Macrophage-mediated clearance of cells undergoing caspase-3-independent death

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide – namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments – are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages – both by CD14-dependent and CD14-independent mechanisms – do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.

Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH

The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC50s of the 2,3-hexanedione (3.3 ± 0.1 mM) and 3,4-hexanedione (3.5 ± 0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC50 of 22.4 ± 0.2 mM). Comparison between the respective IC50s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4 ± 0.3-45.3 ± 0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3 mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the α-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8 mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9 ± 0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4 ± 0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these α-diketones present in diets pose any possible risks to human health. © 2006 Elsevier Ireland Ltd. All rights reserved.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p < 0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p < 0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.