Effect of Solar Particle Event Radiation and Hindlimb Suspension on Gastrointestinal Tract Bacterial Translocation and Immune Activation

The environmental conditions that could lead to an increased risk for the development of an infection during prolonged space flight include: microgravity, stress, radiation, disturbance of circadian rhythms, and altered nutritional intake. A large body of literature exists on the impairment of the immune system by space flight. With the advent of missions outside the Earth''s magnetic field, the increased risk of adverse effects due to exposure to radiation from a solar particle event (SPE) needs to be considered. Using models of reduced gravity and SPE radiation, we identify that either 2 Gy of radiation or hindlimb suspension alone leads to activation of the innate immune system and the two together are synergistic. The mechanism for the transient systemic immune activation is a reduced ability of the GI tract to contain bacterial products. The identification of mechanisms responsible for immune dysfunction during extended space missions will allow the development of specific countermeasures.     

Hindlimb Suspension and SPE-Like Radiation Impairs Clearance of Bacterial Infections

A major risk of extended space travel is the combined effects of weightlessness and radiation exposure on the immune system. In this study, we used the hindlimb suspension model of microgravity that includes the other space stressors, situational and confinement stress and alterations in food intake, and solar particle event (SPE)-like radiation to measure the combined effects on the ability to control bacterial infections. A massive increase in morbidity and decrease in the ability to control bacterial growth was observed using 2 different types of bacteria delivered by systemic and pulmonary routes in 3 different strains of mice. These data suggest that an astronaut exposed to a strong SPE during extended space travel is at increased risk for the development of infections that could potentially be severe and interfere with mission success and astronaut health.     

Analysis of white blood cell counts in mice after gamma- or proton-radiation exposure

In the coming decades human space exploration is expected to move beyond low-Earth orbit. This transition involves increasing mission time and therefore an increased risk of radiation exposure from solar particle event (SPE) radiation. Acute radiation effects after exposure to SPE radiation are of prime importance due to potential mission-threatening consequences. The major objective of this study was to characterize the dose-response relationship for proton and γ radiation delivered at doses up to 2 Gy at high (0.5 Gy/min) and low (0.5 Gy/h) dose rates using white blood cell (WBC) counts as a biological end point. The results demonstrate a dose-dependent decrease in WBC counts in mice exposed to high- and low-dose-rate proton and γ radiation, suggesting that astronauts exposed to SPE-like radiation may experience a significant decrease in circulating leukocytes.     

Effect of solar particle event radiation on gastrointestinal tract bacterial translocation and immune activation

Space flight conditions within the protection of Earth's gravitational field have been shown to alter immune responses, which could lead to potentially detrimental pathology. An additional risk of extended space travel outside the Earth's gravitational field is the effect of solar particle event (SPE) radiation exposure on the immune system. Organisms that could lead to infection include endogenous, latent viruses, colonizing pathogenics, and commensals, as well as exogenous microbes present in the spacecraft or other astronauts. In this report, the effect of SPE-like radiation on containment of commensal bacteria and the innate immune response induced by its breakdown was investigated at the radiation energies, doses and dose rates expected during an extravehicular excursion outside the Earth's gravitational field. A transient increase in serum lipopolysaccharide was observed 1 day after irradiation and was accompanied by an increase in acute-phase reactants and circulating proinflammatory cytokines, indicating immune activation. Baseline levels were reestablished by 5 days postirradiation. These findings suggest that astronauts exposed to SPE radiation could have impaired containment of colonizing bacteria and associated immune activation.     

