Publications

Scholarly Journals--Published

  • Overbey EG, da Silveira WA, Stanbouly S, Nishiyama NC, Roque-Torres GD, Pecaut MJ, Zawieja DC, Wang C, Willey JS, Delp MD, Hardiman G & Mao XW. Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina. Sci Rep. 2019 Sep 16;9(1):13304. doi: 10.1038/s41598-019-49453-x. PMID: 31527661. (09/2019)
  • Rettig TA, Nishiyama NC, Pecaut MJ & Chapes SK. Effects of skeletal unloading on the bone marrow antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice. Life Sci Space Res (Amst). 2019 Aug;22:16-28. doi: 10.1016/j.lssr.2019.06.001. Epub 2019 Jun 14. PMID: 31421845. (06/2019)
  • Rettig TA, Bye BA, Nishiyama NC, Hlavacek S, Ward C, Pecaut MJ, Chapes SK. Effects of skeletal unloading on the antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice. PLoS One. 2019 Jan 17;14(1):e0210284. doi: 10.1371/journal.pone.0210284. eCollection 2019. PMID: 30653556 (01/2019)
  • Rettig TA, Pecaut MJ & Chapes SK. A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing. FASEB Bioadv. 2019 Jan;1(1):6-17. Epub 2018 Aug 30. https://doi.org/10.1096/fba.1017. PMID: 30740592 (01/2019)
  • Mao XW, Sandberg LB, Gridley DS, Herrmann EC, Zhang G, Raghavan R, Zubarev RA, Zhang B, Stodieck LS, Ferguson VL, Bateman TA & Pecaut MJ. Proteomic Analysis of Mouse Brain Subjected to Spaceflight. Int J Mol Sci. 2018 Dec 20;20(1). pii: E7. doi: 10.3390/ijms20010007. PMID: 30577490 (12/2018)
  • Baio J, Martinez AF, Silva I, Hoehn CV, Countryman S, Bailey L, Hasaniya N, Pecaut MJ & Kearns-Jonker M. Cardiovascular progenitor cells cultured aboard the International Space Station exhibit altered developmental and functional properties. NPJ Microgravity. 2018 Jul 26;4:13. doi: 10.1038/s41526-018-0048-x. eCollection 2018. PMID: 30062101 (07/2018)
  • Mao X W, Nishiyama N C, Campbell-Beachler M, Gifford P, Haynes K E, Gridley D S, & Pecaut M J. (2017). Role of NADPH Oxidase as a Mediator of Oxidative Damage in Low-Dose Irradiated and Hindlimb-Unloaded Mice. Radiat Res, , . The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2+/+) C57BL/6 mice or Nox2-/- (B6.129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control; hindlimb unloading (HLU); low-dose/low-dose-rate radiation (LDR); or HLU with LDR simultaneously for 21 days. The mice were then sacrificed one month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04 Gy at 0.01 cGy/h) to the simulate whole-body irradiation, similar to that experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology. The level of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, was measured. Expression of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte end-feet, was quantified. Thirty days after simulated spaceflight, KO mice showed decreased apoptosis (P < 0.05) in the brain compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. The level of 4-HNE protein was significantly elevated in the hippocampus of the LDR with HLU treatment group compared to WT controls (P < 0.05). However, there were no significant differences among groups of Nox2-KO mice at the one-month time point. In contrast to findings in the WT animals, superoxide dismutase (SOD) level and expression of AQP4 were similar among all KO groups. In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2-KO mice. This suggests that Nox2-containing NADPH oxidase may contribute to spaceflight environment-induced oxidative stress. (08/2017) (link)
  • Pecaut M J, Mao X W, Bellinger D L, Jonscher K R, Stodieck L S, . . . Gridley D S. (2017). Is spaceflight-induced immune dysfunction linked to systemic changes in metabolism?. PLoS One, 12(5), e0174174. The Space Shuttle Atlantis launched on its final mission (STS-135) on July 8, 2011. After just under 13 days, the shuttle landed safely at Kennedy Space Center (KSC) for the last time. Female C57BL/6J mice flew as part of the Commercial Biomedical Testing Module-3 (CBTM-3) payload. Ground controls were maintained at the KSC facility. Subsets of these mice were made available to investigators as part of NASA's Bio-specimen Sharing Program (BSP). Our group characterized cell phenotype distributions and phagocytic function in the spleen, catecholamine and corticosterone levels in the adrenal glands, and transcriptomics/metabolomics in the liver. Despite decreases in most splenic leukocyte subsets, there were increases in reactive oxygen species (ROS)-related activity. Although there were increases noted in corticosterone levels in both the adrenals and liver, there were no significant changes in catecholamine levels. Furthermore, functional analysis of gene expression and metabolomic profiles suggest that the functional changes are not due to oxidative or psychological stress. Despite changes in gene expression patterns indicative of increases in phagocytic activity (e.g. endocytosis and formation of peroxisomes), there was no corresponding increase in genes related to ROS metabolism. In contrast, there were increases in expression profiles related to fatty acid oxidation with decreases in glycolysis-related profiles. Given the clear link between immune function and metabolism in many ground-based diseases, we propose a similar link may be involved in spaceflight-induced decrements in immune and metabolic function. (2017) (link)
  • Gridley D S, & Pecaut M J. (2016). Changes in the distribution and function of leukocytes after whole-body iron ion irradiation. , , . High-energy particle radiation could have a considerable impact on health during space missions. This study evaluated C57BL/6 mice on Day 40 after total-body 56Fe26+ irradiation at 0, 1, 2 and 3 gray (Gy). Radiation consistently increased thymus mass (one-way ANOVA: P < 0.005); spleen, liver and lung masses were similar among all groups. In the blood, there was no radiation effect on the white blood cell (WBC) count or major leukocyte types. However, the red blood cell count, hemoglobin, hematocrit and the CD8+ T cytotoxic (Tc) cell count and percentage all decreased, while both the CD4:CD8 (Th:Tc) cell ratio and spontaneous blastogenesis increased, in one or more irradiated groups compared with unirradiated controls (P < 0.05 vs 0 Gy). In contrast, splenic WBC, lymphocyte, B cell and T helper (Th) counts, %B cells and the CD4:CD8 ratio were all significantly elevated, while Tc percentages decreased, in one or more of the irradiated groups compared with controls (P < 0.05 vs 0 Gy). Although there were trends for minor, radiation-induced increases in %CD11b+ granulocytes in the spleen, cells double-labeled with adhesion markers (CD11b+CD54+, CD11b+CD62E+) were normal. Splenocyte spontaneous blastogenesis and that induced by mitogens (PHA, ConA, LPS) was equivalent to normal. In bone marrow, the percentage of cells expressing stem cell markers, Sca-1 and CD34/Sca-1, were low in one or more of the irradiated groups (P < 0.05 vs 0 Gy). Collectively, the data indicate that significant immunological abnormalities still exist more than a month after 56Fe irradiation and that there are differences dependent upon body compartment. (07/2016) (link)
