T-cell activation under hypoxic conditions enhances IFN-gamma secretion.

Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4(+) T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4(+) T-cell cytokines, especially IFN-gamma. The enhancing effects of hypoxia on IFN-gamma secretion were independent of mouse strain, and were also unaffected using CD4(+) T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1alpha. Using T cells from IFN-gamma receptor-deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-gamma expression were not due to effects on IFN-gamma consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45-related factor 2 attenuated the enhancing effect of hypoxia on IFN-gamma secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4(+) T cells.

Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage.

This study evaluated the utility of human blood micronucleated reticulocyte (MNCD71+) frequency measurement as a cytogenetic damage biomarker. The analytical methodology was flow cytometry in conjunction with a previously described three color fluorescence labeling technique that includes anti-CD71 to focus analyses on the most immature fraction of reticulocytes [S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.K. Torous, S. Avlasevich, et al., Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ. Mol. Mutagen. 44 (2004) 427-435]. Blood specimens from 50 self-reported healthy adult volunteers were studied. In addition to MNCD71+ measurements, blood plasma folate and B12 levels were assessed, since these variables tend to influence other indices of cytogenetic damage. Time-course data are also provided for 10 cancer patients undergoing treatment. For these subjects, frequency of MNCD71+ was measured immediately before therapy, and daily during the first week of chemotherapy and/or fractionated radiotherapy. For the group of healthy volunteers, the variables of age, and folate and B12 levels demonstrated no significant effect on MNCD71+ frequency. In addition, no difference was observed between pre-treatment MNCD71+ values for cancer patients compared with healthy volunteers. Regarding chemotherapy and/or partial body radiotherapy, elevated frequencies were observed upon initiation of treatment for 9 of the 10 patients studied. Maximal effects were observed 3-5 days following initiation of therapy. The largest increases in frequency of MNCD71+ (up to 25.9-fold) were observed in those patients exposed to anti-neoplastic drugs, presumably due to the systemic red marrow exposure provided by these agents. Taken together, these data support the hypothesis that the MNCD71+ endpoint represents a valuable biomarker of cytogenetic damage that does not require cell culture or microscopy-based scoring.

Performance of flow cytometric analysis for the micronucleus assay--a reconstruction model using serial dilutions of malaria-infected cells with normal mouse peripheral blood.

To confirm the performance and statistical power of a flow cytometric method for scoring micronucleated erythrocytes, reconstruction experiments were performed. For these investigations, peripheral blood erythrocytes from untreated mice, with a micronucleated erythrocyte frequency of approximately 0.1% were combined with known quantities of Plasmodium berghei (malaria) infected mouse erythrocytes. These cells had an infected erythrocyte frequency of approximately 0.7%, and mimic the DNA content of micronuclei (MN). For an initial experiment, samples with a range of MN/malaria (Mal) content were constructed and analysed in triplicate by flow cytometry until 2000, 20,000 and 200,000 total erythrocytes were acquired. In a second experiment, each specimen was analysed in triplicate until 2000, 20,000, 200,000 and 1,000,000 erythrocytes were acquired. As expected, the sensitivity of the assay to detect small changes in rare erythrocyte sub-population frequencies was directly related to the number of cells analysed. For example, when 2000 cells were scored, increases in MN/Mal frequencies of 3.9- or 2.7-fold were detected as statistically significant. When 200,000 cells were analysed, a 1.2-fold increase was detected. These data have implications for the experimental design and interpretation of micronucleus assays that are based on automated scoring procedures, since previously unattainable numbers of cells can now be readily scored.