F EBRUARY 2015, N O 2
STEM Today
Katherine Johnson Katherine Johnson is a pioneer of the American space movement. She is a research mathematician and physicist who calculated trajectories and orbits for historic missions including the first flight to put a man on the moon. She also helped develop space navigation systems to guide the astronauts. Katherine’s specialty was calculating the trajectories for space shots which determined the timing for launches. "I’d ask (another section at NASA), ’Where do you want (the astronauts) to come down?’ And they’d tell me the spot and I’d work backward from there." An early achievement was correctly computing the ’launch window’ for astronaut Alan Shepard’s Mercury mission. His successful splashdown at sea on May 5, 1961 marked the return of the first American in space.As the work grew more complex, Katherine was tasked with calculations to propel space capsules into orbit around the moon and to send landing units to and from the lunar surface. She also earned kudos for plotting backup navigational charts that would enable astronauts to guide their ships by the stars in case of electronic failures. In 1962 computers were used for the first time to calculate John Glenn’s history making orbit around Earth. But, according to Katherine, NASA officials called on her to verify the numbers. "They knew I had done most of the [the calculations], so they let me do it," she said. When Neil Armstrong took his first step on the moon in July 1969, Katherine Johnson was attending a sorority convention in Pennsylvania and watched the event on television. Few of her sorority sisters knew that she had calculated the trajectory for the Apollo 11 flight to the Moon. Along with fellow team members, Katherine was given a souvenir flag that made the trip with Armstrong and his crew.
— Image Credit:NASA
Gap’s in NASA Human Reserach Roadmap
The effect(s) of SPE-like radiation exposure(s) on immune function Future space missions will involve distant travel and extended stays outside the Earth’s magnetic field that provides protection from solar radiation. Multiple environmental factors have been identified that increase the risk of infection during these missions that include; stress, reduced weight bearing, disturbance of circadian rhythms, and altered nutritional intake , in addition to solar and galactic radiation. These factors, either alone, independently additive, or through synergistic interactions, pose a threat for the development of pathogenic infection by exogenous or endogenous organisms. 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.The risks for developing infections during space flight include reduced weight bearing, stress, radiation, disturbance of circadian rhythms, and altered nutritional intake. Endogenous organisms, which are resident in the astronaut at the start of space flight, consist of latent viruses common in humans (e.g., Epstein-Barr, Herpes simplex and others) or commensal and colonizing pathogenic organisms, while exogenous organisms are present in the spacecraft and other astronauts. Fifteen of 29 Apollo astronauts contracted bacterial or viral infections either during their missions or within a week of returning, although none were severe. A urinary tract infection with Pseudomonas aeruginosa was documented after an Apollo mission. Increased ear and skin infections were noted during early space missions. A reduced ability to clear infections by Klebsiella pneumonia and Pseudomonas aeruginosa has been reported in studies using animal models of space flight.
Resistant mice have been shown to develop infections with the Encephalomyocarditis virus D variant when they were subjected to hind limb unloading . The decrease in resistance to infection was correlated with a drop in type I interferon production . In another model of space flight using murine infection with Listeria monocytogenes, innate immunity based resistance to primary infection was enhanced, but the ability to generate a T-lymphocyte immune response was severely impaired. Decreased antibiotic potency and enhanced microbial virulence associated with space flight could further increase the risk for serious infections in immunocompromised astronauts.