Using electron beam radiation to simulate the dose distribution for whole body solar particle event proton exposure

As a part of the near solar system exploration program, astronauts may receive significant total body proton radiation exposures during a solar particle event (SPE). In the Center for Acute Radiation Research (CARR), symptoms of the acute radiation sickness syndrome induced by conventional radiation are being compared to those induced by SPE-like proton radiation, to determine the relative biological effectiveness (RBE) of SPE protons. In an SPE, the astronaut’s whole body will be exposed to radiation consisting mainly of protons with energies below 50 MeV. In addition to providing for a potentially higher RBE than conventional radiation, the energy distribution for an SPE will produce a relatively inhomogeneous total body dose distribution, with a significantly higher dose delivered to the skin and subcutaneous tissues than to the internal organs. These factors make it difficult to use a ⁶⁰Co standard for RBE comparisons in our experiments. Here, the novel concept of using megavoltage electron beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation is described. In these studies, Monte Carlo simulation was used to determine the dose distribution of electron beam radiation in small mammals such as mice and ferrets as well as large mammals such as pigs. These studies will help to better define the topography of the time-dose-fractionation versus biological response landscape for astronaut exposure to an SPE.     

Effects of dietary antioxidant supplementation on the development of malignant lymphoma and other neoplastic lesions in mice exposed to proton or iron-ion radiation

Malignancy is considered to be a particular risk associated with exposure to the types of ionizing radiation encountered during extended space flight. In the present study, two dietary preparations were evaluated for their ability to prevent carcinogenesis in CBA mice exposed to different forms of space radiation: protons and highly energetic heavy particles (HZE particles). One preparation contained a mixture of antioxidant agents. The other contained the soybean-derived Bowman-Birk protease inhibitor (BBI), used in the form of BBI Concentrate (BBIC). The major finding was that there was a reduced risk of developing malignant lymphoma in animals exposed to space radiation and maintained on diets containing the antioxidant formulation or BBIC compared to the irradiated animals maintained on the control diet. In addition, the two different dietary countermeasures also reduced the yields of a variety of different rare tumor types observed in the animals exposed to space radiation. These results suggest that dietary supplements could be useful in the prevention of malignancies and other neoplastic lesions developing from exposure to space radiation.     

Heavy ions, radioprotectors and genomic instability: implications for human space exploration

The risk associated with space radiation exposure is unique from terrestrial radiation exposures due to differences in radiation quality, including linear energy transfer (LET). Both high- and low-LET radiations are capable of inducing genomic instability in mammalian cells, and this instability is thought to be a driving force underlying radiation carcinogenesis. Unfortunately, during space exploration, flight crews cannot entirely avoid radiation exposure. As a result, chemical and biological countermeasures will be an important component of successful extended missions such as the exploration of Mars. There are currently several radioprotective agents (radioprotectors) in use; however, scientists continue to search for ideal radioprotective compounds—safe to use and effective in preventing and/or reducing acute and delayed effects of irradiation. This review discusses the agents that are currently available or being evaluated for their potential as radioprotectors. Further, this review discusses some implications of radioprotection for the induction and/or propagation of genomic instability in the progeny of irradiated cells.     

Stress, suspension and resistance to infection

Immune function is altered in stressful situations, including space flight. This may result in increased risk of infection. Antiorthostatic suspension has been used to study the effects of space flight-like conditions on immunity. The mechanisms of promoting infection in stressful situations have not been defined, but catecholamines could play a role. In the present study gram negative bacteria grown with catecholamines showed enhanced bacterial growth compared to controls. Additionally, antiorthostatically suspended mice infected with Klebsiella pneumoniae showed decreased survival compared to restrained or normally caged controls. Therefore, stress-induced enhanced bacterial growth and immunosuppression could play a role in suspension-induced enhanced mortality due to infection.     

Radioprotective efficacy of delta-tocotrienol,a vitamin E isoform,is mediated through granulocyte colony-stimulating factor

Aims

The objectives of this study were to determine the cytokine induction by delta tocotrienol (DT3, a promising radiation countermeasure) and to investigate the role of granulocyte colony-stimulating factor (G-CSF) in its radioprotective efficacy against ionizing radiation in mice.

