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In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

It is not yet clear whether immature NK (iNK) cells are bystanders to or rather participate in immune responses to pathogens that may colocalize in areas of NK-cell maturation such as bone marrow or lymph nodes. Mycobacteria, including Bacillus Calmette-Guerin (BCG), have been shown to interact with peripheral NK cells and in vivo may colocalize in areas of iNK-cell development. We studied infection with BCG of human cord blood CD34(+) Lin(-)-derived cultures containing myelomonocytes and iNK cells in vitro. Increased iNK-cell DNAM-1 expression, transient natural cytotoxicity receptor modulation, and production of IFN-γ were observed. Transcriptional receptor modulation was associated to BCG challenge, which determined increased iNK-cell cytotoxic activity against tumor cell lines and also increased killing of immature dendritic cells (iDCs). No requirement for cell contact was recorded for BCG-induced iNK-cell activation, while cytokine production including IL-18, IL-10, GM-CSF, and TGF-β contributed to the observed effects. Thus, iNK cells are affected by mycobacteria in vitro and may contribute to shaping of adaptive mature innate responses through iDC-iNK cross-talk. In addition, iNK-cell activation by BCG may represent a novel additional mechanism contributing to the effects observed upon BCG administration in vivo., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.