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Gap’s in NASA Human Reserach Roadmap The primary components of interplanetary radiation are galactic cosmic rays and solar radiation , which consists of low-energy solar wind and highly energetic solar particle events (SPEs) that originate from magnetically disturbed regions of the sun. SPEs are unpredictable and typically last for no more than several hours, although some may continue for several days. SPEs are composed predominately of low-energy protons with a minor contribution from helium ions (≈ 10%) and an even smaller contribution from heavy ions and electrons (≈ 1%). Astronauts are at greatest risk for radiation exposure from SPEs while performing extravehicular activities. It is predicted that astronauts may receive up to a dose of 2 Gy to the bone marrow and up to 25 Gy to the skin. While it is expected that a 2-Gy bone marrow dose of conventional radiation (electrons, X or γ rays) is likely to have adverse effects on immune function, it is not known whether 2 Gy of protons given at dose rates expected during an SPE (1-50 cGy/h) will have similar effects. Hindlimb Unloading Model The main potential stresses on immune function during space travel include the weightless environment, altered nutritional intake, disturbances in circadian rhythm, situational and confinement stress, and exposure to radiation. The hindlimb unloading model, first used in 1985([3,4]) to model the weightless conditions of spaceflight, is the widely accepted land based model. In addition to mimicking weightless conditions, this model also induces situational and confinement stress and altered nutritional intake. To model the conditions that an astronaut would be exposed to SPE radiation, mice were suspended for 2 days and then irradiated with 2, 1, or 0.5 Gy of proton (70 MeV) or gamma radiation and placed back in suspension and analyzed over 7 days. Similar data was obtained when mice were suspended for 5 days prior to irradiation. Both irradiated and non-irradiated mice were placed in 7.2 cm X 4.1 cm X 4.1 cm enclosures for the same amount of time to control for the effects of this confinement. Certain groups received only suspension or radiation or were untreated. Mice were placed in similar caging with or without hindlimb suspension in the same room. 2 Gy of proton or gamma radiation led to a low but significant increase, 6 and 24 hr later (Figure 1A and B) compared to control animals (F = 21.9, p= 3 X 10 −8 and F = 6.7, p= 0.01, respectively).Hindlimb unloading led to an increase in serum LPS at 6 hr (54 hr total suspension) and the combination of radiation and unloading demonstrated a larger increase (Figure 1A and B). At 4 days post irradiation, the circulating levels of LPS remained significantly elevated for animals that received both gamma radiation and suspension, while the other groups returned to baseline (Figure 1B)(F = 47.2, p= 1.8 X 10 −11 ). Lower doses of radiation, 1 and 0.5 Gy, did not significantly increase circulating levels of LPS either alone or with hindlimb suspension. To determine whether the increase in circulating LPS induced a systemic response, the type I acute phase protein, LPS binding protein (LBP), was measured.LBP is a circulating protein that binds to LPS of Gram-negative bacteria via the lipid A region, which then promotes its binding to CD14. LBP is constitutively present and is induced during various types of infection and inflammatory processes. LBP was increased after radiation (proton) (24 hr) and hindlimb suspension (3 days total suspension) and was increased further when they were combined (Figure 2A) (F = 15.0, p = 1.7X10−6 ). At 4 days post radiation, or a total of 6 days of hindlimb suspension, LBP remained significantly elevated in the group that received both 2 Gy of radiation and suspension (Figure 2B) (F= 5.4, p= 0.014). Similar data was obtained with gamma radiation. This demonstrates that the increase in circulating LPS led to a systemic response and while radiation and suspension appeared additive on day 1 post radiation, the increase in LBP remained on day 4 only for the groups that received both 2 Gy of radiation and hindlimb unloading. Mice receiving 1 and 0.5 Gy of proton or gamma radiation and hindlimb suspension were similar to controls at day 4
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Gap’s in NASA Human Reserach Roadmap
The circulating levels of sCD14 24 hr after 2 Gy of proton or gamma radiation or 3 days after the initiation of suspension were significantly elevated and the combination of radiation and hindlimb unloading further increased sCD14 (Figure 3A and gamma data not shown) compared to control animals (F = 11.9, p = 0.00016). Four days after proton irradiation of suspended mice, the levels of sCD14 continued to be significantly elevated compared to control animals (F =50.3, p = 8.2 X 10−12 ), while mice that received radiation or hindlimb unloading alone were not statistically different from control mice (Figure 3B and proton data not shown). Lower doses of radiation combined with hindlimb suspension did not result in elevated levels of circulating sCD14 four days after irradiation (Data not shown).
