be identified through 7 days that are highly accurate at predicting radiation status and distinguishing different levels of radiation exposure. From a practical perspective, these results suggest that the application of a single reference set of ‘‘radiation response’’ genes would be unlikely to provide the most sensitive screen for radiation exposure over time. Conversely, reference lists of PB genes that are specific for different time points could be applied in the screening for radiation exposure provided that the time of exposure was known. A critical question to be addressed in the development of PB gene expression profiling to detect medical conditions or exposures is the specificity of PB gene expression changes in response to genotoxic stresses. The PB signatures of 3 different doses of radiation displayed 100% accuracy in identifying septic animals as non-irradiated and the PB signature of sepsis was also 100% accurate in identifying irradiated mice as non-septic. These results demonstrate specificity in the PB responses to ionizing radiation and sepsis. These data also provide in vivo validation of a prior report by Boldrick et al. [40] in which human PB mononuclear cells were found to have a stereotypic response to LPS exposure in vitro and specific alterations in gene expression were observed in response to different strains of bacteria [40]. Ramilo et al. also recently reported that distinct patterns of PB gene expression can be identified among patients with different bacterial infections [11]. We found no genes in common between the PB signatures of radiation exposure and the PB signature of gram negative sepsis. Taken together, our results demonstrate that the in vivo PB molecular responses to ionizing radiation and bacterial sepsis are quite distinct and can be utilized to distinguish one condition from the other with a high level of accuracy. Our analyses of expression signatures in human patients demonstrated that it is possible to utilize PB gene expression profiles to distinguish individuals who have been exposed to an environmental hazard, ionizing radiation, within a heterogeneous human population with a high level of accuracy. It will be important to further test the accuracy of this PB predictor of human radiation exposure in a human population exposed to lower dose irradiation (e.g. 0.1–1 cGy), as might be expected via occupational exposures (e.g. radiology technicians, nuclear power plant workers)[41–43]. A potential pitfall in the clinical application of PB gene expression profiling would be that variations in PB gene expression in people would be such that it might be difficult to distinguish the effects of a given exposure or medical condition from expected background alterations in gene expression [12]. However, Whitney et al. [12] showed that the alterations in PB gene expression observed in patients with lymphoma or bacterial infection was significantly greater than the relatively narrow variation observed in healthy individuals [12]. Our study confirms that PB gene expression profiles can be successfully applied to detect a specific exposure in a heterogeneous human population and that inter-individual differences in PB gene expression do not significantly confound the utility of this approach. We further showed that unique PB gene expression profiles can be identified which distinguish chemotherapy-treated patients versus patients who had not received chemotherapy with an overall accuracy of 81% and 78%, respectively. Similar to the PB signature of radiation, the PB signature of chemotherapy demonstrated accuracy and specificity in distinguishing healthy individuals and pre-irradiated patients (100% and 92% accuracy, respectively). However, the accuracy of the PB signature of chemotherapy was more limited when tested against patients who received radiation conditioning (62%). This observation provides the basis for further investigation as to which families of genes may Radiation Signatures be represented in both the PB molecular response to radiation and chemotherapy. However, since all 12 of the post-irradiation patients whose status was mispredicted by the PB chemotherapy signature had received combination chemotherapy within the prior year, the true specificity of this PB signature of chemotherapy cannot be addressed via this comparison. We are currently enrolling additional patients to this study who have not undergone prior chemotherapy to further test the specificity of a PB metagene of chemotherapy treatment. Peripheral blood is a readily accessible source of tissue which has the potential to provide a window to the presence of disease or exposures. Early studies applying PB gene expression analysis have demonstrated that this approach is sensitive for the detection of patterns of gene expression in association with a variety of medical conditions [3–12,27]. It remains to be determined whether PB gene expression profiles can be successfully applied in medical practice or public health screening for the early detection of specific diseases or environmental exposures. Our results demon- strate that PB gene expression profiles can be identified in mice and humans which are specific, accurate over time, and not confounded by inter-individual differences. Supporting Information Table S1 Found at: doi:10.1371/journal.pone.0001912.s001 (0.43 DOC) Table S2 Found at: doi:10.1371/journal.pone.0001912.s002 (0.09 DOC) Table S3 Found at: doi:10.1371/journal.pone.0001912.s003 (0.20 DOC) Table S4 Found at: doi:10.1371/journal.pone.0001912.s004 (0.49 DOC) Table S5 Found at: doi:10.1371/journal.pone.0001912.s005 (0.08 DOC) Table S6 Found at: doi:10.1371/journal.pone.0001912.s006 (0.03 DOC) Table S7 Found at: doi:10.1371/journal.pone.0001912.s007 (0.08 DOC) Table S8 Found at: doi:10.1371/journal.pone.0001912.s008 (0.08 DOC) MB MB MB MB MB MB MB MB Acknowledgments The authors wish to acknowledge Lorinda O’Connor and Ashley Morris for their support in sample acquisition. Author Contributions Conceived and designed the experiments: JC NC JN. Performed the experiments: HD GM SM HH AS ZW. Analyzed the data: JC HD JN SM ZW. Contributed reagents/materials/analysis tools: GM ZW. Wrote the paper: JC HD NC GG JN SM. PLoS ONE | www.plosone.org 8 April 2008 | Volume 3 | Issue 4 | e1912