Removal of organic content from diesel exhaust particles alters cellular responses of primary human bronchial epithelial cells cultured at an air-liquid interface

Background Exposure to air pollutants, including diesel particulate matter, has been linked to adverse respiratory health effects. Inhaled diesel particulate matter contains adsorbed organic compounds. It is not clear whether the adsorbed organics or the residual components are more deleterious to airway cells. Using a physiologically relevant model, we investigated the role of diesel organic content on mediating cellular responses of primary human bronchial epithelial cells (HBECs) cultured at an air-liquid interface (ALI). Methods Primary HBECs were cultured and differentiated at ALI for at least 28 days. To determine which component is most harmful, we compared primary HBEC responses elicited by residual (with organics removed) diesel emissions (DE) to those elicited by neat (unmodified) DE for 30 and 60 minutes at ALI, with cigarette smoke condensate (CSC) as the positive control, and filtered air as negative control. Cell viability (WST-1 cell proliferation assay), inflammation (TNF-α, IL-6 and IL-8 ELISA) and changes in gene expression (qRT-PCR for HO-1, CYP1A1, TNF-α and IL-8 mRNA) were measured. Results Immunofluorescence and cytological staining confirmed the mucociliary phenotype of primary HBECs differentiated at ALI. Neat DE caused a comparable reduction in cell viability at 30 or 60 min exposures, whereas residual DE caused a greater reduction at 60 min. When corrected for cell viability, cytokine protein secretion for TNF-α, IL-6 and IL-8 were maximal with residual DE at 60 min. mRNA expression for HO-1, CYP1A1, TNF-α and IL-8 was not significantly different between exposures. Conclusion This study provides new insights into epithelial cell responses to diesel emissions using a physiologically relevant aerosol exposure model. Both the organic content and residual components of diesel emissions play an important role in determining bronchial epithelial cell response in vitro. Future studies should be directed at testing potentially useful interventions against the adverse health effects of air pollution exposure.

Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Purpose: Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. Methods: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. Results: Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). Conclusions: GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

A potent nonporphyrin class of photodynamic therapeutic agent: Cellular localisation, cytotoxic potential and influence of hypoxia

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics. © 2005 Cancer Research UK.