying and prioritizing knowledge gaps in this field. Currently, the OPBC focuses on research projects that address the major heterogeneity in breast reconstruction practice after nipple-sparing mastectomy.