Eric S. Diffenderfer

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 Earths 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.

Leukocyte Activity Is Altered in a Ground Based Murine Model of Microgravity and Proton Radiation Exposure

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.

Kinetics of Neutrophils in Mice Exposed to Radiation and/or Granulocyte Colony-Stimulating Factor Treatment

Astronauts have the potential to develop the hematopoietic syndrome as a result of exposure to radiation from a solar particle event (SPE) during exploration class missions. This syndrome is characterized by a reduction in the number of circulating blood cells (cytopenias). In the present study the effects of SPE-like proton and γ radiation on the kinetics of circulating neutrophils were evaluated during a one-month time period using mice as a model system. The results revealed that exposure to a 2 Gy dose of either SPE-like proton or γ radiation significantly decreased the number of circulating neutrophils, with two nadirs observed on day 4 and day 16 postirradiation. Low circulating neutrophil count (neutropenia) is particularly important because it can increase the risk of astronauts developing infections, which can compromise the success of the mission. Thus, two granulocyte colony-stimulating factors (G-CSFs), filgrastim and pegfilgrastim were evaluated as countermeasures for this endpoint. Both forms of G-CSF significantly increased neutrophil counts in irradiated mice, however, the effect of pegfilgrastim was more potent and lasted longer than filgrastim. Using the expression of CD11b, CD18 and the production of reactive oxygen species (ROS) as markers of neutrophil activation, it was determined that the neutrophils in the irradiated mice treated with pegfilgrastim were physiologically active. Thus, these results suggest that pegfilgrastim could be a potential countermeasure for the reduced number of circulating neutrophils in irradiated animals.

Acute Hematological Effects of Solar Particle Event Proton Radiation in the Porcine Model

Acute radiation sickness (ARS) is expected to occur in astronauts during large solar particle events (SPEs). One parameter associated with ARS is the hematopoietic syndrome, which can result from decreased numbers of circulating blood cells in those exposed to radiation. The peripheral blood cells are critical for an adequate immune response, and low blood cell counts can result in an increased susceptibility to infection. In this study, Yucatan minipigs were exposed to proton radiation within a range of skin dose levels expected for an SPE (estimated from previous SPEs). The proton-radiation exposure resulted in significant decreases in total white blood cell count (WBC) within 1 day of exposure, 60% below baseline control value or preirradiation values. At the lowest level of the blood cell counts, lymphocytes, neutrophils, monocytes and eosinophils were decreased up to 89.5%, 60.4%, 73.2% and 75.5%, respectively, from the preirradiation values. Monocytes and lymphocytes were decreased by an average of 70% (compared to preirradiation values) as early as 4 h after radiation exposure. Skin doses greater than 5 Gy resulted in decreased blood cell counts up to 90 days after exposure. The results reported here are similar to studies of ARS using the nonhuman primate model, supporting the use of the Yucatan minipig as an alternative. In addition, the high prevalence of hematologic abnormalities resulting from exposure to acute, whole-body SPE-like proton radiation warrants the development of appropriate countermeasures to prevent or treat ARS occurring in astronauts during space travel.

Comparison of secondary neutron dose in proton therapy resulting from the use of a tungsten alloy MLC or a brass collimator system

PURPOSE To apply the dual ionization chamber method for mixed radiation fields to an accurate comparison of the secondary neutron dose arising from the use of a tungsten alloy multileaf collimator (MLC) as opposed to a brass collimator system for defining the shape of a therapeutic proton field. METHODS Hydrogenous and nonhydrogenous ionization chambers were constructed with large volumes to enable measurements of absorbed doses below 10(-4) Gy in mixed radiation fields using the dual ionization chamber method for mixed-field dosimetry. Neutron dose measurements were made with a nominal 230 MeV proton beam incident on a closed tungsten alloy MLC and a solid brass block. The chambers were cross-calibrated against a (60)Co-calibrated Farmer chamber in water using a 6 MV x-ray beam and Monte Carlo simulations were performed to account for variations in ionization chamber response due to differences in secondary neutron energy spectra. RESULTS The neutron and combined proton plus γ-ray absorbed doses are shown to be nearly equivalent downstream from either a closed tungsten alloy MLC or a solid brass block. At 10 cm downstream from the distal edge of the collimating material the neutron dose from the closed MLC was (5.3 ± 0.4) × 10(- 5) Gy/Gy. The neutron dose with brass was (6.4 ± 0.7) × 10(- 5) Gy/Gy. Further from the secondary neutron source, at 50 cm, the neutron doses remain close for both the MLC and brass block at (6.9 ± 0.6) × 10(- 6) Gy/Gy and (6.3 ± 0.7) × 10(- 6) Gy/Gy, respectively. CONCLUSIONS The dual ionization chamber method is suitable for measuring secondary neutron doses resulting from proton irradiation. The results of measurements downstream from a closed tungsten alloy MLC and a brass block indicate that, even in an overly pessimistic worst-case scenario, secondary neutron production in a tungsten alloy MLC leads to absorbed doses that are nearly equivalent to those seen from brass collimators. Therefore, the choice of tungsten alloy in constructing the leaves of a proton MLC is appropriate, and does not lead to a substantial increase in the secondary neutron dose to the patient compared to that generated in a brass collimator.

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.

