Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis.

BACKGROUND: In healthy lungs, deposited micrometer-sized particles are efficiently phagocytosed by macrophages present on airway surfaces; however, uptake of nanoparticles (NP) by macrophages appears less effective and is largely unstudied in lung disease. Using mouse models of allergic asthma and chronic obstructive pulmonary disease (COPD), we investigated NP uptake after challenge with common biogenic ambient air microparticles. METHODS: Bronchoalveolar lavage (BAL) cells from diseased mice (allergic asthma: ovalbumin [OVA] sensitized and COPD: Scnn1b-transgenic [Tg]) and their respective healthy controls were exposed ex vivo first to 3-µm fungal spores of Calvatia excipuliformis and then to 20-nm gold (Au) NP. Electron microscopic imaging was performed and NP uptake was assessed by quantitative morphometry. RESULTS: Macrophages from diseased mice were significantly larger compared to controls in OVA-allergic versus sham controls and in Scnn1b-Tg versus wild type (WT) mice. The percentage of macrophages containing AuNP tended to be lower in Scnn1b-Tg than in WT mice. In all animal groups, fungal spores were localized in macrophage phagosomes, the membrane tightly surrounding the spore, whilst AuNP were found in vesicles largely exceeding NP size, co-localized in spore phagosomes and occasionally, in the cytoplasm. AuNP in vesicles were located close to the membrane. In BAL from OVA-allergic mice, 13.9 ± 8.3% of all eosinophils contained AuNP in vesicles exceeding NP size and close to the membrane. CONCLUSIONS: Overall, AuNP uptake by BAL macrophages occurred mainly by co-uptake together with other material, including micrometer-sized ambient air particles like fungal spores. The lower percentage of NP containing macrophages in BAL from Scnn1b-Tg mice points to a change in the macrophage population from a highly to a less phagocytic phenotype. This likely contributes to inefficient macrophage clearance of NP in lung disease. Finally, the AuNP containing eosinophils in OVA-allergic mice show that other inflammatory cells present on airway surfaces may substantially contribute to NP uptake.

Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease.

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of ≤ 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2±4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0±5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3±32.2% AuNP were on the epithelium and 58.3±41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5±4.8% AuNP were luminal, 21.4±14.2% within epithelial cells and 63.0±18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5±5.0% AuNP were luminal, 2.2±1.6% within epithelial cells and 82.8±0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.

Electric pulses augment reporter gene expression in the beating heart.

BACKGROUND: Gene therapy of the heart has been attempted in a number of clinical trials with the injection of naked DNA, although quantitative information on myocellular transfection rates is not available. The present study aimed to quantify the efficacy of electropulsing protocols that differ in pulse duration and number to stimulate transfection of cardiomyocytes and to determine the impact on myocardial integrity. METHODS: Reporter plasmid for constitutive expression of green fluorescent protein (GFP) was injected into the left ventricle of beating hearts of adult, male Lewis rats. Four electrotransfer protocols consisting of repeated long pulses (8 × 20 ms), trains of short pulses (eight trains of either 60 or 80 × 100 µs) or their combination were compared with control procedures concerning the degree of GFP expression and the effect on infiltration, fibrosis and apoptosis. RESULTS: All tested protocols produced GFP expression at the site of plasmid injection. Continuous pulses were most effective and increased the number of GFP-positive cardiomyocytes by more than 300-fold compared to plasmid injection alone (p < 0.05). Concomitantly, the incidence of macrophage infiltration, fibrosis and cell death was increased. Trains of short pulses reduced macrophage infiltration and fibrosis by four- and two-fold, respectively, although they were 20-fold less efficient in stimulating cardiomyocyte transfection. GFP expression co-related to delivered electric energy, infiltration and fibrosis, although not apoptosis. CONCLUSIONS: The data imply that electropulsing of the myocardium promotes the overexpression of exogenous protein in mature cardiomyocytes in relation to an injury component. Fractionation of pulses is indicated as a option for sophisticated gene therapeutic approaches to the heart.

Rasagiline interferes with neurodegeneration in the Prph2/rds mouse.

PURPOSE: Rasagiline (N-propargyl-1(R)-aminoindan) is a second-generation propargylamine with neuroprotective effects. We used the Prph2/rds mouse to assess the effect of rasagiline on photoreceptor cell death and to examine the possible modulation of different pathways of programmed cell death. METHODS: The animals were orally treated with various doses of rasagiline from Postnatal Day 1 to 56. Methodological approaches consisted of morphometric analyses of the outer nuclear layer thickness and investigation of apoptotic events using TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay, immunohistochemistry, and immunoblot staining. The expression of programmed cell death marker genes involved in photoreceptor degeneration was studied by quantitative real-time polymerase chain reaction. RESULTS: In the Prph2/rds mouse, treatment resulted in a significant dose-dependent neuroprotection at Postnatal Day 56 and a delay in the induction of apoptotic events at Postnatal Day 14. Programmed cell death marker gene expression showed that several mechanisms were involved in photoreceptor degeneration. Furthermore, rasagiline did not only target apoptosis but also other pathways such as autophagy and inflammation. CONCLUSION: This study showed for the first time significant neuroprotective effects of rasagiline in the retina of Prph2/rds mice through caspase-dependent pathways. However, the activation of caspase-independent programmed cell death pathways that are not affected by rasagiline eventually led to retinal degeneration, but in a delayed manner.

Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina.

BACKGROUND: Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death. METHODS: C57/BL6 mice were injected with different doses of MNU, and function was determined by analysing optokinetic reflex measurements and cued water maze results at several time points post-injection. Morphometric measurements were also taken from H&E-stained paraffin eye sections. TUNEL-positive cells and caspase-3 and -6 were detected by immunohistochemistry. To assess the molecular changes leading to cell death, qRT-PCR from neurosensory retina mRNA was performed. RESULTS: The application of MNU led to an instant decrease in function and a delayed decrease in the thickness of the retinal outer nuclear layer. These responses were observed in the absence of any structural changes in the retinal pigment epithelium. The degeneration of the photoreceptor cell layer was highest with 60 mg/kg MNU. The assessment of TUNEL-positive cells visualised cell death after treatment, but no detectable caspase-3 activity was observed concomitant with these changes. qRT-PCR revealed the possible involvement of the inflammatory mediator caspase-1 and endoplasmic reticulum stress-mediated apoptosis by caspase-12. CONCLUSION: MNU leads to the dose-dependent degeneration of photoreceptor cells in mice by caspase-3-independent pathways and is, therefore, a suitable model to study retinal degeneration in an animal model.