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Gap’s in NASA Human Reserach Roadmap
Bacterial translocation increases circulating levels of LPS, as well as other bacterial components, such as DNA, some of which can activate innate immune sensors. To measure whether the increase in bacterial product translocation induced by radiation and/or hindlimb unloading led to the induction of a systemic cytokine response, circulating levels of interferon (IFN)-α, TNF-α, and IL-6 were measured. Radiation (2 Gy of gamma or proton) and hindlimb unloading alone led to an increase in circulating (IFN)-α and IL-6 and at least additive levels were observed in mice treated with both (Figure 4 and proton data not shown) ((F= 7.9, p = 0.0019 and F = 15.2, p = 0.00018, respectively). The combination of irradiation and hindlimb unloading led to an increase in TNF-a (F =6.8, p =0.0045). Lower doses of radiation in combination with hindlimb suspension did not lead to significant increases in circulating TNF-α, IL-6, and IFN-α (Data not shown).
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Gap’s in NASA Human Reserach Roadmap
To determine mechanisms of increased LPS translocation, immunohistochemical staining for the tight junction protein Claudin-3 was performed on terminal ileum 4 days after irradiation and/or 6 days after hindlimb unloading. A significant increase in the number of breaks and reductions in staining were observed in 2 Gy proton or gamma radiation treated and hindlimb suspended animals (Figure 5). The frequency of breaks was not significantly increased in ileum from radiation treated or suspended animals compared to untreated.
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Gap’s in NASA Human Reserach Roadmap
Hindlimb suspension alone led to an increase in circulating LPS that decreased with increasing amounts of time in suspension (Figure 1B). This may reflect contributions from various elements of microgravity modeling, including fluid shifts, situational and confinement stress, altered nutritional intake, and changes in circadian rhythms. The addition of SPE-like radiation to hindlimb suspension both increased circulating LPS and extended the duration of the elevated LPS to 4 days post irradiation (Figure 1), while radiation alone resulted in circulating LPS levels returning to baseline by 2 days post irradiation. Associated markers of immune activation, LBP and sCD14, were also elevated at 24 hr and remained elevated 4 days after irradiation when mice were also hindlimb unloaded. A major difficulty of studying immunologic dysfunction during space travel involves the model systems needed to perform the analyses on Earth. The hindlimb unloading model is the accepted best system and has been directly compared to animals subjected to spaceflight[3,4]. Numerous alterations to immune function have been observed after short or long space missions in humans and animals and in animals during land-based models of spaceflight. Consistent observations include altered distributions of immune cells and variations in cytokines released in response to stimulations. This includes an increase in antiinflammatory cytokines and a decrease in TNF-α in LPS stimulated spleen cells , reductions in interferon-γ and IL-2 following PMA and ionomycin stimulation of peripheral blood cells of astronauts , and reduced NK cell number and functionality. These alterations to immune function are very similar to those observed in settings of increased immune activation caused by exposure to pathogen associated molecular patterns (PAMPs), such as LPS. Specific examples include; a reduction in proinflammatory cytokine production by myeloid cells, a reduction in antigenspecific T cell effector cytokine responses, and a reduction in circulating NK cells.
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FRED WALLACE HAISE, JR. NASA ASTRONAUT (FORMER) Haise was lunar module pilot for Apollo 13, April 11-17, 1970. Apollo 13 was scheduled for a ten-day mission for the first landing in the hilly, upland Fra Mauro region of the moon. The original flight plan, however, was modified en route to the moon due to a failure of the service module cryogenic oxygen system which occurred at approximately 55 hours into the flight. Haise and fellow crewmen, James A. Lovell (spacecraft commander) and John L. Swigert (command module pilot), working closely with Houston ground controllers, converted their lunar module "Aquarius" into an effective lifeboat. Their emergency activation and operation of lunar module systems conserved both electrical power and water in sufficient supply to assure their safety and survival while in space and for the return to earth. Pseudomonas aeruginosa, the bacterium caused astronaut Fred Haise to become sick during the Apollo 13 mission to the moon in 1970.