  • Mao X W, Nishiyama N C, Pecaut M J, Campbell-Beachler M, Gifford P, . . . Gridley D S. (2016). Simulated Microgravity and Low-Dose/Low-Dose-Rate Radiation Induces Oxidative Damage in the Mouse Brain. Radiat Res, 185(6), 647-57. Microgravity and radiation are stressors unique to the spaceflight environment that can have an impact on the central nervous system (CNS). These stressors could potentially lead to significant health risks to astronauts, both acutely during the course of a mission or chronically, leading to long-term, post-mission decrements in quality of life. The CNS is sensitive to oxidative injury due to high concentrations of oxidizable, unsaturated lipids and low levels of antioxidant defenses. The purpose of this study was to evaluate oxidative damage in the brain cortex and hippocampus in a ground-based model for spaceflight, which includes prolonged unloading and low-dose radiation. Whole-body low-dose/low-dose-rate (LDR) gamma radiation using (57)Co plates (0.04 Gy at 0.01 cGy/h) was delivered to 6 months old, mature, female C57BL/6 mice (n = 4-6/group) to simulate the radiation component. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of the microgravity component. Mice were hindlimb suspended and/or irradiated for 21 days. Brains were isolated 7 days or 9 months after irradiation and hindlimb unloading (HLU) for characterization of oxidative stress markers and microvessel changes. The level of 4-hydroxynonenal (4-HNE) protein, an oxidative specific marker for lipid peroxidation, was significantly elevated in the cortex and hippocampus after LDR + HLU compared to controls (P < 0.05). The combination group also had the highest level of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression compared to controls (P < 0.05). There was a significant decrease in superoxide dismutase (SOD) expression in the animals that received HLU only or combined LDR + HLU compared to control (P < 0.05). In addition, 9 months after LDR and HLU exposure, microvessel densities were the lowest in the combination group, compared to age-matched controls in the cortex (P < 0.05). Our data provide the first evidence that prolonged exposure to simulated microgravity and LDR radiation is associated with increased oxidative stress biomarkers that may increase the likelihood of brain injury and reduced antioxidant defense. NOX2-containing nicotinamide adenosine dinucleotide phosphate (NADPH oxidase) may contribute to spaceflight environment-induced oxidative stress. (06/2016) (link)
  • Gridley D S, Mao X W, Tian J, Cao J D, Perez C, . . . Pecaut M J. (2015). Genetic and Apoptotic Changes in Lungs of Mice Flown on the STS-135 Mission in Space. In Vivo, 29(4), 423-433. AIM: The goal of the study was to evaluate changes in lung status due to spaceflight stressors that include radiation above levels found on Earth. MATERIALS AND METHODS: Within hours after return from a 13-day mission in space onboard the Space Shuttle Atlantis, C57BL/6 mice (FLT group) were euthanized; mice housed on the ground in similar animal enclosure modules served as controls (AEM group). Lung tissue was collected to evaluate the expression of genes related to extracellular matrix (ECM)/adhesion and stem cell signaling. Pathway analysis was also performed. In addition, immunohistochemistry for stem cell antigen-1 (SCA-1), the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis, and staining for histological characteristics were performed. RESULTS: There were 18/168 genes significantly modulated in lungs from the FLT group (p<0.05 vs. AEM); 17 of these were up-regulated and one was down-regulated. The greatest effect, namely a 5.14-fold increase, was observed on Spock1 (also known as Spark/osteonectin), encoding a multi-functional protein that has anti-adhesive effects, inhibits cell proliferation and regulates activity of certain growth factors. Additional genes with increased expression were cadherin 3 (Cdh3), collagen, type V, alpha 1 (Col5a1), integrin alpha 5 (Itga5), laminin, gamma 1 (Lamc1), matrix metallopeptidase 14 (Mmp14), neural cell adhesion molecule 1 (Ncam1), transforming growth factor, beta induced (Tgfbi), thrombospondin 1 (Thbs1), Thbs2, versican (Vcan), fibroblast growth factor receptor 1 (Fgfr1), frizzled homolog 6 (Fzd6), nicastrin (Ncstn), nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 (Nfatc4), notch gene homolog 4 (Notch4) and vang-like 2 (Vangl2). The down-regulated gene was Mmp13. Staining for SCA-1 protein showed strong signal intensity in bronchiolar epithelial cells of FLT mice (p<0.05 vs. AEM). TUNEL positivity was also significantly higher in the FLT mice (p<0.05 vs. AEM), but no consistent histological differences were noted. CONCLUSION: The results demonstrate that spaceflight-related stress had a significant impact on lung integrity, indicative of tissue injury and remodeling. (2015)
  • Pecaut Michael J, Baqai Farnaz P, & Gridley Daila S. (2014). Impact of total-body irradiation on the response to a live bacterial challenge. Int J Radiat Biol, 90(7), 515-526. Purpose: Concern regarding radiation effects on human health continues to increase worldwide. Given that infection is a major cause of morbidity and mortality after exposure, the aim of this study was to evaluate decrements in immune cell populations using a mammalian model subjected to a live bacterial infection. Materials and methods: C57BL/6 mice were exposed to total-body irradiation (TBI) with 3 Gy protons (70 cGy/min). One, 2, 4, 8 or 16 days later, subsets of mice were injected intraperitoneally with live Escherichia coli [055: K59(B5)]. Control groups received no radiation and vehicle (no bacteria). The mice were euthanized for analyses 90-120 min after injection of the bacteria. Results: There were no unexpected effects of radiation or E. coli alone. Despite dramatic radiation-induced decreases in all leukocyte populations in both the blood and spleen, irradiated mice were still able to respond to an immune challenge based on capacity to generate an oxidative burst and secrete inflammatory cytokines, i.e., tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). However, these responses were generally elevated above control values. Conclusions: Together, these results suggest the possibility for enhanced inflammation-associated tissue injury and increased risk for chronic inflammation. (07/2014) (link)
  • Mehrotra Shalini, Pecaut Michael J, & Gridley Daila S. (2014). Minocycline Modulates Cytokine and Gene Expression Profiles in the Brain After Whole-body Exposure to Radiation. In Vivo, 28(1), 21-32. An effective countermeasure against radiation damage to normal tissues is urgently needed. The major goal of the present study was to determine if minocycline could modify the immunomodulatory effects of radiation on the brain. C57BL/6 mice were treated with minocycline intraperitoneally for 5 days beginning immediately before total-body exposure to 0, 1, 2 and 3 Gray (Gy)(60) Co gamma-rays. Brains were collected on days 4 and 32 post-irradiation for cytokine and gene analyses. Minocycline treatment significantly increased the levels of interleukin (IL)-10, IL-15 and vascular endothelial growth factor (VEGF) in the brain on day 4 in one or more irradiated groups compared to radiation-alone (p < 0.05). IL-10 is anti-inflammatory, IL-15 can prevent apoptosis and VEGF is nuroprotective. On day 32, the drug decreased IL-1 beta in the 2- Gy group (p < 0.05 vs. 2-Gy alone); this cytokine is implicated in immune-related central nervous system pathologies. Microarray analysis of brains on day 32 showed that while radiation increased expression of inflammatory genes such as Il1f10,Il17, Tnfrsf11b, Tnfsf12, Il12b and Il1f8, these were no longer up-regulated in the minocycline-treated groups. Similarly, the pro-apoptotic gene Bik and nitric oxide synthase producer (Nostrin) were no longer up-regulated in the drug-treated groups. Pathway analysis based on gene data suggested that catenin-beta 1 and tumor suppressor-related transcription regulation were significantly activated by radiation and/or minocycline (activation z-score > 2.0). Overall, the data warrant further testing of minocycline as a potential neuroprotectant against radiation-induced damage. (01/2014)