Main methods

Multiplex Luminex was used to analyze cytokines induced by DT3 and other tocols (gamma-tocotrienol and tocopherol succinate) in CD2F1 mice. Mice were injected with an optimal dose of DT3 and a G-CSF antibody, and their 30-day survival against cobalt-60 gamma-irradiation was monitored. The neutralization of G-CSF by the administration of a G-CSF-specific antibody in DT3-injected mice was investigated by multiplex Luminex.

Key findings

Our data demonstrate that DT3 induced high levels of various cytokines comparable to other tocols being developed as radiation countermeasures. DT3 significantly protected mice against ionizing radiation, and the administration of a G-CSF neutralizing antibody to DT3-treated animals resulted in the complete abrogation of DT3's radioprotective efficacy and neutralization of G-CSF in peripheral blood.

Significance

Our study findings suggest that G-CSF induced by DT3 mediates its radioprotective efficacy against ionizing radiation in mice.     

Acute biological effects of simulating the whole-body radiation dose distribution from a solar particle event using a porcine model

In a solar particle event (SPE), an unshielded astronaut would receive proton radiation with an energy profile that produces a highly inhomogeneous dose distribution (skin receiving a greater dose than internal organs). The novel concept of using megavoltage electron-beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation has been described previously. Here, Yucatan minipigs were used to determine the effects of a superficial, SPE-like proton dose distribution using megavoltage electrons. In these experiments, dose-dependent increases in skin pigmentation, ulceration, keratinocyte necrosis and pigment incontinence were observed. Five of 18 animals (one each exposed to 7.5 Gy and 12.5 Gy radiation and three exposed to 25 Gy radiation) developed symptomatic, radiation-associated pneumonopathy approximately 90 days postirradiation. The three animals from the highest dose group showed evidence of mycoplasmal pneumonia along with radiation pneumonitis. Moreover, delayed-type hypersensitivity was found to be altered, suggesting that superficial irradiation of the skin with ionizing radiation might cause immune dysfunction or dysregulation. In conclusion, using total doses, patterns of dose distribution, and dose rates that are compatible with potential astronaut exposure to SPE radiation, animals experienced significant toxicities that were qualitatively different from toxicities previously reported in pigs for homogeneously delivered radiation at similar doses.     

Countermeasures against space radiation induced oxidative stress in mice

Of particular concern for the health of astronauts during space travel is radiation from protons and high atomic number (Z), high energy particles (HZE particles). Space radiation is known to induce oxidative stress in astronauts after extended space flight. In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by proton and HZE particle radiation in the plasma of CBA mice and the protective effect of dietary supplement agents. The results indicate that exposure to proton and HZE particle radiation significantly decreased the plasma level of total antioxidants in the irradiated CBA mice. Dietary supplementation with l-selenomethionine (SeM) or a combination of selected antioxidant agents (which included SeM) could partially or completely prevent the decrease in the total antioxidant status in the plasma of animals exposed to proton or HZE particle radiation. These findings suggest that exposure to space radiation may compromise the capacity of the host antioxidant defense system; this adverse biological effect can be prevented at least partially by dietary supplementation with agents expected to have effects on antioxidant activities.     

Emphasis of biological research for space radiation]

The paper summarized issues, current status and the recent topics in biological research of space radiation. Researches to estimate a risk associated with space radiation exposure during a long-term manned space flight, such as in the International Space Station, is emphasized because of the large uncertainty of biological effects and a complexity of the radiation environment in space. The Issues addressed are; 1) biological effects and end points in low dose radiation, 2) biological effects under low dose rate and long-term radiation exposure, 3) modification of biological responses to radiation under space environments, 4) various aspects of biological end points vs. cellular and molecular mechanisms, 5) estimation of human risk associated with radiation exposure in space flight, 6) regulations for radiation exposure limits for space workers. The paper also summarized and introduced recent progress in space related radiation researches with various biological systems.     