— Image Credit:NASA
Gap’s in NASA Human Reserach Roadmap Effect of Solar Particle Event Radiation on Gastrointestinal Tract Bacterial Translocation and Immune Activation Male and female outbred ICR mice, 5-6 weeks of age, were obtained from Harlan Laboratories (Livermore, CA). For irradiation, the mice were placed in aerated plastic chambers (AMAC 530C) with dimensions of 7.30 cm X 4.13 cm X 4.13 cm. The chambers allowed the mice to turn around easily (reverse nose to tail direction). The mice were exposed to total-body radiation with 50 or 70 MeV protons delivered in a spread-out Bragg peak (SOBP) at doses of 50 cGy, 1 Gy and 2 Gy using the horizontal clinical beam line at LLUMC. The GI tract contains over 1012 bacteria whose functions include carbohydrate fermentation and absorption, repression of pathogenic microbial growth, metabolic activity, and continuous and dynamic effects on the gut and systemic immune system. The control of bacteria and bacterial product passage across the GI mucosa, known as translocation, is an important function that can be disturbed in multiple diseases. The effect of SPE-like radiation on GI containment of bacterial products was analyzed using proton radiation at energies (50 and 70 MeV SOBP), doses (0.5, 1 and 2 Gy) and dose rates (50 cGy/h) possible during a typical strong SPE. For comparison, the same total doses of γ and proton radiation at were delivered at a high dose rate (50 cGy/min). It is expected that the maximum dose delivered to the bone marrow during an SPE would not exceed 2 Gy; thus the maximum dose delivered in this study was 2 Gy. SPE-Like Radiation Exposure Causes Bacterial Product Translocation Across the GI Tract Increased levels of circulating LPS were found 24 h after a 2-Gy low-dose-rate (50 cGy/h) proton exposure (Fig. 1A). A similar increase was observed for high-dose-rate (50 cGy/min) protons (Fig. 1A) and for 2 Gy of γ radiation at low and high dose rates (Fig. 1B). No increase in circulating LPS was detected 5 days after irradiation (Fig. 1C and data not shown). To exclude the possibility that oral bacteria-derived LPS was contaminating our samples, blood from the tail vein was compared to cheek pouch-derived blood with similar results. Two different assays were used to measure serum LPS, an end point kinetic Limulus amebocyte lysate (LAL) assay and a more sensitive assay of recombinant Factor C, which is the first component of the LAL response. Serum diluted 1:5 had produced significant and variable inhibition of LPS after heat inactivation, while a 1:20 dilution had minimal inhibition. Because the cutoff value for the Factor C assay at this dilution was 40 pg/ml of LPS (0.2 EU/ml), any sample with less than 40 pg/ml of LPS was set to 40 pg/ml. Similar results were found with the end point LAL assay, but because a 1:5 dilution was needed to measure serum LPS levels, the results were variable. Authors studied both male and female mice and used both 50 MeV and 70 MeV protons and found no differences in the increases in circulating LPS or in the other assays and hence combined the data for the analyses. Control animals placed in boxes for the same amount of time as the irradiated animals but not irradiated were similar to control mice. The low-dose-rate groups started their radiation exposure 4 h before the high-dose-rate groups, but, since we did not see any difference between low and high dose rates at 24 h after completion of irradiation and since shamirradiated controls for the total time for both low and high dose rate were the same, we concluded that the additional time required for the low-dose-rate irradiation did not alter the measurements of effect. There were no increases in the levels of circulating LPS at radiation doses below 2 Gy compared to the boxed control groups.