  • Mao X W, Pecaut M J, Stodieck L S, Ferguson V L, Bateman T A, Bouxsein M L, & Gridley D S. (2014). Biological and metabolic response in STS-135 space-flown mouse skin. Free Radical Research, 48(8), 890-897. There is evidence that space flight condition-induced biological damage is associated with increased oxidative stress and extracellular matrix (ECM) remodeling. To explore possible mechanisms, changes in gene expression profiles implicated in oxidative stress and in ECM remodeling in mouse skin were examined after space flight. The metabolic effects of space flight in skin tissues were also characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Female C57BL/6 mice were flown in the STS-135 using animal enclosure modules (AEMs). Within 3-5 h after landing, the mice were euthanized and skin samples were harvested for gene array analysis and metabolic biochemical assays. Many genes responsible for regulating production and metabolism of reactive oxygen species (ROS) were significantly ( p < 0.05) altered in the flight group, with fold changes >1.5 compared to AEM control. For ECM profile, several genes encoding matrix and metalloproteinases involved in ECM remodeling were significantly up-/down-regulated following space flight. To characterize the metabolic effects of space flight, global biochemical profiles were evaluated. Of 332 named biochemicals, 19 differed significantly ( p < 0.05) between space flight skin samples and AEM ground controls, with 12 up-regulated and 7 down-regulated including altered amino acid, carbohydrate metabolism, cell signaling, and transmethylation pathways. Collectively, the data demonstrated that space flight condition leads to a shift in biological and metabolic homeostasis as the consequence of increased regulation in cellular antioxidants, ROS production, and tissue remodeling. This indicates that astronauts may be at increased risk for pathophysiologic damage or carcinogenesis in cutaneous tissue. [ABSTRACT FROM AUTHOR] Copyright of Free Radical Research is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) (2014) (link)
  • Mao X W, Pecaut M J, Cao J D, Moldovan M, & Gridley D S. (2013). Low-dose Radiation Modifies Skin Response to Acute Gamma-rays and Protons. In Vivo, 27(6), 695-700. The goal of the present study was to obtain pilot data on the effects of protracted low-dose/low-dose-rate (LDR) gamma-rays on the skin, both with and without acute gamma or proton irradiation (IR). Six groups of C57BL/6 mice were examined: a) 0 Gy control, b) LDR, c) Gamma, d) LDR+Gamma, e) Proton, and f) LDR+Proton. LDR radiation was delivered to a total dose of 0.01 Gy (0.03 cGy/h), whereas the Gamma and Proton groups received 2 Gy (0.9 Gy/min and 1.0 Gy/min, respectively). Assays were performed 56 days after exposure. Skin samples from all irradiated groups had activated caspase-3, indicative of apoptosis. The significant (p<0.05) increases in immunoreactivity in the Gamma and Proton groups were not present when LDR pre-exposure was included. However, the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay for DNA fragmentation and histological examination of hematoxylin and eosin-stained sections revealed no significant differences among groups, regardless of radiation regimen. The data demonstrate that caspase-3 activation initially triggered by both forms of acute radiation was greatly elevated in the skin nearly two months after whole-body exposure. In addition, LDR gamma-ray priming ameliorated this response. (11/2013)
  • Mao X W, Pecaut M J, Stodieck L S, Ferguson V L, Bateman T A, . . . Gridley D S. (2013). Spaceflight Environment Induces Mitochondrial Oxidative Damage in Ocular Tissue. Radiat Res, 180(4), 340-350. A recent report shows that more than 30% of the astronauts returning from Space Shuttle missions or the International Space Station (ISS) were diagnosed with eye problems that can cause reduced visual acuity. We investigate here whether spaceflight environment-associated retinal damage might be related to oxidative stress-induced mitochondrial apoptosis. Female C57BL/6 mice were flown in the space shuttle Atlantis (STS-135), and within 3-5 h of landing, the spaceflight and ground-control mice, similarly housed in animal enclosure modules (AEMs) were euthanized and their eyes were removed for analysis. Changes in expression of genes involved in oxidative stress, mitochondrial and endothelial cell biology were examined. Apoptosis in the retina was analyzed by caspase-3 immunocytochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Levels of 4-hydroxynonenal (4-HNE) protein, an oxidative specific marker for lipid peroxidation were also measured. Evaluation of spaceflight mice and AEM ground-control mice showed that expression of several genes playing central roles in regulating the mitochondria-associated apoptotic pathway were significantly altered in mouse ocular tissue after spaceflight compared to AEM ground-control mice. In addition, the mRNA levels of several genes, which are responsible for regulating the production of reactive oxygen species were also significantly up-regulated in spaceflight samples compared to AEM ground-control mice. Further more, the level of HNE protein was significantly elevated in the retina after spaceflight compared to controls. Our results also revealed that spaceflight conditions induced significant apoptosis in the retina especially inner nuclear layer (INL) and ganglion cell layer (GCL) compared to AEM ground controls. The data provided the first evidence that spaceflight conditions induce oxidative damage that results in mitochondrial apoptosis in the retina. This data suggest that astronauts may be at increased risk for late retinal degeneration. (C) 2013 by Radiation Research Society (10/2013) (link)
  • Gridley D S, Mao X W, Cao J D, Bayeta E J M, & Pecaut M J. (2013). Protracted low-dose radiation priming and response of liver to acute gamma and proton radiation. Free Radical Research, 47(10), 811-820. This study evaluated liver from C57BL/6 mice irradiated with low-dose/low-dose-rate (LDR) gamma-rays (0.01 Gy, 0.03 cGy/h), with and without subsequent exposure to acute 2 Gy gamma or proton radiation. Analyses were performed on day 56 post-exposure. Expression patterns of apoptosis-related genes were strikingly different among irradiated groups compared with 0 Gy (p < 0.05). Two genes were affected in the Gamma group, whereas 10 were modified in the LDR + Gamma group. In Proton and LDR + Proton groups, there were six and 12 affected genes, respectively. Expression of genes in the Gamma (Traf3) and Proton (Bak1, Birc2, Birc3, Mcl1) groups was no longer different from 0 Gy control group when mice were pre-exposed to LDR gamma-rays. When each combined regimen was compared with the corresponding group that received acute radiation alone, two genes in the LDR + Gamma group and 17 genes in the LDR + Proton group were modified; greatest effect was on Birc2 and Nol3 (>5-fold up-regulated by LDR + Protons). Oxygen radical production in livers from the LDR + Proton group was higher in LDR, Gamma, and LDR + Gamma groups (p < 0.05 vs. 0 Gy), but there were no differences in phagocytosis of E. coli. Sections stained with hematoxylin and eosin (H&E) suggested more inflammation, with and without necrosis, in some irradiated groups. The data demonstrate that response to acute radiation is dependent on radiation quality and regimen and that some LDR gamma-ray-induced modifications in liver response were still evident nearly 2 months after exposure. (10/2013) (link)
  • Gridley D S, Mao X W, Stodieck L S, Ferguson V L, Bateman T A, . . . Pecaut M J. (2013). Changes in Mouse Thymus and Spleen after Return from the STS-135 Mission in Space. PLoS One, 8(9), 14. Our previous results with flight (FLT) mice showed abnormalities in thymuses and spleens that have potential to compromise immune defense mechanisms. In this study, the organs were further evaluated in C57BL/6 mice after Space Shuttle Atlantis returned from a 13-day mission. Thymuses and spleens were harvested from FLT mice and ground controls housed in similar animal enclosure modules (AEM). Organ and body mass, DNA fragmentation and expression of genes related to T cells and cancer were determined. Although significance was not obtained for thymus mass, DNA fragmentation was greater in the FLT group (P<0.01). Spleen mass alone and relative to body mass was significantly decreased in FLT mice (P<0.05). In FLT thymuses, 6/84 T cell-related genes were affected versus the AEM control group (P<0.05; up: IL10, Il18bp, Il18r1, Spp1; down: Ccl7, IL6); 15/84 cancer-related genes had altered expression (P<0.05; up: Casp8, FGFR2, Figf, Hgf, IGF1, Itga4, Ncam1, Pdgfa, Pik3r1, Serpinb2, Sykb; down: Cdc25a, E2F1, Mmp9, Myc). In the spleen, 8/84 cancer-related genes were affected in FLT mice compared to AEM controls (P<0.05; up: Cdkn2a; down: Birc5, Casp8, Ctnnb1, Map2k1, Mdm2, NFkB1, Pdgfa). Pathway analysis (apoptosis signaling and checkpoint regulation) was used to map relationships among the cancer-related genes. The results showed that a relatively short mission in space had a significant impact on both organs. The findings also indicate that immune system aberrations due to stressors associated with space travel should be included when estimating risk for pathologies such as cancer and infection and in designing appropriate countermeasures. Although this was the historic last flight of NASA's Space Shuttle Program, exploration of space will undoubtedly continue. (09/2013) (link)
