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Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

Background On long-duration spaceflight, most astronauts experience persistent immune dysregulation and the reactivation of latent herpesviruses, including varicella zoster virus (VZV). To understand the clinical risk of these perturbations to astronauts, we paralleled the immunology and virology work-up of astronauts to otherwise healthy terrestrial persons with acute herpes zoster. Methods Blood samples from 42 zoster patients – confirmed positive by PCR for VZV DNA in saliva (range from 100 to >285 million copies/mL) were analyzed for peripheral leukocyte distribution, T cell function, and plasma cytokine profiles via multi-parametric flow cytometry and multiplex bead-based immune-array assays. Patient findings were compared to normal value ranges specific for each assay that were defined in-house previously from healthy adult test subjects. Results Compared to the healthy adult ranges, the zoster patients possess (1) a higher proportion of constitutively activated T-cells, (2) a T-cell population skewed towards a more experienced maturation state, (3) depressed general T-cell function, and (4) a higher concentration of 20 of 22 measured plasma cytokines. Discussion The pattern of immune dysregulation in zoster patients is similar to that of astronauts during spaceflight who shed VZV DNA in their saliva. Because future deep space exploration missions will be of an unprecedented duration, prolonged immune depression and chronic viral reactivation threaten to manifest overt disease in exploration class astronauts.

Over the course of a mission to the International Space Station (ISS) crew members are exposed to a number of stressors that can potentially alter the composition of their microbiomes and may have a negative impact on astronauts’ health. Here we investigated the impact of long-term space exploration on the microbiome of nine astronauts that spent six to twelve months in the ISS. We present evidence showing that the microbial communities of the gastrointestinal tract, skin, nose and tongue change during the space mission. The composition of the intestinal microbiota became more similar across astronauts in space, mostly due to a drop in the abundance of a few bacterial taxa, some of which were also correlated with changes in the cytokine profile of crewmembers. Alterations in the skin microbiome that might contribute to the high frequency of skin rashes/hypersensitivity episodes experienced by astronauts in space were also observed. The results from this study demonstrate that the composition of the astronauts’ microbiome is altered during space travel. The impact of those changes on crew health warrants further investigation before humans embark on long-duration voyages into outer space.

Background Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella, Megasphera, and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria, Absconditabacteria, and Abiotrophia and a negative correlation between Oribacterium, Veillonella, and Haemophilus. There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions.

The International Space Station (ISS) has continued to evolve from an operational perspective and multiple studies have monitored both stress and the immune system of ISS astronauts. Alterations were ascribed to a potentially synergistic array of factors, including microgravity, radiation, psychological stress, and circadian misalignment. Comparing similar data across 12 years of ISS construction and operations, we report that immunity, stress, and the reactivation of latent herpesviruses have all improved in ISS astronauts. Major physiological improvements seem to have initiated approximately 2012, a period coinciding with improvements onboard ISS including cargo delivery and resupply frequency, personal communication, exercise equipment and protocols, food quality and variety, nutritional supplementation, and schedule management. We conclude that spaceflight associated immune dysregulation has been positively influenced by operational improvements and biomedical countermeasures onboard ISS. Although an operational challenge, agencies should therefore incorporate, within vehicle design limitations, these dietary, operational, and stress-relieving countermeasures into deep space mission planning. Specific countermeasures that have benefited astronauts could serve as a therapy augment for terrestrial acquired immunodeficiency patients.