Efficacy of radiation countermeasures depends on radiation quality

The detonation of a nuclear weapon or a nuclear accident represent possible events with significant exposure to mixed neutron/γ-radiation fields. Although radiation countermeasures generally have been studied in subjects exposed to pure photons (γ or X rays), the mechanisms of injury of these low linear energy transfer (LET) radiations are different from those of high-LET radiation such as neutrons, and these differences may affect countermeasure efficacy. We compared 30-day survival in mice after varying doses of pure γ and mixed neutron/γ (mixed field) radiation (MF, Dn/Dt = 0.65), and also examined peripheral blood cells, bone marrow cell reconstitution, and cytokine expression. Mixed-field-irradiated mice displayed prolonged defects in T-cell populations compared to mice irradiated with pure γ photons. In mouse survival assays, the growth factor granulocyte colony-stimulating factor (G-CSF) was effective as a (post-irradiation) mitigator against both γ-photons and mixed-field radiation, while the thrombopoietin (TPO) mimetic ALXN4100TPO was effective only against γ irradiation. The results indicate that radiation countermeasures should be tested against radiation qualities appropriate for specific scenarios before inclusion in response plans.     

Progenitors Mobilized by Gamma-Tocotrienol as an Effective Radiation Countermeasure

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.     

Inhibition of Intestinal Epithelial Apoptosis Improves Survival in a Murine Model of Radiation Combined Injury

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.     

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30 d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2 Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68(+) cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.     

Effect of antiorthostatic suspension on interferon-alpha/beta production by the mouse

Mice were suspended in a model that simulates weightlessness that occurs during prolonged space flight. After 1 and 2 weeks of suspension in an antiorthostatic (head-down tilt) position, the mice were challenged with polyriboinosinic-polyribocytidylic acid to induce interferon-alpha/beta. Interferon production was severely reduced in mice that had been suspended. When mice were allowed to recover in cages for a week following removal from suspension, they recovered their full interferon-production capacity. Mice suspended in an orthostatic (horizontal) position did not have their interferon production capabilities affected, which indicates that stress per se was not a major component in the effects of antiorthostatic suspension on interferon induction.     

Response of primary human fibroblasts exposed to solar particle event protons

Solar particle events (SPEs) present a major radiation-related risk for manned exploratory missions in deep space. Within a short period the astronauts may absorb doses that engender acute effects, in addition to the risk of late effects, such as the induction of cancer. Using primary human cells, we studied clonogenic survival and the induction of neoplastic transformation after exposure to a worst case scenario SPE. We simulated such an SPE with monoenergetic protons (50, 100, 1000 MeV) delivered at a dose rate of 1.65 cGy min?1 in a dose range from 0 to 3 Gy. For comparison, we exposed the cells to a high dose rate of 33.3 cGy min?1. X rays (100 kVp, 8 mA, 1.7 mm Al filter) were used as a reference radiation. Overall, we observed a significant sparing effect of the SPE dose rate on cell survival. High-dose-rate protons were also more efficient in induction of transformation in the dose range below 30 cGy. However, as dose accumulated at high dose rate, the transformation levels declined, while at the SPE dose rate, the number of transformants continued to increase up to about 1 Gy. These findings suggest that considering dose-rate effects may be important in evaluating the biological effects of exposure to space radiation. Our analyses of the data based on particle fluence showed that lethality and transforming potential per particle clearly increased with increasing linear energy transfer (LET) and thus with the decreasing energy of protons. Further, we found that the biological response was determined not only by LET but also type of radiation, e.g. particles and photons. This suggests that using γ or X rays may not be ideal for assessing risk associated with SPE exposures.     

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.     

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b<Superscript>+</Superscript>Gr-1<Superscript>+</Superscript> myeloid cells without compromising CD8<Superscript>+</Superscript> T cell immune responses

Background

Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.

Methods

We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.

Results

We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b⁺Gr-1⁺ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8⁺ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.

Conclusions

Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.