  • Mehrotra S, Pecaut M J, & Gridley D S. (2013). Effects of Minocycline on Hematopoietic Recovery after Whole-body Irradiation. In Vivo, 27(1), 11-28. Background/Aim: We previously found that minocycline enhanced the levels of several leukocyte populations and had the capacity to induce secretion of certain cytokines early after irradiation. In the current study we further determined the drug's effect on hematopoietic recovery. Materials and Methods: Minocycline was injected intraperitoneally into C57BL/6 mice for 5 days, beginning immediately before exposure to Co-60 gamma-rays ( 1, 2, 3 Gy). Thirty-two days post-irradiation, spleen and blood were collected to quantify cell populations, cytokines in splenic T-cell supernatants after anti-CD3 activation, and chromosomic status based on spectral karyotyping. Results: While radiation resulted in significantly lower B-cell counts at 3 Gy in both blood and spleen, minocycline treatment increased the counts and/or percentages of splenic B-cells at 2 Gy and 3 Gy. In spleen supernatants, the drug-alone increased the levels of cytokines, including interleukin-1 alpha (IL-1 alpha) and IL-6 that are radioprotective, as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF that accelerate neutrophil recovery. In addition, minocycline suppressed the production of interferon-gamma that can prevent hematopoiesis. Dose-dependent radiation-induced chromosomic abnormalities were present in splenic leukocytes. Conclusion: The data indicate that minocycline exerts a relatively long-term effect on parameters that influence hematopoietic recovery. Further testing of this drug as a countermeasure for acute radiation syndrome, is necessary to determine its full potential. (01/2013)
  • Gridley D S, Rizvi A, Makinde A Y, Luo-Owen X, Mao X W, . . . Pecaut M J. (2013). Space-relevant radiation modifies cytokine profiles, signaling proteins and Foxp3(+) T cells. Int J Radiat Biol, 89(1), 26-35. Purpose:The major goal was to evaluate effects of various radiation regimens on leukocyte populations relatively long-term after whole-body irradiation. Materials and methods:C57BL/6 mice were exposed to-low-dose/low-dose rate (LDR) Co-57 gamma-rays (0.01 Gy, 0.03 cGy/h), with and without acute 2 Gy proton (1 Gy/min) or gamma-ray (0.9 Gy/min) irradiation; analyses were done on days 21 and 56 post-exposure. Results:Numerous radiation-induced changes were noted at one or both time points. Among the most striking differences (P < 0.05) were:(i) High percentage of CD4(+)CD25(+)Foxp(3+) T cells in spleens from the Proton vs. LDR, Gamma and LDR + Proton groups (day 56); (ii) high interleukin-2 (IL-2) in spleen supernatants from the LDR and LDR + Proton groups vs. 0 Gy (day 56), whereas IL-10 was high in the LDR + Gamma group vs. 0 Gy (day 56); (iii) difference in transforming growth factor-beta 1 (TGF-beta 1) in spleen supernatants from Proton and LDR + Proton groups vs. Gamma and LDR + Gamma groups (both days); (iv) low TGF-beta 1 in blood from LDR + Proton vs. LDR + Gamma group (day 21); and (v) high level of activated cJun N-terminal kinase (JNK) in CD4(+) T cells from LDR + Proton vs. LDR + Gamma group (day 21). Conclusions:The findings demonstrate that at least some immune responses to acute 2 Gy radiation were dependent on radiation quality time of assessment, and pre-exposure to LDR gamma-rays. (01/2013) (link)
  • Mehrotra S, Pecaut M J, Freeman T L, Crapo J D, Rizvi A, . . . Gridley D S. (2012). Analysis of a Metalloporphyrin Antioxidant Mimetic (MnTE-2-PyP) as a Radiomitigator: Prostate Tumor and Immune Status. Technology in Cancer Research & Treatment, 11(5), 447-457. Due to radiation-induced immune depression and development of pathologies such as cancer, there is increasing urgency to identify radiomitigators that are effective when administered after radiation exposure. The main goal of this study was to determine the radiomitigation capacity of MnTE-2-PyP[Mn(III) tetrakis (N-ethylpyridinium-2-yl) porphyrin], a superoxide dismutase (SOD) mimetic, and evaluate leukocyte parameters in spleen and blood. C57BL/6 mice were total-body exposed to 2 Gy gamma-rays (Co-60), i.e., well below a lethal dose, followed by subcutaneous implantation of 5 x 10(5) RM-9 prostate tumor cells and initiation of MnTE-2-PyP treatment (day 0); interval between each procedure was 1-2h. The drug was administered daily (12 times). Tumor progression was monitored and immunological analyses were performed on a subset per group on day 12. Animals treated with MnTE-2-PyP alone had significantly slower tumor growth compared to mice that did not receive the drug (P < 0.05), while radiation alone had no effect. Treatment of tumor-bearing mice with MnTE-2-PyP alone significantly increased spleen mass relative to body mass; the numbers of splenic white blood cells (WBC) and lymphocytes (B and T), as well as circulating WBC, granulocytes, and platelets, were high compared to one of more of the other groups (P < 0.05). The results show that MnTE-2-PyP slowed RM-9 tumor progression and up-regulated immune parameters, but mitigation of the effects of 2 Gy total-body irradiation were minimal. (10/2012) (link)
  • Lloyd S A, Bandstra E R, Willey J S, Riffle S E, Tirado-Lee L, . . . Bateman T A. (2012). Effect of proton irradiation followed by hindlimb unloading on bone in mature mice: A model of long-duration spaceflight. Bone, 51(4), 756-764. Bone loss associated with microgravity unloading is well documented; however, the effects of spaceflight-relevant types and doses of radiation on the skeletal system are not well defined. In addition, the combined effect of unloading and radiation has not received much attention. In the present study, we investigated the effect of proton irradiation followed by mechanical unloading via hindlimb suspension (HLS) in mice. Sixteen-week-old female C57BL/6 mice were either exposed to 1 Gy of protons or a sham irradiation procedure (n = 30/group). One day later, half of the mice in each group were subjected to four weeks of HLS or normal loading conditions. Radiation treatment alone (IRR) resulted in approximately 20% loss of trabecular bone volume fraction (BV/TV) in the tibia and femur, with no effect in the cortical bone compartment. Conversely, unloading induced substantially greater loss of both trabecular bone (60-70% loss of BV/TV) and cortical bone (approximately 20% loss of cortical bone volume) in both the tibia and femur, with corresponding decreases in cortical bone strength. Histological analyses and serum chemistry data demonstrated increased levels of osteoclast-mediated bone resorption in unloaded mice, but not IRR. HLS + IRR mice generally experienced greater loss of trabecular bone volume fraction, connectivity density, and trabecular number than either unloading or irradiation alone. Although the duration of unloading may have masked certain effects, the skeletal response to irradiation and unloading appears to be additive for certain parameters. Appropriate modeling of the environmental challenges of long duration spaceflight will allow for a better understanding of the underlying mechanisms mediating spaceflight-associated bone loss and for the development of effective countermeasures. (C) 2012 Elsevier Inc. All rights reserved. (10/2012) (link)