Long duration spaceflights are associated with profound dysregulation of the immune system and latent viral reactivations. However, little is known on the impact of long duration spaceflight on innate immunity which raises concerns on crewmembers' ability to fight infections during a mission. The aim of this study was to determine the effects of spaceflight on plasma antimicrobial proteins (AMPs) and how these changes impact latent herpesvirus reactivations. Plasma, saliva and urine samples were obtained from 23 crewmembers before, during and after a 6-month mission on the International Space Station (ISS). Plasma AMP concentrations were determined by ELISA, and saliva Epstein-Barr virus (EBV) and varicella zoster virus (VZV) and urine cytomegalovirus (CMV) DNA levels were quantified by Real-Time PCR. There was a non-significant increase in plasma HNP1-3 and LL-37 during the early and middle stages of the missions, which was significantly associated with changes in viral DNA during and after spaceflight. Plasma HNP1-3 and Lysozyme increased at the late mission stages in astronauts who had exhibited EBV and VZV reactivations during the early flight stages. Following return to Earth and during recovery, HNP1-3 and lysozyme concentrations were associated with EBV and VZV viral DNA levels, reducing the magnitude of viral reactivation. Reductions in plasma LL-37 upon return were associated with greater CMV reactivation. This study shows that biomarkers of innate immunity appeared to be partially restored after 6-months in space and suggests that following adaptation to the space environment, plasma HNP1-3 and lysozyme facilitate the control of EBV and VZV reactivation rate and magnitude in space and upon return on earth. However, the landing-associated decline in plasma LL-37 may enhance the rate of CMV reactivation in astronauts following spaceflight, potentially compromising crewmember health after landing.

This pilot study compared placental growth factor (PIGF) levels in populations with high versus low risk for cardiovascular disease. Previous experiments from our laboratory (Sundaresan et al. 2005, 2009) revealed that the angiogenic factor PIGF was up regulated in modeled microgravity conditions in human lymphocytes leading to possible atherogenesis and pathogenesis in microgravity. Since the findings came from microgravity analog experiments, there is a strong link to its usefulness in the microgravity field as a biomarker. It is important to understand, that these findings came from both studies on expression levels of this cardiovascular marker in human lymphocytes in microgravity (in vitro microgravity analog), and a follow up gene expression study in hind limb suspended mice (in vivo microgravity analog). The relevance is enhanced because in life on earth, PIGF is an inflammatory biomarker for cardiovascular disease. Studies on the levels of PIGF would help to reduce the risk and prevention of heart failures in astronauts. If we can use this marker to predict and reduce the risk of cardiac events in astronauts and pilots, it would significantly help aerospace medicine operations. The investigations here confirmed that in a cardiovascular stressed population such as coronary artery disease (CAD) and acute coronary syndrome (ACS) patients, PIGF could be overexpressed. We desired to re-evaluate this marker in patients with cardiovascular disease in our own study. PIGF is a marker of inflammation and a predictor of short-term and long-term adverse outcome in ACS. In addition, elevated PIGF levels may be associated with increased risk for CAD.PIGF levels were determined in thirty-one patients undergoing cardiovascular catheterization for reasons other than ACS and in thirty-three low-risk asymptomatic subjects. Additional data on traditional cardiovascular risk factors for both populations were also compiled and compared. We found that PIGF levels were significantly higher in the high-risk population as compared to low-risk population. Also we were able to ascertain that PIGF levels were inversely correlated with HDL-cholesterol but directly correlated with the triglyceride levels. With further validation, PIGF may prove a useful addition to the armamentarium of noninvasive biomarkers for cardiovascular disease including a new area of stressful physiological conditions such as microgravity.

As the international space community plans for manned missions to Mars, spaceflight-associated immune dysregulation has been identified as a potential risk to the health and safety of the flight crew. There is a need to determine whether salivary antimicrobial proteins, which act as a first line of innate immune defense against multiple pathogens, are altered in response to long-duration (>6 mo) missions. We collected 7 consecutive days of whole and sublingual saliva samples from eight International Space Station (ISS) crewmembers and seven ground-based control subjects at nine mission time points, ~180 and ~60 days before launch (L−180/L−60), on orbit at flight days ~10 and ~90 (FD10/FD90) and ~1 day before return (R−1), and at R+0, R+18, R+33, and R+66 days after returning to Earth. We found that salivary secretory (s)IgA, lysozyme, LL-37, and the cortisol-to-dehydroepiandrosterone ratio were elevated in the ISS crew before (L−180) and during (FD10/FD90) the mission. “Rookie” crewmembers embarking on their first spaceflight mission had lower levels of salivary sIgA but increased levels of α-amylase, lysozyme, and LL-37 during and after the mission compared with the “veteran” crew who had previously flown. Latent herpesvirus reactivation was distinct to the ~6-mo mission crewmembers who performed extravehicular activity (“spacewalks”). Crewmembers who shed at least one latent virus had higher cortisol levels than those who did not shed. We conclude that long-duration spaceflight alters the concentration and/or secretion of several antimicrobial proteins in saliva, some of which are related to crewmember flight experience, biomarkers of stress, and latent viral reactivation. NEW & NOTEWORTHY Spaceflight-associated immune dysregulation may jeopardize future exploration-class missions. Salivary antimicrobial proteins act as a first line of innate immune defense. We report here that several of these proteins are elevated in astronauts during an International Space Station mission, particularly in those embarking on their first space voyage. Astronauts who shed a latent herpesvirus also had higher concentrations of salivary cortisol compared with those who did not shed. Stress-relieving countermeasures are needed to preserve immunity and prevent viral reactivation during prolonged voyages into deep space.

