Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines.

Purpose.: To formulate conventional contact lenses that incorporate nanosphere-encapsulated antibiotic and demonstrate that the lenses provide for sustained antibacterial activity. Methods.: A copolymer composed of pullulan and polycaprolactone (PCL) was used to synthesize core-shell nanospheres that encapsulated ciprofloxacin. Bactericidal activity of the nanosphere-encapsulated ciprofloxacin (nanosphere/cipro) was tested by using liquid cultures of either S taphylococcus aureus or P seudomonas a eru g i nosa. Nanosphere/cipro was then incorporated into HEMA-based contact lenses that were tested for growth inhibition of S. aureus or P. aeruginosa in liquid cultures inoculated daily with fresh bacteria. Lens designs included thin or thick lenses incorporating nanosphere/cipro and ciprofloxacin-HCl-soaked Acuvue lenses (Acuvue; Johnson & Johnson Vision Care, Inc., Jacksonville, FL). Results.: Less than 2 μg/mL of nanosphere/cipro effectively inhibited the proliferation of cultures inoculated with 10⁷ or 10⁸ bacteria/mL of S. aureus and P. aeruginosa, respectively. HEMA-based contact lenses polymerized with nanosphere/cipro were transparent, effectively inhibited the proliferation of greater than 10⁷/mL of bacteria added daily over 3 days of culture, and killed up to 5 × 10⁹ total microbes in a single inoculation. A thicker lens design provided additional inhibition of bacterial growth for up to 96 hours. Conclusions.: Core-shell nanospheres loaded with an antibiotic can be incorporated into a conventional, transparent contact lens and provide for sustained and effective bactericidal activity and thereby provide a new drug delivery platform for widespread use in treating ocular disorders.

Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin-binding domains plus a C-terminal sterol Δ¹⁴ reductase domain. LBR expression increases during neutrophil differentiation, and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus, LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (erythroid, myeloid, and lymphoid [EML]-derived promyelocytes) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. In this study, we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EML-derived promyelocytes, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full-length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ¹⁴ reductase domain of LBR plays a critical role in cholesterol biosynthesis and that this process is essential to both myeloid cell growth and functional maturation.

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.

Printing devices such as photocopiers and printers emit predominantly nanoparticles, which may aggregate with time to form PM_0.25–2.0 particles. To date, there are no reports on cytotoxic or genotoxic effects of PM_0.25–2.0 particles emitted from photocopiers. This study's objective was to investigate the ability of PM_0.25–2 fraction emitted from photocopiers, to induce pro-inflammatory cytokines, DNA damage and apoptosis in different human-derived cell lines. Three cell types, i.e. a THP-1 line, primary human nasal and small airway epithelial cells, were used. The airborne PM_0.25–2.0 size fraction collected from a photocopy center was characterized for its physicochemical and morphological properties, dispersed in culture media and cells were treated with 30, 100 or 300 µg/ml doses. Levels of 13 cytokines and chemokines in the culture medium harvested at 6 and 24 h of treatment were measured using Luminex cytokine kits. In cells harvested at the same timepoints, DNA damage in cells was studied by Comet assay, and apoptosis was measured by cytofluorimetry using an Annexin V staining kit. The results indicate that in THP-1 cells, several cytokines (IL-6, IL-8, TNFα and IL-1β) were significantly elevated. Only IL-8 was significantly elevated in the primary nasal and small airway cells. Cells exposed to PM_0.25–2.0 underwent apoptosis in a dose-dependent manner, but no significant differences were found in the extent of DNA damage at either timepoint. Airborne PM_0.25–2.0 collected at one photocopier center was capable of inducing several pro-inflammatory cytokines and apoptosis, but no genotoxicity, in all cell lines suggesting a role for PM_0.25–2.0 in our previously documented airway inflammation in human volunteers. Further toxicological evaluations of these particles across different toner manufacturers are warranted.