  • Mehrotra S, Pecaut M J, & Gridley D S. (2012). Analysis of Minocycline as a Countermeasure Against Acute Radiation Syndrome. In Vivo, 26(5), 743-758. Background/Aim: To evaluate the impact of an antibiotic, minocycline, on several immune parameters in response to radiation in a mouse model. Materials and Methods: C57BL/6 mice were treated with minocycline (i.p.) for 5 days, beginning immediately before radiation with 1-3 Gy Co-60 gamma-rays. Spleen and blood were collected on day 4 post-irradiation. Cell populations were determined in the blood and spleen. Splenocytes were activated with anti-CD3 antibody for 48 h and cytokines were quantified. Results: Minocycline increased the counts and/or percentages of splenic macrophages, granulocytes, natural killer, T- and CDS+ T-cells (p<0.05 versus radiation alone). Minocycline significantly increased the expression of interleukin-1 alpha and beta, which are radioprotective, as well as the ones of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, which accelerate neutrophil recovery (p<0.05 versus radiation alone), while suppressing cytokines that could prevent hematopoiesis, e.g. macrophage inflammatory protein-1 alpha, tumor necrosis factor-alpha and interferon-gamma. Conclusion: These data indicate that minocycline should be further tested for use in restoration of the hematopoietic system after radiation exposure. (09/2012)
  • Luo-Owen X, Pecaut M J, Rizvi A, & Gridley D S. (2012). Low-Dose Total-Body gamma Irradiation Modulates Immune Response to Acute Proton Radiation (vol 177, pg 251, 2012). Radiation Research, 177(6), 827-827. (06/2012)
  • Luo-Owen X, Pecaut M J, Rizvi A, & Gridley D S. (2012). Low-Dose Total-Body gamma Irradiation Modulates Immune Response to Acute Proton Radiation (vol 177, pg 251, 2012). Radiation Research, 177(6), 827-827. (06/2012)
  • Haerich P, Eggers C, & Pecaut M J. (2012). Investigation of the Effects of Head Irradiation with Gamma Rays and Protons on Startle and Pre-Pulse Inhibition Behavior in Mice. Radiation Research, 177(5), 685-692. With the increased international emphasis on manned space exploration, there is a growing need to understand the impact of the spaceflight environment on health and behavior. One particularly important aspect of this environment is low-dose radiation. In the present studies, we first characterized the gamma- and proton-irradiation dose effect on acoustic startle and pre-pulse inhibition behaviors in mice exposed to 0-5 Gy brain-localized irradiation, and assessed these effects 2 days later. Subsequently, we used 2 Gy to assess the time course of gamma- and proton-radiation effects on startle reactivity 0-8 days after exposure. Exposures targeted the brain to minimize the impact of peripheral inflammation-induced sickness behavior. The effects of radiation on startle were subtle and acute. Radiation reduced the startle response at 2 and 5 Gy. Following a 2-Gy exposure, the response reached a minimum at the 2-day point. Proton and gamma-ray exposures did not differ in their impact on startle. We found there were no effects of radiation on pre-pulse inhibition of the startle response. (C) 2012 by Radiation Research Society (05/2012) (link)
  • Luo-Owen X, Pecaut M J, Rizvi A, & Gridley D S. (2012). Low-Dose Total-Body gamma Irradiation Modulates Immune Response to Acute Proton Radiation. Radiation Research, 177(3), 251-264. Luo-Owen, X., Pecaut, M. J., Rizvi, A. and Gridley, D. S. Low-Dose Total-Body gamma Irradiation Modulates Immune Response to Acute Proton Radiation. Radiat. Res. 177, 251-264 (2012). Health risks due to exposure to low-dose/low-dose-rate radiation alone or when combined with acute irradiation are not yet clearly defined. This study quantified the effects of protracted exposure to low-dose/low-dose-rate gamma rays with and without acute exposure to protons on the response of immune and other cell populations. C57BL/6 mice were irradiated with Co-57 (0.05 Gy at 0.025 cGy/h); subsets were subsequently exposed to high-dose/high-dose-rate proton radiation (250 MeV; 2 or 3 Gy at 0.5 Gy/min). Analyses were performed at 4 and 17 days postexposure. Spleen and thymus masses relative to body mass were decreased on day 4 after proton irradiation with or without pre-exposure to gamma rays; by day 17, however, the decrease was attenuated by the priming dose. Proton dose-dependent decreases, either with or without pre-exposure to gamma rays, occurred in white blood cell, lymphocyte and granulocyte counts in blood but not in spleen. A similar pattern was found for lymphocyte subpopulations, including CD3(+) T, CD19(+) B, CD4(+) T, CD8(+) T and NK1.1(+) natural killer (NK) cells. Spontaneous DNA synthesis by leukocytes after proton irradiation was high in blood on day 4 and high in spleen on day 17; priming with gamma radiation attenuated the effect of 3 Gy in both body compartments. Some differences were also noted among groups in erythrocyte and thrombocyte characteristics. Analysis of splenocytes activated with anti-CD3/anti-CD28 antibodies showed changes in T-helper 1 (Th1) and Th2 cytokines. Overall, the data demonstrate that pre-exposure of an intact mammal to low-dose/low-dose-rate gamma rays can attenuate the response to acute exposure to proton radiation with respect to at least some cell populations. (C) 2012 by Radiation Research Society (03/2012) (link)
  • Rizvi A, Pecaut M J, & Gridley D S. (2011). Low-dose Gamma-rays and Simulated Solar Particle Event Protons Modify Splenocyte Gene and Cytokine Expression Patterns. Journal of Radiation Research, 52(6), 701-711. The goal was to investigate the T helper (Th) response in splenocytes of mice exposed to low-dose/low-dose-rate (LDR) gamma-rays, simulated solar particle event protons (sSPE), or combination of both. C57BL/6 mice were exposed to LDR gamma-radiation (Co-57) to a total dose of 0.05 Gray (Gy) at 0.024 cGy/h, either with or without subsequent exposure to 2 Gy sSPE protons. Expression of genes related to Th cells was evaluated immediately after exposure (day 0). On day 21, intra- and extracellular cytokine production was assessed after activation with anti-CD3 monoclonal antibodies (mAb) or phorbol 12-myristate 13-acetate/ionophore (PMA/I). Five genes were significantly modulated on day 0 in one or more of the irradiated groups compared to controls (p < 0.05): Ccl11, Ccr5, Cd80, Inha, and Il9. On day 21, numbers of cells positive for interferon-gamma were high in the LDR + sSPE group versus 0 Gy and LDR gamma-rays (p < 0.05), but there was no difference in IL-2 and TNF-alpha. Levels of secreted cytokines after anti-CD3 mAb activation were high for 5 (MIP-1 alpha, GM-CSF, IFN-gamma, TNF-alpha, IL-13) and low for 2 (IL-7, IL-9) in all irradiated groups. Priming with LDR photons had a significant effect on IFN-gamma and IL-17 compared to sSPE protons alone; IL-2 was low only in the LDR + sSPE group. The cytokine patterns after anti-PMA/I activation were different compared to anti-CD3 mAb and with fewer differences among groups. The data show that total-body exposure to space-relevant radiation has profound effects on Th cell status and that priming with LDR gamma-rays can in some cases modulate the response to sSPE. (11/2011) (link)
  • Pecaut M J, & Gridley D S. (2011). Impact of Head-Only Iron Ion Radiation on the Peripheral LPS Response. In Vivo, 25(6), 903-916. Although there is a large body of evidence indicating that radiation can have a dramatic impact on both immune and brain function, there is very little known about its effect on communication between these critical two systems. In this study, mice were exposed to head-localized irradiation with 5 Gy (56)Fe(26+) ions and assessed for immune function. Mice were inoculated with lipopolysaccharide 37-38 days post-irradiation. Subsets of mice were euthanized 1, 7, or 14 days later. Radiation significantly impacted the response to an immune challenge in terms of splenic and circulating leukocyte counts and lymphocyte distributions; the effect was especially pronounced on granulocytes and B-cells (p<0.05). However, there were no interactions in spontaneous or mitogen-induced blastogenesis of activated T-cell proportions, brain interleukin-1 beta, or circulating corticosterone levels. These data demonstrate that head-localized iron ion radiation modified the peripheral response to a potent bacterial component associated with septic shock. (11/2011)