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Gap’s in NASA Human Reserach Roadmap
SPE-Like Radiation Induces a Transient Acute-Phase Response An increase in circulating LPS can induce the release of acute-phase reactants that serve multiple functions, including inhibiting pathogen growth, promoting opsonization, and recruiting immune cells. LPS binding protein (LBP) is an early acute-phase reactant that is required for the innate immune responses to LPS. Circulating LBP was increased 1 day after low-dose-rate proton irradiation (Fig. 2A). As observed for LPS, the increase in LBP also occurred with high-dose-rate proton radiation and with both low- and high-doserate Îł radiation; a statistically significant increase was noted only with the highest dose of 2 Gy (Fig. 2A, B). By 5 days postirradiation, the levels returned to and remained at the level observed in the corresponding unirradiated boxed controls. No differences in serum LBP in controls restrained in boxes for 4, 2 or 1 h, corresponding to 2 Gy, 1 Gy and 50 cGy, respectively, and unrestrained mice were observed.
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Gap’s in NASA Human Reserach Roadmap
SPE-Like Radiation Induces Elevation of Circulating Soluble CD14 CD14 is the LPS binding receptor that presents LPS to the signaling receptor complex comprised of myeloid differentiation protein 2 (MD-2) and Toll-like receptor 4 (TLR4). It is found in both glycosylphosphatidylinositol (GPI)-linked cell surface and soluble forms. Increased levels of sCD14 are found during infection and sepsis, and the delivery of sCD14 with LPS reduces the lethal effects of LPS. It is also the most sensitive measure of increased bacterial translocation. One day after exposure to 2 Gy proton or ĂŁ radiation given at low or high dose rates, a significant increase in the serum levels of sCD14 occurred that returned to control levels by 5 days postirradiation (Fig. 3 and data not shown). The control group shown was restrained in boxes for 4 h, which corresponded to the time the 2-Gy lowdoserate groups were restrained.
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Gap’s in NASA Human Reserach Roadmap SPE-Like Radiation Induces a Transient Increase in Circulating Proinflammatory Cytokines The observation that 2 Gy of proton or γ radiation led to a transient increase in bacterial translocation leading to activation of an acute-phase response led us to investigate whether a systemic effect on the innate immune system occurred. The proinflammatory cytokines TNF-α, IL-1 β and IL-6 were measured in serum, and a small transient increase 1 day after proton or γ irradiation at low and high dose rates was found that returned to baseline levels 5 days after irradiation (Fig. 4 and data not shown). An astronaut exposed to an SPE could receive over 25 Gy of radiation to the skin. Authors assessed whether proton or reference γ radiation delivered at low or high dose rates led to measurable levels of proinflammatory cytokines in the skin. No significant increase was observed in the skin 2 days after irradiation (data not shown).
SPE-Like Radiation Induces Transient Disruption of Tight Junctions in the GI Tract Disease states associated with bacterial translocation are associated with a breakdown in the integrity of the epithelial layer of the GI tract. Terminal ileum was obtained from control mice or mice irradiated with 2 Gy high- or low-dose-rate protons or γ rays 2 or 7 days postirradiation. Immunohistochemistry for the tight junction protein Claudin-3 demonstrated occasional regions of reduced staining and interruptions along the epithelial barrier at 2 days postirradiation (Fig. 5). Such disruptions were observed at a frequency of 0.71 to 1.12 occurrences per 1,000 enterocytes and were similar at high or low dose rate in proton- or γ-irradiated tissue (Table 1). Claudin-3 staining in control and irradiated tissue was not significantly different on day 7.
