Apoptosis and expression of apoptosis-related genes in mouse intestinal tissue after whole-body proton exposure.

Energetic protons are the most abundant particle type in space and can pose serious health risks to astronauts during long-duration missions. The health effects of proton exposure are also a concern for cancer patients undergoing radiation treatment with accelerated protons. To investigate the damage induced by energetic protons in vivo to radiosensitive organs, 6-week-old BALB/c male mice were subjected to 250 MeV proton radiation at whole-body doses of 0.1, 1, and 2 Gy. The gastrointestinal (GI) tract of each exposed animal was dissected 4 h post-irradiation, and the isolated small intestinal tissue was analyzed for histopathological and gene expression changes. Histopathologic observation of the tissue using standard hematoxylin and eosin (H&E) staining methods to screen for morphologic changes showed a marked increase in apoptotic lesions for even the lowest dose of 0.1 Gy, similar to X- or γ rays. The percentage of apoptotic cells increased dose-dependently, but the dose response appeared supralinear, indicating hypersensitivity at low doses. A significant decrease in surviving crypts and mucosal surface area, as well as in cell proliferation, was also observed in irradiated mice. Gene expression analysis of 84 genes involved in the apoptotic process showed that most of the genes affected by protons were common between the low (0.1 Gy) and high (1 and 2 Gy) doses. However, the genes that were distinctively responsive to the low or high doses suggest that high doses of protons may cause apoptosis in the small intestine by direct damage to the DNA, whereas low doses of protons may trigger apoptosis through a different stress response mechanism.

Proteomic Analysis of Mouse Brain Subjected to Spaceflight.

There is evidence that spaceflight poses acute and late risks to the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched from the Kennedy Space Center (KSC) on a 13-day mission. Within 3⁻5 h after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the gray and/or white matter. While there was no overlap between the white and gray matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

Prevention of Mammary Tumor Development through Neuroimmunomodulation in the Spleen and Lymph Nodes of Old Female Sprague-Dawley Rats by L-Deprenyl.

BACKGROUND: Development of mammary tumors is an age-associated phenomenon that is likely due to deficits in the neuroendocrine-immune interactions. Previously, we demonstrated that L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, can enhance immune responses and restore noradrenergic (NA) innervation in the spleens of rats with carcinogen-induced and spontaneously developing mammary tumors. OBJECTIVES: To investigate whether (1) treatment of early middle-aged female rats would prevent the spontaneous development of mammary tumors accompanied by restoration of immunity in the spleen and draining lymph nodes (DLN) and sympathetic NA innervation in the spleen and (2) deprenyl can influence the proliferation of estrogen receptor (ER)-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and Hs 578T) human breast cancer cells. METHODS: Early middle-aged (8- to 9-month-old) female Sprague-Dawley rats were treated with 0, 1.0 or 2.5 mg of deprenyl/kg body weight (BW) daily i.p. for 12 months. Cells of ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines were incubated with media or 10(-3) to 10(-8) M deprenyl for 1, 2, 4 or 6 days to examine the proliferation of cells. RESULTS: Tumor incidence increased in saline-treated old female rats, while deprenyl treatment significantly reduced the incidence of mammary tumors in these rats. Saline-treated tumor-bearing rats exhibited reduced splenic NA innervation and norepinephrine (NE) content, splenic interleukin (IL)-2 and interferon (IFN)-γ levels and NK cell activity as well as DLN IL-2 and IFN-γ levels compared to young female rats without tumors. In contrast, treatment with 2.5 mg/kg of deprenyl enhanced IL-2 and IFN-γ production in both the spleen and DLN as well as splenic natural killer (NK) cell activity. Deprenyl treatment also increased concanavalin A (Con A)-induced proliferation of T lymphocytes in the DLN. Deprenyl-induced changes in immune responses were accompanied by enhanced NA innervation and NE content in the spleen. In vitro incubation of various concentrations of deprenyl with ER+ human breast cancer cell lines partly inhibited the proliferation of cells, while it had no effect on the ER- breast cancer cells. CONCLUSIONS: These results suggest that (1) development of mammary tumors is mediated through the loss of immunity and sympathetic NA nerve fibers accompanied by reduced NE levels in the spleen, (2) the prevention of mammary tumor development by deprenyl may involve the reversal of the tumor-associated decline in sympathetic NA activity and cell-mediated immune responses in the spleen and DLN and (3) the antitumor effects of deprenyl may be partially mediated through ER-dependent intracellular signaling pathways.