Brian Crucian,1 Adriana Babiak-Vazquez,2 Smith Johnston,1 Duane L Pierson,1 C Mark Ott,1 Clarence Sams1 1Biomedical Research and Environmental Sciences Division, NASA-Johnson Space Center, 2Epidemiology/Lifetime Surveillance of Astronaut Health, KBR-Wyle, Houston, TX, USA Background: The environment of spaceflight may elevate an astronaut’s clinical risk for specific diseases. The purpose of this study was to derive, as accurately as currently possible, an assessment of in-flight clinical “incidence” data, based on observed clinical symptoms in astronauts on board the International Space Station (ISS).Methods: Electronic medical records were examined from 46 long-duration ISS crew members, each serving approximately a 6-month mission on board the ISS, constituting 20.57 total flight years. Incidence for immunological-related adverse health events or relevant clinical symptoms was tabulated in a non-identifiable fashion. Event categories included infectious diseases, allergies, and rashes/hypersensitivities. A subsequent re-evaluation of more notable events, either of prolonged duration or unresponsive to treatment, was performed.Results: For the disease/symptom categories used in this evaluation, the ISS incidence rate was 3.40 events per flight year. Skin rashes were the most reported event (1.12/flight year) followed by upper respiratory symptoms (0.97/flight year) and various other (non-respiratory) infectious processes. During flight, 46% of crew members reported an event deemed “notable”. Among the notable events, 40% were classified as rashes/hypersensitivities. Characterization of on-orbit rashes manifested as redness with irritation, and could present on a variety of body locations.Conclusion: Based on reported symptoms, astronauts experience adverse medical events of varying severity during long-duration spaceflights. The data suggests caution, from both a vehicle design and biomedical countermeasures perspective, as space agencies plan for prolonged deep space exploration missions. Keywords: spaceflight, immunity, clinical incidence, astronauts, gravity, clinical risk, disease

Latent viral reactivation is a commonly reported manifestation of immune system dysregulation during spaceflight. As physical fitness and exercise training have been shown to benefit multiple arms of the immune system, we hypothesized that higher levels of preflight physical fitness and/or maintaining fitness during a mission would protect astronauts from latent viral reactivation. Standardized tests of maximal strength, muscular endurance, flexibility, and cardiorespiratory fitness (CRF) were performed in 22 international space station (ISS) crewmembers before and after a ~6-month mission. Reactivation of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) was determined in crewmembers and ground-based controls before, during, and after spaceflight. Crewmembers with higher CRF before spaceflight had a 29% reduced risk of latent viral reactivation compared to crew with lower CRF. Higher preflight upper body muscular endurance was associated with a 39% reduced risk of viral reactivation, a longer time to viral reactivation, and lower peak viral DNA concentrations, particularly for EBV and VZV. Latent viral reactivation rates were highest in crew with lower preflight CRF and higher levels of CRF deconditioning on return to Earth. We conclude that physical fitness may protect astronauts from latent viral reactivation during long duration spaceflight missions.