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Gap’s in NASA Human Reserach Roadmap
Astronauts could receive up to 2 Gy to the bone marrow from an SPE. This dose of proton radiation delivered at an energy and dose rate possible during an SPE results in a transient increase in the release of LPS into the circulation and potentially pathological changes to the GI tract. In this experiments, the increases in LPS were small, less than twofold, but were statistically significant. An increase in the acute-phase reactant LPS binding protein, which is required for LPS signaling, was also observed, as was a transient elevation in sCD14. Immunohistological analysis of GI tract tissue demonstrated occasional disruptions in the epithelial cell barrier as measured by a loss in tight junctions. Finally, the serum proinflammatory cytokines TNF-α, IL-1 β and IL-6 increased 1 day after irradiation, demonstrating systemic activation of the innate immune system. The expression of proinflammatory cytokines induced by translocation of bacterial products across the GI tract can have multiple effects on immune function. The induction of inflammatory cytokines occurs in a finely tuned time-dependent equilibrium. They can have multiple and opposite effects in the same subject depending on the time of expression. The aberrant expression of proinflammatory cytokines can result in unusual and unwanted regulation of an immune response. In an astronaut exposed to an SPE with resulting release of proinflammatory cytokines, exposure to a pathogenic insult could result in an altered immune response, resulting in incomplete protection from infection or immunopathology from an exaggerated response.
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Gap’s in NASA Human Reserach Roadmap Reference: 1.Zhou Y, Ni H, Li M, Sanzari JK, Diffenderfer ES, et al. (2012) Effect of Solar Particle Event Radiation and Hindlimb Suspension on Gastrointestinal Tract Bacterial Translocation and Immune Activation. PLoS ONE 7(9): e44329. doi:10.1371/journal.pone.0044329. 2.Houping Ni,Klara Balint,Yu Zhou,Daila S. Gridley,Casey Maks,Ann R. Kennedy, and Drew Weissman (2011) Effect of Solar Particle Event Radiation on Gastrointestinal Tract Bacterial Translocation and Immune Activation. Radiat Res. 2011 April ; 175(4): 485-492. doi:10.1667/RR2373.1. 3.Musacchia XJ (1985) The use of suspension models and comparison with true weightlessness: ’a resume’. Physiologist 28: S237-240. 4.Morey-Holton E, Globus RK, Kaplansky A, Durnova G (2005) The hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. Adv Space Biol Med 10: 7-40.
5.NASA/GSFC Radiation Effects and Analysis. http://radhome.gsfc.nasa.gov/top.htm 6.Low-Dose Total-Body γ Irradiation Modulates Immune Response to Acute Proton Radiation,Xian Luo-Owen, Michael J. Pecaut, Asma Rizvi, and Daila S. Gridley,Radiation Research 2012 177 (3), 251-264. 7.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: 1935U1942, 2009. First published April 2, 2009; doi:10.1152/japplphysiol.91361.2008. 8.S. Mehrotra, M.Sc.,M. J. Pecaut, Ph.D.,T. L. Freeman, B.S.,J. D. Crapo, M.D.,A. Rizvi, Ph.D.,X. Luo-Owen, Ph.D.,J. M. Slater, B.S.,D. S. Gridley, Ph.D., Analysis of a Metalloporphyrin Antioxidant Mimetic (MnTE-2-PyP) as a Radiomitigator: Prostate Tumor and Immune Status,www.tcrt.org, DOI: 10.7785/tcrt.2012.500260. 9.Yingtai Chena, Yumin Lib, Hong Zhangc, Yi Xiec, Xuezhong Chend, Jinyu Rene, Xiaowei,Zhangf, Zijiang Zhud, Hongliang Liud, and Yawei Zhangg, Early effects of low dos 12 C 6+ ion or X-ray irradiation on human peripheral blood lymphocytes,Adv Space Res. 2010 April 1; 45(7): 832-838. doi:10.1016/j.asr.2009.09.024. 10.Pecaut, M. J., Dutta-Roy, R., Smith, A. L., Jones, T. A., Nelson, G. A. and Gridley, D. S. Acute Effects of Iron-Particle Radiation on Immunity. Part I: Population Distributions. Radiat. Res. 165, 68-77 (2006). 11. William T. Shearer, MD, PhD, Shaojie Zhang, PhD, James M. Reuben, PhD, Bang-Ning Lee, PhD, and Janet S. Butel, PhD,Effects of radiation and latent virus on immune responses in a space flight model,2005 American Academy of Allergy, Asthma and Immunology, doi:10.1016/j.jaci.2005.03.003.
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