Efficient reprogramming of human cord blood CD34+ cells into induced pluripotent stem cells with OCT4 and SOX2 alone.

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

The biobehavioral and neuroimmune impact of low-dose ionizing radiation.

In the clinical setting, repeated exposures (10-30) to low-doses of ionizing radiation (≤200 cGy), as seen in radiotherapy for cancer, causes fatigue. Almost nothing is known, however, about the fatigue inducing effects of a single exposure to environmental low-dose ionizing radiation that might occur during high-altitude commercial air flight, a nuclear reactor accident or a solar particle event (SPE). To investigate the short-term impact of low-dose ionizing radiation on mouse biobehaviors and neuroimmunity, male CD-1 mice were whole body irradiated with 50 cGy or 200 cGy of gamma or proton radiation. Gamma radiation was found to reduce spontaneous locomotor activity by 35% and 36%, respectively, 6 h post irradiation. In contrast, the motivated behavior of social exploration was un-impacted by gamma radiation. Examination of pro-inflammatory cytokine gene transcripts in the brain demonstrated that gamma radiation increased hippocampal TNF-α expression as early as 4 h post-irradiation. This was coupled to subsequent increases in IL-1RA (8 and 12 h post irradiation) in the cortex and hippocampus and reductions in activity-regulated cytoskeleton-associated protein (Arc) (24 h post irradiation) in the cortex. Finally, restraint stress was a significant modulator of the neuroimmune response to radiation blocking the ability of 200 cGy gamma radiation from impairing locomotor activity and altering the brain-based inflammatory response to irradiation. Taken together, these findings indicate that low-dose ionizing radiation rapidly activates the neuroimmune system potentially causing early onset fatigue-like symptoms in mice.

Reactive oxygen species mediated tissue damage in high energy proton irradiated mouse brain.

Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. We recently reported that the high energy protons induce cell death through activation of apoptotic signaling genes; caspase 3 and 8 (Baluchamy et al. J Biol Chem 285:24769-24774, 2010). In this study, we investigated the effect of different doses of protons in in vivo mouse system, particularly, brain tissues. A significant dose-dependent induction of reactive oxygen species and lipid peroxidation and reduction of antioxidants; glutathione and superoxide dismutase were observed in proton irradiated mouse brain as compared to control brain. Furthermore, histopathology studies on proton irradiated mouse brain showed significant tissue damage as compared to control brain. Together, our in vitro and in vivo results suggest that proton irradiation alters oxidant and antioxidant levels in the cells to cause proton mediated DNA/tissue damage followed by apoptotic cell death.

Induction of cell death through alteration of oxidants and antioxidants in lung epithelial cells exposed to high energy protons.

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.

Stem cell antigen-1 positive cell-based systemic human growth hormone gene transfer strategy increases endosteal bone resorption and bone loss in mice.

BACKGROUND: The present study assesses the effect of the stem cell antigen-1 positive (Sca-1(+) ) cell-based human growth hormone (hGH) ex vivo gene transfer strategy on endosteal bone mass in the mouse. METHODS: Sublethally irradiated recipient mice were transplanted with Sca-1(+) cells transduced with lentiviral vectors expressing hGH or ß-galactosidase control genes. Bone parameters were assessed by micro-computed tomography and histomorphometry. RESULTS: This hGH strategy drastically increased hGH mRNA levels in bone marrow cells and serum insulin-like growth factor-I (IGF-I) (by nearly 50%, p < 0.002) in hGH recipient mice. Femoral trabecular bone volume of the hGH mice was significantly reduced by 35% (p < 0.002). The hGH mice also had decreased trabecular number (by 26%; p < 0.0001), increased trabecular separation (by 38%; p < 0.0002) and reduced trabecular connectivity density (by 64%; p < 0.001), as well as significantly more osteoclasts (2.5-fold; p < 0.05) and greater osteoclastic surface per bone surface (2.6-fold; p < 0.01). CONCLUSIONS: Targeted expression of hGH in cells of marrow cavity through the Sca-1(+) cell-based gene transfer strategy increased circulating IGF-I and decreased endosteal bone mass through an increase in resorption in recipient mice. These results indicate that high local levels of hGH or IGF-I in the bone marrow microenvironment enhanced resorption, which is consistent with previous findings in transgenic mice with targeted bone IGF-I expression showing that high local IGF-I expression increased bone remodeling, favoring a net bone loss. Thus, GH and/or IGF-I would not be an appropriate transgene for use in this Sca-1(+) cell-based gene transfer strategy to promote endosteal bone formation. Published 2011 John Wiley & Sons, Ltd.