  • Gridley D S, Pecaut M J, Green L M, Sanchez M C, & Kadhim M A. (2011). Strain-related Differences and Radiation Quality Effects on Mouse Leukocytes: Gamma-rays and Protons (with and without Aluminum Shielding). In Vivo, 25(6), 871-880. Increasing evidence indicates that radiation-induced genomic instability plays an important role in the development of cancer. However, radiation quality and genetic background can influence the outcome. The goal of this study was to quantify radiation-induced changes in lymphocyte populations in mouse strains known to differ in susceptibility to genomic instability (C57BL/6, resistant; CBA/Ca, susceptible). The effects of whole-body exposure to gamma-rays and protons, with and without aluminum shielding, were compared. Total radiation doses of 0, 0.1, 0.5, and 2.0 Gy were delivered and subsets of mice from each group were euthanized on days I and 30 after exposure for spleen and bone marrow analyses. In the spleen on day 1, lymphocyte counts were decreased (p<0.05) in C57, but not CBA, mice irradiated with 2 Gy. By day 30 in the C57 strain, counts were still low in the group exposed to 2 Gy shielded protons. Some strain- and radiation-dependent differences were also noted in percentages of specific lymphocyte populations (T, B, NK) and the CD4:CD8 ratio. In bone marrow, percentages of stem/progenitor cells (CD34(+), Ly-6A/E(+), CD34(+)Ly-6A/E(+)) were generally highest I day after 2 Gy irradiation, regardless of strain and radiation type. Based on dUTP incorporation, bone marrow cells from C57 mice had consistently higher levels of DNA damage on day 30 after irradiation with doses less than 2 Gy, regardless of quality. Annexin V binding supported the conclusion that C57 bone marrow cells were more susceptible to radiation-induced apoptosis. Overall, the data indicate that leukocytes of CBA mice are less sensitive to the effects of high-linear energy transfer radiation (shielded protons) than C57 mice, a phenomenon consistent with increased possibility for genomic instability and progression to a malignant cell phenotype after sublethal damage. (11/2011)
  • Gridley D S, & Pecaut M J. (2011). Genetic background and lymphocyte populations after total-body exposure to iron ion radiation. International Journal of Radiation Biology, 87(1), 8-23. Purpose: Particle radiations could significantly impact astronaut health during space missions. This study quantified the effects of iron ion radiation on lymphocytes in two strains of mice differing in susceptibility to radiation-induced acute myeloid leukemia (AML) and thymic lymphoma (TL): C57BL/6 (AML resistant, TL sensitive) and CBA/Ca (AML sensitive, TL resistant). Materials and methods: The animals (n = 60/strain) were irradiated with (56)Fe(26+) (1 GeV) to total doses of 0, 0.5, 2 and 3 Gray (Gy) at an average dose rate of 1 Gy/min and euthanised on days 4 and 30 thereafter; blood, spleen, and bone marrow were collected for flow cytometry analyses. Cells expressing the following molecules were quantified: Cluster of differentiation (CD) 4, CD8, CD25, CD34, CD71, B220 (isoform of CD45 on B cells), NK1.1 (marker on natural killer or NK cells, C57B mice), panNK (marker on NK cells, CBA mice), and Sca1 (stem cell antigen 1). Results: Exposure to radiation resulted in different distribution patterns in lymphocyte populations and leukocytes expressing activation and progenitor markers in the two mouse strains. Significant main effects were dependent upon strain, as well as radiation dose, body compartment, and time of assessment. Especially striking differences were noted on day 4 after 3 Gy irradiation, including in the CD4:CD8 ratio [blood, C57 (2.83 +/- 0.25) vs. CBA (6.19 +/- 0.24); spleen, C57 (2.29 +/- 0.12) vs. CBA (4.98 +/- 0.22)], %CD25(+) mononuclear cells in bone marrow [C57 (5.62 +/- 1.19) vs. CBA (12.45 +/- 0.93)] and % CD34(+)Sca1(+) cells in bone marrow [CD45(lo) gate, C57 (2.72 +/- 0.74) vs. CBA (21.44 +/- 0.73)]. Conclusion: The results show that genetic background, as well as radiation dose and time post-exposure, had a profound impact on lymphocyte populations, as well as other leukocytes, after exposure to iron ion radiation. (01/2011) (link)
  • Rizvi A, Pecaut M J, Slater J M, Subramaniam S, & Gridley D S. (2011). Low-dose gamma-rays modify CD4(+) T cell signalling response to simulated solar particle event protons in a mouse model. International Journal of Radiation Biology, 87(1), 24-35. Purpose: Astronauts on missions are exposed to low-dose/low-dose-rate (LDR) radiation and could receive high doses during solar particle events (SPE). This study investigated T cell function in response to LDR radiation and simulated SPE (sSPE) protons, alone and in combination. Materials and methods: C57BL/6 mice received LDR gamma-radiation (Co-57) to a total dose of 0.01 Gray (Gy) at 0.179 mGy/h, either with or without subsequent exposure to 1.7 Gy sSPE protons delivered over 36 h. Mice were euthanised on days 4 and 21 post-exposure. T cells with cluster of differentiation 4 (CD4(+)) were negatively isolated from spleens and activated with anti-CD3 antibody. Cells and supernatants were evaluated for survival/signalling proteins and cytokines. Results: The most striking effects were noted on day 21. In the survival pathway, nuclear factor-kappaB (NF-kappa B; total and active forms) and p38 mitogen activated protein kinase (p38MAPK; total) were significantly increased and cJun N-terminal kinase (JNK; total and active) was decreased when mice were primed with LDR gamma-rays prior to sSPE exposure (P < 0.001). Evaluation of the T cell antigen receptor (TCR) signalling pathway revealed that LDR gamma-ray exposure normalised the high sSPE proton-induced level of lymphocyte specific protein tyrosine kinase (Lck; total and active) on day 21 ( < 0.001 for sSPE vs. LDR + sSPE), while radiation had no effect on active zeta-chain-associated protein kinase 70 (Zap-70). There was increased production of interleukin-2 (IL-2) and IL-4 and decreased transforming growth factor-beta 1 in the LDR + sSPE group compared to the sSPE group. Conclusion: The data demonstrate, for the first time, that protracted exposure to LDR gamma-rays can significantly modify the effects of sSPE protons on critical survival/signalling proteins and immunomodulatory cytokines produced by CD4(+) T cells. (01/2011) (link)
  • Gridley D S, Xian L O, Rizvi A, Makinde A Y, Pecaut M J, Mao X W, & Slater J M. (2010). Low-dose Photon and Simulated Solar Particle Event Proton Effects on Foxp3(+) T Regulatory Cells and Other Leukocytes. Technology in Cancer Research & Treatment, 9(6), 637-649. Radiation is a major factor in the spaceflight environment that has carcinogenic potential. Astronauts on missions are continuously exposed to low-dose/low-dose-rate (LDR) radiation and may receive relatively high doses during a solar particle event (SPE) that consists primarily of protons. However, there are very few reports in which LDR photons were combined with protons. In this study, C57BL/6 mice were exposed to 1.7 Gy simulated SPE (sSPE) protons over 36 h, both with and without pre-exposure to 0.01 Gray (Gy) LDR gamma-rays at 0.018 cGy/h. Apoptosis in skin samples was determined by immunohistochemistry immediately post-irradiation (day 0). Spleen mass relative to body mass, white blood cells (WBC), major leukocyte populations, lymphocyte subsets (T, Th, Tc, B, NK), and CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells were analyzed on days 4 and 21. Apoptosis in skin samples was evident in all irradiated groups; the LDR+sSPE mice had the greatest expression of activated caspase-3. On day 4 post-irradiation, the sSPE and LDR+sSPE groups had significantly lower WBC counts in blood and spleen compared to non-irradiated controls (p < 0.05 vs. 0 Gy). CD4(+)CD25(+)Foxp3(+) Treg cell numbers in spleen were decreased at day 4, but proportions were increased in the sSPE and LDR+sSPE groups (p < 0.05 vs. 0 Gy). By day 21, lymphocyte counts were still low in blood from the LDR+sSPE mice, especially due to reductions in B, NK, and CD8(+) T cytotoxic cells. The data demonstrate, for the first time, that pre-exposure to LDR photons did not protect against the adverse effects of radiation mimicking a large solar storm. The increased proportion of immunosuppressive CD4(+)CD25(+)Foxp3(+) Treg and persistent reduction in circulating lymphocytes may adversely impact immune defenses that include removal of sub-lethally damaged cells with carcinogenic potential, at least for a period of time post-irradiation. (12/2010)
