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Warren H.,Cancer Immunology Research Unit
Current Protocols in Immunology | Year: 2013

Natural killer (NK) cells are an important effector cell of innate immunity. Their interaction with susceptible target cells triggers NK cell cytotoxicity and the release of cytokines. Immunofluorescence flow cytometry-based assays are now the preferred methods for measuring NK cell responses. For these assays, assessment is made on NK cells (CD3-CD56+ CD16+) within the viable lymphocyte gate, defined by the parameters of size (FSC) and granularity (SSC). Accordingly, NK cells that have not dissociated from target cells at the time of measurement, or that have undergone target cell-induced apoptosis, are excluded from the viable lymphocyte gate and therefore from analysis. This unit describes a protocol for assessing NK cell function in response to various target cells (natural killing, antibody-dependent cell cytotoxicity, and NK cell alloreactivity) based on the loss of NK cells from the lymphocyte gate. This target-induced NK loss (TINKL) should provide a sensitive measure of NK cell responses in a clinical laboratory setting. © 2013 by John Wiley & Sons, Inc. Source

Warren H.S.,Cancer Immunology Research Unit | Warren H.S.,Australian National University
Journal of Immunological Methods | Year: 2011

The interaction of natural killer cells with susceptible target cells triggers NK cell activation, eliciting not only NK cell cytotoxicity and cytokine secretion, but also NK cell death. This study shows that following target cell interaction there is a substantial loss of NK cells, the extent of which correlates with measures of NK cell cytotoxicity assessed by the target cell release of 51Cr and by the externalisation of the lysosomal marker LAMP-1 (CD107a) which is assessed on the remaining NK cells. This is the case for the killing of K562 (natural killing) and the CD20 mAb (Rituximab)-mediated killing of RAJI cells and autologous B cells (antibody-dependent cell cytotoxicity). This target-induced NK loss (TINKL) provides a sensitive and specific measure of NK cell responses appropriate to a clinical laboratory setting. © 2011 Elsevier B.V. Source

West N.P.,Griffith University | Horn P.L.,Australian Institute of Sport | Barrett S.,Griffith University | Warren H.S.,Cancer Immunology Research Unit | And 9 more authors.
e-SPEN Journal | Year: 2014

Background and aims: The immune mechanisms by which probiotics reduce susceptibility to upper respiratory tract illness is uncertain. The aim of this study was to examine purported cell-mediated immune mechanisms that might explain the reduction in respiratory illness observed following daily supplementation with Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) and a combined Lactobacillus acidophilus NCFM & B. animalis subsp. lactis BI-07 (NCFM & Bi-07). Methods: A cohort of 144 healthy physically active individuals were allocated to daily supplementation consumed as a beverage with Bl-04 (n=46) supplemented at a dosage of 2.0×109 colony forming units (cfu) per day, NCFM & Bi-07 (n=47) at a dosage of 5.0×109CFU per day each, or a placebo (n=51) over 150d. Markers included plasma cytokines, metalloproteinases and neurotrophins, peripheral blood leucocyte numbers, antibody-dependent and antibody-independent NK cell activity (NKCA), and peripheral blood mononuclear cell (PBMC) phagocytosis. Results: A total of 125 subjects were included in the final analysis. No significant effects were observed on cytokines, on white cell differentials, NKCA or PBMC phagocytosis from pre- to post-supplementation. The biomarkers that increased significantly from pre- to post-supplementation were the concentration of plasma macrophage inflammatory protein (MIP)-1δ which was higher in the Bl-04 than placebo group (Bl-04 25%±11%, placebo-3.3%±9.4%; mean±SD, P=0.003) while the concentration of plasma matrix metallo-proteinase (MMP)-1 decreased by 11%±16% in the NCFM & Bi-07 group and increased by 21%±17% in the placebo group, which was a significant 26% difference (8-41%; P=0.02). Conclusion: Probiotic supplementation had little effect on parameters of the innate immune system. Mechanisms explaining the beneficial effect of Bl-04 or NCFM & Bi-07 supplementation on respiratory illness remain unclear. © 2014 European Society for Clinical Nutrition and Metabolism. Source

Warren H.S.,Cancer Immunology Research Unit | Warren H.S.,Australian National University | Wu F.,Cancer Immunology Research Unit | Wu F.,University of Canberra | And 6 more authors.
Journal of Immunological Methods | Year: 2013

In this technical note we provide data useful for the clinical application of the target-induced Natural Killer (NK) loss (TINKL) assay. The TINKL assay is a sensitive flow cytometry-based assay for measuring NK cell function. Loss of NK cells from the lymphocyte gate occurs following culture with K562 (the prototypic target cell for natural killing) and antibody-coated target cells (for antibody-dependent killing). By analysis of multiple samples of PBMC from single donors we document the intra-experimental variability and the inter-experimental variability of the assay. The intra-experimental coefficient of variation (CV) was on average 11% for natural killing and 3% for antibody-dependent killing, compared to 14% and 9% respectively for the inter-experimental variation. Analysis of a 123 normal healthy adults shows large variability in the functional capacity of NK cells in the population both for natural killing (CV 33%) and antibody-dependent killing (CV 27%). © 2013 Elsevier B.V. Source

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