The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the remaining four genes (XPC, ERCC2, XRCC1, and MLH1) in either the experimental or control animals.

Psychosocial Stress and Age Influence Depression and Anxiety-Related Behavior, Drive Tumor Inflammatory Cytokines and Accelerate Prostate Cancer Growth in Mice.

Prostate cancer (PCa) prevalence is higher in older men and poorer coping with psychosocial stressors effect prognosis. Yet, interactions between age, stress and PCa progression are underexplored. Therefore, we characterized the effects of age and isolation combined with restraint (2 h/day) for 14 days post-tumor inoculation on behavior, tumor growth and host defense in the immunocompetent, orthotopic RM-9 murine PCa model. All mice were tumor inoculated. Isolation/restraint increased sympathetic and hypothalamic-pituitary-adrenal cortical activation, based on elevated serum 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratios and corticosterone levels, respectively. Elevated zero maze testing revealed age-related differences in naïve C57Bl/6 mice, and increased anxiety-like behavior in tumor-bearing mice. In open field testing, old stressed mice were less active throughout the 30-min test than young non-stressed and stressed, and old non-stressed mice, suggesting greater anxiety in old stressed mice. Old (18 month) mice demonstrated more depression-like behavior than young mice with tail suspension testing, without effects of isolation/restraint stress. Old mice developed larger tumors, despite similar tumor expression of tumor vascular endothelial growth factor or transforming growth factor-beta1 across age. Tumor chemokine/cytokine expression, commonly prognostic for poorer outcomes, were uniquely age- and stress-dependent, underscoring the need for PCa research in old animals. Macrophages predominated in RM-9 tumors. Macrophages, and CD4+ and CD4+FoxP3+ T-cell tumor infiltration were greater in young mice than in old mice. Stress increased macrophage infiltration in old mice. Conversely, stress reduced intratumoral CD4+ and CD4+FoxP3+ T-cell numbers in young mice. CD8+ T-cell infiltration was similar across treatment groups. Our findings support that age- and psychological stress interacts to affect PCa outcomes by interfering with neural-immune mechanisms and affecting behavioral responses.

Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.

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 < or = 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-alpha2a (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.

Expression profile of DNA damage signaling genes in 2 Gy proton exposed mouse brain.

Exposure of living systems to radiation results in a wide assortment of lesions, the most significant of is damage to genomic DNA which alter specific cell functions including cell proliferation. The radiation induced DNA damage investigation is one of the important area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes such as damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2 Gy proton exposed mouse brain tissues as compared to control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed cells undergo severe DNA damage which in turn destabilize the chromatin stability.

Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina.

UNLABELLED: The aim of this study was to examine the induction of oxidative stress and apoptosis-associated gene expression profiles in retina after proton irradiation exposure at 0.5 to 4 Gy. MATERIALS AND METHODS: One eye of each Sprague-Dawley rat (6 per group) was irradiated with a conformal proton beam to total doses of 0, 0.5, 1 and 4 Gy. Retinal tissues were isolated for characterization of gene expression profiles 6 hours after proton radiation. RESULTS: For oxidative stress, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly up-regulated (Fmo2, Gpx2, Noxa1 and Sod3) compared to controls. Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated following irradiation (Fas, Faslg, Trp63 and Trp73). TUNEL assay and caspase-3 immunocytochemical analysis revealed increased apoptotic immunoreactivity following irradiation. CONCLUSION: The data revealed that exposure to proton radiation induced oxidative stress-associated apoptosis. In response to ionizing radiation, the expression of genes involved in pathways mediating apoptosis may be differentially regulated in different dose regimens.

Stem cell antigen-1+ cell-based bone morphogenetic protein-4 gene transfer strategy in mice failed to promote endosteal bone formation.