  • Pecaut M J, & Gridley D S. (2010). The impact of mouse strain on iron ion radio-immune response of leukocyte populations. International Journal of Radiation Biology, 86(5), 409-419. Materials and methods: The mice were whole-body irradiated with 0, 50, 200, or 300 cGy (05/2010) (link)
  • Lebsack T W, Fa V, Woods C C, Gruener R, Manziello A M, . . . DeLuca D. (2010). Microarray Analysis of Spaceflown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors. Journal of Cellular Biochemistry, 110(2), 372-381. The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P <= 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha 2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space. J. Cell. Biochem. 110: 372-381, 2010. (C) 2010 Wiley-Liss, Inc. (05/2010) (link)
  • Tian J, Pecaut M J, Slater J M, & Gridley D S. (2010). Spaceflight modulates expression of extracellular matrix, adhesion, and profibrotic molecules in mouse lung. Journal of Applied Physiology, 108(1), 162-171. Tian J, Pecaut MJ, Slater JM, Gridley DS. Spaceflight modulates expression of extracellular matrix, adhesion, and profibrotic molecules in mouse lung. J Appl Physiol 108: 162-171, 2010. First published October 22, 2009; doi:10.1152/japplphysiol.00730.2009.NASA has reported pulmonary abnormalities in astronauts on space missions, but the molecular changes in lung tissue remain unknown. The goal of the present study was to explore the effects of spaceflight on expression of extracellular matrix (ECM), cell adhesion, and pro-fibrotic molecules in lungs of mice flown on Space Shuttle Endeavour (STS-118). C57BL/6Ntac mice housed in animal enclosure modules during a 13-day mission in space (FLT) were killed within hours after return; ground controls were treated similarly for comparison (GRD). Analysis of genes associated with ECM and adhesion molecules was performed according to quantitative RTPCR. The data revealed that FLT lung samples had statistically significant transcriptional changes, i.e., at least 1.5-fold, in 25 out of 84 examined genes (P < 0.05); 15 genes were upregulated and 10 were downregulated. The genes that were upregulated by more than twofold were Ctgf, Mmp2, Ncam1, Sparc, Spock1, and Timp3, whereas the most downregulated genes were Lama1, Mmp3, Mmp7, vcam-1, and Sele. Histology showed profibrosis-like changes occurred in FLT mice, more abundant collagen accumulation around blood vessels, and thicker walls compared with lung samples from GRD mice. Immunohistochemistry was used to compare expression of six selected proteins associated with fibrosis. Immunoreactivity of four proteins (MMP-2, CTGF, TGF-beta 1, and NCAM) was enhanced by spaceflight, whereas, no difference was detected in expression of MMP-7 and MMP-9 proteins between the FLT and GRD groups. Taken together, the data demonstrate that significant changes can be readily detected shortly after return from spaceflight in the expression of factors that can adversely influence lung function. (01/2010) (link)
  • Tian J, Pecaut M J, Coutrakon G B, Slater J M, & Gridley D S. (2009). Response of Extracellular Matrix Regulators in Mouse Lung after Exposure to Photons, Protons and Simulated Solar Particle Event Protons. Radiation Research, 172(1), 30-41. This study compared the effects of photons (gamma rays), protons and simulated solar particle event protons (sSPE) on the expression of profibrotic factors/extracellular matrix (ECM) regulators in lung tissue after whole-body irradiation. TGF-beta 1, matrix metalloproteinase 2 and 9 (MMP-2, -9), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1, -2) were assessed on days 4 and 21 in lungs from C57BL/6 mice exposed to 0 Gy or 2 Gy photons (0.7 Gy/min), protons (0.9 Gy/min) and sSPE (0.056 Gy/h). RT-PCR, histological and immunohistochemical techniques were used. The most striking changes included (1) up-regulation of TGF-beta 1 by photons and sSPE, but not protons, at both times, (2) MMP-2 enhancement by photons and sSPEs, (3) TIMP-1 up-regulation by photons at both times, and (4) more collagen accumulation after exposure to either photons or sSPE than after exposure to protons. The findings demonstrate that expression of important ECM regulators was highly dependent upon the radiation regimen as well as the time after exposure. The data further suggest that irradiation during an SPE may increase an astronaut's risk for pulmonary complications. The greater perturbations after photon exposure compared to proton exposure have clinical implications and warrant further investigation. (C) 2009 by Radiation Research Society (07/2009) (link)
  • Baqai F P, Gridley D S, Slater J M, Luo-Owen X, Stodieck L S, . . . Pecaut M J. (2009). Effects of spaceflight on innate immune function and antioxidant gene expression. Journal of Applied Physiology, 106(6), 1935-1942. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, Chapes SK, Pecaut MJ. Effects of spaceflight on innate immune function and antioxidant gene expression. J Appl Physiol 106: 1935-1942, 2009. First published April 2, 2009; doi:10.1152/japplphysiol.91361.2008.-Spaceflight conditions have a significant impact on a number of physiological functions due to psychological stress, radiation, and reduced gravity. To explore the effect of the flight environment on immunity, C57BL/6NTac mice were flown on a 13-day space shuttle mission (STS-118). In response to flight, animals had a reduction in liver, spleen, and thymus masses compared with ground (GRD) controls (P < 0.005). Splenic lymphocyte, monocyte/macrophage, and granulocyte counts were significantly reduced in the flight (FLT) mice (P < 0.05). Although spontaneous blastogenesis of splenocytes in FLT mice was increased, response to lipopolysaccharide (LPS), a B-cell mitogen derived from Escherichia coli, was decreased compared with GRD mice (P < 0.05). Secretion of IL-6 and IL-10, but not TNF-alpha, by LPS-stimulated splenocytes was increased in FLT mice (P < 0.05). Finally, many of the genes responsible for scavenging reactive oxygen species were upregulated after flight. These data indicate that exposure to the spaceflight environment can increase anti-inflammatory mechanisms and change the ex vivo response to LPS, a bacterial product associated with septic shock and a prominent Th1 response. (06/2009) (link)
  • Gridley D S, Rizvi A, Luo-Owen X, Makinde A Y, & Pecaut M J. (2009). Low dose, low dose rate photon radiation modifies leukocyte distribution and gene expression in CD4(+) T cells. J Radiat Res (Tokyo), 50(2), 139-50. A better understanding of low dose radiation effects is needed to accurately estimate health risks. In this study, C57BL/6 mice were gamma-irradiated to total doses of 0, 0.01, 0.05, and 0.1 Gy ((57)Co; ~0.02 cGy/h). Subsets per group were euthanized at the end of irradiation (day 0) and on days 4 and 21 thereafter. Relative spleen mass and splenic white blood cell (WBC) counts, major leukocyte populations, and spontaneous DNA synthesis were consistently higher in the irradiated groups on day 0 compared to 0 Gy controls, although significance was not always obtained. In the spleen, all three major leukocyte types were significantly elevated on day 0 (P < 0.05). By day 21 post-irradiation the T, B, and natural killer (NK) cell counts, as well as CD4(+) T cells and CD4:CD8 T cell ratio, were low especially in the 0.01 Gy group. Although blood analyses showed no significant differences in leukocyte counts or red blood cell and platelet characteristics, the total T cells, CD4(+) T cells, and NK cells were increased by day 21 after 0.01 Gy (P < 0.05). Gene analysis of CD4(+) T cells negatively isolated from spleens on day 0 after 0.1 Gy showed significantly enhanced expression of Il27 and Tcfcp2, whereas Inha and Socs5 were down-regulated by 0.01 Gy and 0.1 Gy, respectively (P < 0.05). A trend for enhancement was noted in two additional genes (Il1r1 and Tbx21) in the 0.1 Gy group (P < 0.1). The data show that protracted low dose photons had dose- and time-dependent effects on CD4(+) T cells after whole-body exposure. (03/2009) (link)
