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1.  Identification of distinct human invariant natural killer T-cell response phenotypes to alpha-galactosylceramide 
BMC Immunology  2008;9:71.
Human CD1d-restricted, invariant natural killer T cells (iNKT) are a unique class of T lymphocytes that recognise glycolipid antigens such as α-galactosylceramide (αGalCer) and upon T cell receptor (TCR) activation produce both Th1 and Th2 cytokines. iNKT cells expand when cultured in-vitro with αGalCer and interleukin 2 (IL-2) in a CD1d-restricted manner. However, the expansion ratio of human iNKT cells varies between individuals and this has implications for attempts to manipulate this pathway therapeutically. We have studied a panel of twenty five healthy human donors to assess the variability in their in-vitro iNKT cell expansion responses to stimulation with CD1d ligands and investigated some of the factors that may influence this phenomenon.
Although all donors had comparable numbers of circulating iNKT cells their growth rates in-vitro over 14 days in response to a range of CD1d ligands and IL-2 were highly donor-dependent. Two reproducible donor response patterns of iNKT expansion were seen which we have called 'strong' or 'poor' iNKT responders. Donor response phenotype did not correlate with age, gender, frequency of circulating iNKT, or with the CD1d ligand utilised. Addition of exogenous recombinant human interleukin 4 (IL-4) to 'poor' responder donor cultures significantly increased their iNKT proliferative capacity, but not to levels equivalent to that of 'strong' responder donors. However in 'strong' responder donors, addition of IL-4 to their cultures did not significantly alter the frequency of iNKT cells in the expanded CD3+ population.
(i) in-vitro expansion of human iNKT cells in response to CD1d ligand activation is highly donor variable, (ii) two reproducible patterns of donor iNKT expansion were observed, which could be classified into 'strong' and 'poor' responder phenotypes, (iii) donor iNKT response phenotypes did not correlate with age, gender, frequency of circulating iNKT cells, or with the CD1d ligand utilised, (iv) addition of IL-4 to 'poor' but not 'strong' responder donor cultures significantly increased their in-vitro iNKT cell expansion to αGalCer.
PMCID: PMC2613383  PMID: 19055753
2.  ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages 
BMC Immunology  2008;9:35.
We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy.
We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).
We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.
PMCID: PMC2483257  PMID: 18627610
3.  Direct cell-to-cell spread of a pathogenic yeast 
BMC Immunology  2007;8:15.
Cryptococcosis, a fatal fungal infection of the central nervous system, is one of the major killers of AIDS patients and other immunocompromised hosts. The causative agent, Cryptococcus neoformans, has a remarkable ability to 'hide' and proliferate within phagocytic cells of the human immune system. This intracellular phase is thought to underlie the ability of the pathogen to remain latent for long periods of time within infected individuals.
We now report that Cryptococcus is able to undergo 'lateral transfer' between phagocytes, moving directly from infected to uninfected macrophages. This novel process was observed in both C. neoformans serotypes (A and D) and occurs in both immortalised cell lines and in primary human macrophages. Lateral transfer is independent of the initial route of uptake, since both serum-opsonised and antibody-opsonised C. neoformans are able to undergo direct cell-to-cell transfer.
We provide the first evidence for lateral transfer of a human fungal pathogen. This rare event may occur repeatedly during latent cryptococcal infections, thereby allowing the pathogen to remain concealed from the immune system and protecting it from exposure to antifungal agents.
PMCID: PMC1976318  PMID: 17705831

Results 1-3 (3)