Latent herpes virus reactivation has been demonstrated in astronauts during shuttle (10-16 days) and International Space Station (≥180 days) flights. Following reactivation, viruses are shed in the body fluids of astronauts. Typically, shedding of viral DNA is asymptomatic in astronauts regardless of mission duration; however, in some cases, live/infectious virus was recovered by tissue culture that was associated with atopic-dermatitis or skin lesions during and after spaceflight. Hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes activation during spaceflight occurs as indicated by increased levels of stress hormones including cortisol, dehydroepiandrosterone, epinephrine, and norepinephrine. These changes, along with a decreased cell mediated immunity, contribute to the reactivation of latent herpes viruses in astronauts. Currently, 47/89 (53%) astronauts from shuttle-flights and 14/23 (61%) astronauts from ISS missions shed one or more herpes viruses in saliva/urine samples. Astronauts shed Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and herpes-simplex-1 (HSV-1) in saliva and cytomegalovirus (CMV) in urine. Larger quantities and increased frequencies for these viruses were found during spaceflight as compared to before or after flight samples and their matched healthy controls. The shedding did not abate during the longer ISS missions, but rather increased in frequency and amplitude. These findings coincided with the immune system dysregulation observed in astronauts from shuttle and ISS missions. VZV shedding increased from 41% in space shuttle to 65% in ISS missions, EBV increased 82 to 96%, and CMV increased 47 to 61%. In addition, VZV/CMV shed ≤30 days after ISS in contrast to shuttle where VZV/CMV shed up to 5 and 3 days after flight respectively. Continued shedding of infectious-virus post-flight may pose a potential risk for crew who may encounter newborn infants, sero-negative adults or any immunocompromised individuals on Earth. Therefore, developing spaceflight countermeasures to prevent viral reactivation is essential. Our spaceflight-developed technologies for saliva collection/rapid viral detection have been extended to include clinical applications including zoster patients, chicken pox, post-herpetic neuralgia, multiple sclerosis, and various neurological disorders. These protocols are employed in various clinics and hospitals including the CDC and Columbia University in New York, as well as overseas in Switzerland and Israel.

Stress factors experienced during space include microgravity, sleep deprivation, radiation, isolation, and microbial contamination, all of which can promote immune suppression (1, 2). Under these conditions, the risk of infection from opportunistic pathogens increases significantly, particularly during long-term missions (3). If infection occurs, it is important that the infectious agent should not be antibiotic resistant. Minimizing the occurrence of antibiotic resistance is, therefore, highly desirable. To facilitate this, it is important to better understand the long-term response of bacteria to the microgravity environment. This study demonstrated that the use of antibiotics as a preventive measure could be counterproductive and would likely result in persistent resistance to that antibiotic. In addition, unintended resistance to other antimicrobials might also occur as well as permanent genome changes that might have other unanticipated and undesirable consequences., The long-term response of microbial communities to the microgravity environment of space is not yet fully understood. Of special interest is the possibility that members of these communities may acquire antibiotic resistance. In this study, Escherichia coli cells were grown under low-shear modeled microgravity (LSMMG) conditions for over 1,000 generations (1000G) using chloramphenicol treatment between cycles to prevent contamination. The results were compared with data from an earlier control study done under identical conditions using steam sterilization between cycles rather than chloramphenicol. The sensitivity of the final 1000G-adapted strain to a variety of antibiotics was determined using Vitek analysis. In addition to resistance to chloramphenicol, the adapted strain acquired resistance to cefalotin, cefuroxime, cefuroxime axetil, cefoxitin, and tetracycline. In fact, the resistance to chloramphenicol and cefalotin persisted for over 110 generations despite the removal of both LSMMG conditions and trace antibiotic exposure. Genome sequencing of the adapted strain revealed 22 major changes, including 3 transposon-mediated rearrangements (TMRs). Two TMRs disrupted coding genes (involved in bacterial adhesion), while the third resulted in the deletion of an entire segment (14,314 bp) of the genome, which includes 14 genes involved with motility and chemotaxis. These results are in stark contrast with data from our earlier control study in which cells grown under the identical conditions without antibiotic exposure never acquired antibiotic resistance. Overall, LSMMG does not appear to alter the antibiotic stress resistance seen in microbial ecosystems not exposed to microgravity.