  • Gridley D S, Rizvi A, Xian L O, Makinde A Y, & Pecaut M J. (2009). Low Dose, Low Dose Rate Photon Radiation Modifies Leukocyte Distribution and Gene Expression in CD4(+) T Cells. Journal of Radiation Research, 50(2), 139-150. A better understanding of low dose radiation effects is needed to accurately estimate health risks. In this study, C57BL/6 mice were gamma-irradiated to total doses of 0, 0.01, 0.05, and 0.1 Gy (Co-57; similar to 0.02 cGy/h). Subsets per group were euthanized at the end of irradiation (day 0) and on days 4 and 21 thereafter. Relative spleen mass and splenic white blood cell (WBC) counts, major leukocyte populations, and spontaneous DNA synthesis were consistently higher in the irradiated groups on day 0 compared to 0 Gy controls, although significance was not always obtained. In the spleen, all three major leukocyte types were significantly elevated on day 0 (P < 0.05). By day 21 post-irradiation the T, B, and natural killer (NK) cell counts, as well as CD4(+) T cells and CD4:CD8 T cell ratio, were low especially in the 0.01 Gy group. Although blood analyses showed no significant differences in leukocyte counts or red blood cell and platelet characteristics, the total T cells, CD4(+) T cells, and NK cells were increased by day 21 after 0.01 Gy (P < 0.05). Gene analysis of CD4(+) T cells negatively isolated from spleens on day 0 after 0.1 Gy showed significantly enhanced expression of Il27 and Tcfcp2, whereas Inha and Socs5 were down-regulated regulated by 0.01 Gy and 0.1 Gy, respectively (P < 0.05). A trend for enhancement was noted in two additional genes (Illr1 and Tbx2l) in the 0.1 Gy group (P < 0.1). The data show that protracted low dose photons had dose- and time-dependent effects on CD4(+) T cells after whole-body exposure. (03/2009) (link)
  • Ortega M T, Pecaut M J, Gridley D S, Stodieck L S, Ferguson V, & Chapes S K. (2009). Shifts in bone marrow cell phenotypes caused by spaceflight. Journal of Applied Physiology, 106(2), 548-555. Ortega MT, Pecaut MJ, Gridley DS, Stodieck LS, Ferguson V, Chapes SK. Shifts in bone marrow cell phenotypes caused by spaceflight. J Appl Physiol 106: 548-555, 2009. First published December 4, 2008; doi: 10.1152/japplphysiol.91138.2008.-Bone marrow cells were isolated from the humeri of C57BL/6 mice after a 13-day flight on the space shuttle Space Transportation System (STS)-118 to determine how spaceflight affects differentiation of cells in the granulocytic lineage. We used flow cytometry to assess the expression of molecules that define the maturation/activation state of cells in the granulocytic lineage on three bone marrow cell subpopulations. These molecules included Ly6C, CD11b, CD31 (platelet endothelial cell adhesion molecule-1), Ly6G (Gr-1), F4/80, CD44, and c-Fos. The three subpopulations were small agranular cells [region (R) 1], larger granular cells (R2), which were mostly neutrophils, and very large, very granular cells (R3), which had properties of macrophages. Although there were no composite phenotypic differences between total bone marrow cells isolated from spaceflight and ground-control mice, there were subpopulation differences in Ly6C (R1 and R3), CD11b (R2), CD31 (R1, R2, and R3), Ly6G (R3), F4/80 (R3), CD44(high) (R3), and c-Fos (R1, R2, and R3). In particular, the elevation of CD11b in the R2 subpopulation suggests neutrophil activation in response to landing. In addition, decreases in Ly6C, c-Fos, CD44(high), and Ly6G and an increase in F4/80 suggest that the cells in the bone marrow R3 subpopulation of spaceflight mice were more differentiated compared with ground- control mice. The presence of more differentiated cells may not pose an immediate risk to immune resistance. However, the reduction in less differentiated cells may forebode future consequences for macrophage production and host defenses. This is of particular importance to considerations of future long-term spaceflights. (02/2009) (link)
  • Gridley D S, Slater J M, Luo-Owen X, Rizvi A, Chapes S K, . . . Pecaut M J. (2009). Spaceflight effects on T lymphocyte distribution, function and gene expression. Journal of Applied Physiology, 106(1), 194-202. Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK, Stodieck LS, Ferguson VL, Pecaut MJ. Spaceflight effects on T lymphocyte distribution, function and gene expression. J Appl Physiol 106: 194-202, 2009. First published November 6, 2008; doi: 10.1152/japplphysiol.91126.2008.-The immune system is highly sensitive to stressors present during spaceflight. The major emphasis of this study was on the T lymphocytes in C57BL/6NTac mice after return from a 13-day space shuttle mission (STS-118). Spleens and thymuses from flight animals (FLT) and ground controls similarly housed in animal enclosure modules (AEM) were evaluated within 3-6 h after landing. Phytohemagglutinin-induced splenocyte DNA synthesis was significantly reduced in FLT mice when based on both counts per minute and stimulation indexes ( P < 0.05). Flow cytometry showed that CD3(+) T and CD19(+) B cell counts were low in spleens from the FLT group, whereas the number of NK1.1(+) natural killer (NK) cells was increased ( P < 0.01 for all three populations vs. AEM). The numerical changes resulted in a low percentage of T cells and high percentage of NK cells in FLT animals ( P < 0.05). After activation of spleen cells with anti-CD3 monoclonal antibody, interleukin-2 (IL-2) was decreased, but IL-10, interferon-gamma, and macrophage inflammatory protein-1 alpha were increased in FLT mice ( P < 0.05). Analysis of cancer-related genes in the thymus showed that the expression of 30 of 84 genes was significantly affected by flight ( P < 0.05). Genes that differed from AEM controls by at least 1.5-fold were Birc5, Figf, Grb2, and Tert (upregulated) and Fos, Ifnb1, Itgb3, Mmp9, Myc, Pdgfb, S100a4, Thbs, and Tnf (downregulated). Collectively, the data show that T cell distribution, function, and gene expression are significantly modified shortly after return from the spaceflight environment. (01/2009) (link)
  • Gridley D S, Pecaut M J, Rizvi A, Coutrakon G B, Luo-Owen X, Makinde A Y, & Slater J M. (2009). Low-dose, low-dose-rate proton radiation modulates CD4+T cell gene expression. International Journal of Radiation Biology, 85(3), 250-261. Purpose: To evaluate cluster of differentiation 4+ (CD4+) T cell gene expression and related parameters after whole-body exposure to proton radiation as it occurs in the spaceflight environment. Materials and methods: C57BL/6 mice were irradiated to total doses of 0, 0.01, 0.05, and 0.1 gray (Gy) at 0.1cGy/h. On day 0 spleens were harvested from a subset in the 0, 0.01 and 0.1Gy groups; (CD4+) T cells were isolated; and expression of 84 genes relevant to T helper (Th) cell function was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). Remaining mice were euthanized on days 0, 4, and 21 for additional analyses. Results: Genes with 2-fold difference and p0.05 compared to 0Gy were noted. After 0.01Gy, five genes were up-regulated (Ccr5, Cd40, Cebpb, Igsf6, Tnfsf4) and three were down-regulated (Il4ra, Mapk8, Nfkb1). After 0.1Gy there were nine up-regulated genes (Ccr4, Cd40, Cebpb, Cxcr3, Socs5, Stat4, Tbx21, Tnfrsf4, Tnfsf4); none were down-regulated. On day 0 after 0.01Gy, CD4+ T cell counts and CD4:CD8 ratio were low in the spleen (p0.05). Spontaneous DNA synthesis in both spleen and blood was lowest in the 0.01Gy group on day 0; on days 4 and 21 all p values were 0.1. Conclusion: The data show that the pattern of gene expression in CD4+ T cells after protracted low-dose proton irradiation was significantly modified and highly dependent upon total dose. The findings also suggest that low-dose radiation, especially 0.01Gy, may enhance CD4+ T cell responsiveness. (2009) (link)