BACKGROUND: This study assessed whether a Sca-1+ cell-based ex vivo gene transfer strategy, which has been shown to promote robust endosteal bone formation with a modified fibroblast growth factor-2 (FGF2) gene, can be extended to use with bone morphogenetic protein (BMP)2/4 hybrid gene. METHODS: Sublethally irradiated recipient mice were transplanted with lentiviral (LV)-BMP2/4-transduced Sca-1+ cells. Bone parameters were monitored by pQCT and microCT. Gene expression was assessed by the real-time reverse transcriptase-polymerase chain reaction. RESULTS: Recipient mice of LV-BMP2/4-transduced Sca-1+ cells yielded high engraftment and increased BMP4 mRNA levels in marrow cells; but exhibited only insignificant increases in serum and bone alkaline phosphatase activity compared to control mice. pQCT and microCT analyses of femurs showed that, with the exception of small changes in trabecular bone mineral density and cortical bone mineral content in LV-BMP2/4 mice, there were no differences in measured bone parameters between mice of the LV-BMP2/4 group and controls. The lack of large endosteal bone formation effects with the BMP4 strategy could not be attributed to ineffective engraftment or expansion of BMP4-expressing Sca-1+ cells, an inability of the transduced cells to secrete active BMP4 proteins, or to use of the LV-based vector. CONCLUSIONS: Sca-1+ cell-based BMP4 ex vivo strategy did not promote robust endosteal bone formation, raising the possibility of intrinsic differences between FGF2- and BMP4-based strategies in their ability to promote endosteal bone formation. It emphasizes the importance of choosing an appropriate bone growth factor gene for delivery by this Sca-1+ cell-based ex vivo systemic gene transfer strategy to promote bone formation.

Response of extracellular matrix regulators in mouse lung after exposure to photons, protons and simulated solar particle event protons.

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-beta1, 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-beta1 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.

Shifts in bone marrow cell phenotypes caused by spaceflight.

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.

Spaceflight effects on T lymphocyte distribution, function and gene expression.

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-1alpha 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.

Effects of spaceflight on innate immune function and antioxidant gene expression.

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.

Effect of a metalloporphyrin antioxidant (MnTE-2-PyP) on the response of a mouse prostate cancer model to radiation.

BACKGROUND: Metalloporphyrin antioxidants can protect tissues against radiation-induced damage. However, for effective use in radiotherapy as normal tissue radioprotectants, they must not protect the cancer. The major objectives were to evaluate the effects of Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) on tumor response to radiation and to explore mechanisms responsible for the observed effects. MATERIALS AND METHODS: C57BL/6 mice were subcutaneously (s.c.) injected with RM-9 prostate tumor cells on day 0 and grouped according to treatment with MnTE-2-PyP (s.c. 6 mg/kg/day beginning on day 1 for 16 maximum days), 10 Gray (Gy) single fraction radiation on day 7, a combination of both or neither. Subsets per group and non-tumor bearing controls were evaluated for leukocyte populations, red blood cell (RBC) and platelet characteristics and cytokines on day 12; the remaining mice were followed for tumor growth. RESULTS: Although radiation alone significantly slowed tumor growth and the addition of MnTE-2-PyP resulted in slightly slower tumor progression, the difference between radiation and radiation plus drug was not statistically significant. However, the treatment with drug alone significantly elevated T (helper, Th and cyotoxic, Tc) and natural killer (NK) cells in the spleen, B-cells in the blood and spleen, and the capacity to produce interleukin-2. The addition of the drug to radiation did not ameliorate the depression seen in all the major leukocyte types, but did protect against radiation-induced decreases in RBC counts, hemoglobin and hematocrit. Vascular endothelial growth factor (VEGF) increased in the plasma from both the irradiated groups and a trend for increased transforming growth factor-beta1 (TGF-beta1) was noted with radiation alone. CONCLUSION: MnTE-2-PyP did not protect RM-9 prostate tumors against radiation damage and was not toxic under the conditions used. The drug-induced enhancement of certain immune parameters suggests that MnTE-2-PyP may be beneficial not only as a normal tissue radioprotectant, but also as a facilitator of antitumor immunity.

Low-dose, low-dose-rate proton radiation modulates CD4(+) T cell gene expression.

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 0.1 cGy/h. On day 0 spleens were harvested from a subset in the 0, 0.01 and 0.1 Gy 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 p < 0.05 compared to 0 Gy were noted. After 0.01 Gy, five genes were up-regulated (Ccr5, Cd40, Cebpb, Igsf6, Tnfsf4) and three were down-regulated (Il4ra, Mapk8, Nfkb1). After 0.1 Gy there were nine up-regulated genes (Ccr4, Cd40, Cebpb, Cxcr3, Socs5, Stat4, Tbx21, Tnfrsf4, Tnfsf4); none were down-regulated. On day 0 after 0.01 Gy, CD4(+) T cell counts and CD4:CD8 ratio were low in the spleen (p < 0.05). Spontaneous DNA synthesis in both spleen and blood was lowest in the 0.01 Gy 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 0.01 Gy, may enhance CD4(+) T cell responsiveness.