Relative biological effectiveness of simulated solar particle event proton radiation to induce acute hematological change in the porcine model

The present study was undertaken to determine relative biological effectiveness (RBE) values for simulated solar particle event (SPE) radiation on peripheral blood cells using Yucatan minipigs and electron-simulated SPE as the reference radiation. The results demonstrated a generally downward trend in the RBE values with increasing doses of simulated SPE radiation for leukocytes in the irradiated animals. The fitted RBE values for white blood cells (WBCs), lymphocytes, neutrophils, monocytes and eosinophils were above 1.0 in all three radiation dose groups at all time-points evaluated, and the lower limits of the 95% confidence intervals were > 1.0 in the majority of the dose groups at different time-points, which together suggest that proton-simulated SPE radiation is more effective than electron-simulated SPE radiation in reducing the number of peripheral WBCs, lymphocytes, neutrophils, monocytes and eosinophils, especially at the low end of the 5–10 Gy dose range evaluated. Other than the RBE values, the responses of leukocytes to electron-simulated SPE radiation and proton-simulated SPE radiation exposure are highly similar with respect to the time-course, the most radiosensitive cell type (the lymphocytes), and the shape of the dose–response curves, which is generally log-linear. These findings provide additional evidence that electron-simulated SPE radiation is an appropriate reference radiation for determination of RBE values for the simulated SPE radiations, and the RBE estimations using electron-simulated SPE radiation as the reference radiation are not complicated by other characteristics of the leukocyte response to radiation exposure.

Ionizing Radiation Selectively Reduces Skin Regulatory T Cells and Alters Immune Function

The skin serves multiple functions that are critical for life. The protection from pathogens is achieved by a complicated interaction between aggressive effectors and controlling functions that limit damage. Inhomogeneous radiation with limited penetration is used in certain types of therapeutics and is experienced with exposure to solar particle events outside the protection of the Earth’s magnetic field. This study explores the effect of ionizing radiation on skin immune function. We demonstrate that radiation, both homogeneous and inhomogeneous, induces inflammation with resultant specific loss of regulatory T cells from the skin. This results in a hyper-responsive state with increased delayed type hypersensitivity in vivo and CD4+ T cell proliferation in vitro. The effects of inhomogeneous radiation to the skin of astronauts or as part of a therapeutic approach could result in an unexpected enhancement in skin immune function. The effects of this need to be considered in the design of radiation therapy protocols and in the development of countermeasures for extended space travel.

Broad-Spectrum Antibiotic or G-CSF as Potential Countermeasures for Impaired Control of Bacterial Infection Associated with an SPE Exposure during Spaceflight

A major risk for astronauts during prolonged space flight is infection as a result of the combined effects of microgravity, situational and confinement stress, alterations in food intake, altered circadian rhythm, and radiation that can significantly impair the immune system and the body’s defense systems. We previously reported a massive increase in morbidity with a decrease in the ability to control a bacterial challenge when mice were maintained under hindlimb suspension (HS) conditions and exposed to solar particle event (SPE)-like radiation. HS and SPE-like radiation treatment alone resulted in a borderline significant increase in morbidity. Therefore, development and testing of countermeasures that can be used during extended space missions in the setting of exposure to SPE radiation becomes a serious need. In the present study, we investigated the efficacy of enrofloxacin (an orally bioavailable antibiotic) and Granulocyte colony stimulating factor (G-CSF) (Neulasta) on enhancing resistance to Pseudomonas aeruginosa infection in mice subjected to HS and SPE-like radiation. The results revealed that treatment with enrofloxacin or G-CSF enhanced bacterial clearance and significantly decreased morbidity and mortality in challenged mice exposed to suspension and radiation. These results establish that antibiotics, such as enrofloxacin, and G-CSF could be effective countermeasures to decrease the risk of bacterial infections after exposure to SPE radiation during extended space flight, thereby reducing both the risk to the crew and the danger of mission failure.

Monte Carlo modeling in CT-based geometries: dosimetry for biological modeling experiments with particle beam radiation

The space radiation environment imposes increased dangers of exposure to ionizing radiation, particularly during a solar particle event (SPE). These events consist primarily of low energy protons that produce a highly inhomogeneous dose distribution. Due to this inherent dose heterogeneity, experiments designed to investigate the radiobiological effects of SPE radiation present difficulties in evaluating and interpreting dose to sensitive organs. To address this challenge, we used the Geant4 Monte Carlo simulation framework to develop dosimetry software that uses computed tomography (CT) images and provides radiation transport simulations incorporating all relevant physical interaction processes. We found that this simulation accurately predicts measured data in phantoms and can be applied to model dose in radiobiological experiments with animal models exposed to charged particle (electron and proton) beams. This study clearly demonstrates the value of Monte Carlo radiation transport methods for two critically interrelated uses: (i) determining the overall dose distribution and dose levels to specific organ systems for animal experiments with SPE-like radiation, and (ii) interpreting the effect of random and systematic variations in experimental variables (e.g. animal movement during long exposures) on the dose distributions and consequent biological effects from SPE-like radiation exposure. The software developed and validated in this study represents a critically important new tool that allows integration of computational and biological modeling for evaluating the biological outcomes of exposures to inhomogeneous SPE-like radiation dose distributions, and has potential applications for other environmental and therapeutic exposure simulations.