While no illnesses or infections have been linked to altered immune response due to space flight, it is apparent that changes in immunity can potentially impact the crew during space flight. Neither the extent of space flight-induced immunological changes nor their consequences to the crew can be fully determined at the present time. Factors such as the physical and psychological stressors associated with space flight appear to be the major contributors to the observed immune alterations, although direct effects of weihtlessness on cells of the immune system or their regulation cannot be ruled out. The complex interplay of these immune changes with the unique environmental challenges present in the spacecraft must be fully understood to minimize the impacts on flight operations. Much more study of immune factors during space flight and the fine balance between immune regulation and clinical response in healthy normal individuals will be required to make realistic projections of crewmember risks during and after extended space flight. The ground-based study of analog populations and correlation of immune dysregulation and clinical events will shed more light on this scenario. This information will provide the basis for mission planners and flight surgeons to design systems that minimize the potential for adverse events and increase productivity, comfort, and safety of the crew during flight and upon return to Earth.

Maintaining astronaut health during space travel is paramount for further human exploration of the solar system beyond Earth’s orbit. Of concern are potential dysregulations in immunity, which could increase the likelihood of cancer and latent viral reactivation. Natural killer (NK) cells are critical effectors of the innate immune system, and their function and phenotype are important to immunosurveillance of nascent tumors and latent viral infections. We compared changes in NK cell phenotype and function in eight crew members who completed an ~6-mo mission to the International Space Station (ISS) with healthy controls who remained on Earth. Assessments were made before (180 and 60 days before launch), during [flight day + 90 days (FD+90) and 1 day before return (R−1)], and after the mission (at R+0, R+18, R+33, and R+66). These samples, plus an additional in-flight sample (FD+180), were collected from a crew member who spent 340 days (~1 yr) on the ISS. NK cell cytotoxic activity (NKCA) against K562 leukemia targets in vitro was reduced by ~50% at FD+90 in ISS crew but not controls. This decrease was more pronounced in “rookie” compared with “veteran” crew members. The ~1-yr mission crew member did not show declines in NKCA against K562 until late in the mission (R−1 and R+0). NK cell numbers, expression of activating and inhibitory receptors, target cell binding, and expression and degranulation of perforin and granzyme B were unaltered with spaceflight. Similarly, when we exposed an immortalized NK cell line (NK-92) to sera collected at different mission time points (before, during, and after flight), there was no effect on NKCA. This is the first study to report impaired NK cell function during long-duration space travel. Countermeasures may be needed to mitigate immune system impairment in exploration class mission crew during long-duration spaceflight missions. NEW & NOTEWORTHY Immune system impairment may inhibit future human space exploration missions to Mars. Natural killer (NK) cells are key components of immunity and vital for tumor surveillance and the prevention of latent virus reactivation. We report that NK cell function is impaired in astronauts during an ~6-mo orbital space mission compared with preflight levels and ground-based controls. Declines in NK cell function were more marked in first-time “rookie” fliers. Countermeasures are needed to preserve NK cell-mediated immunity during spaceflight.

Salivary antimicrobial proteins (sAMPs) protect the upper respiratory tract (URTI) from invading microorganisms and have been linked with URTI infection risk in athletes. While high training volume is associated with increased URTI risk, it is not known if fitness affects the sAMP response to acute exercise. This study compared the sAMP responses to various exercising workloads of highly fit experienced cyclists with those who were less fit. Seventeen experienced cyclists (nine highly fit; eight less fit) completed three 30-min exercise trials at workloads corresponding to −5, +5 and +15 % of the individual blood lactate threshold. Saliva samples were collected pre- and post-exercise to determine the concentration and secretion of α-amylase, human neutrophil proteins 1–3 (HNP1–3) lactoferrin, LL-37, lysozyme, and salivary SIgA. The concentration and/or secretion of all sAMPs increased post-exercise, but only α-amylase was sensitive to exercise workload. Highly fit cyclists had lower baseline concentrations of α-amylase, HNP1–3, and lactoferrin, although secretion rates did not differ between the groups. Highly fit cyclists did, however, exhibit greater post-exercise increases in the concentration and/or secretion of a majority of measured sAMPs (percentage difference between highly fit and less fit in parentheses), including α-amylase concentration (+107 %) and secretion (+148 %), HNP1–3 concentration (+97 %) and secretion (+158 %), salivary SIgA concentration (+181 %), lactoferrin secretion (+209 %) and LL-37 secretion (+138 %). We show for the first time that fitness level is a major determinant of exercise-induced changes in sAMPs. This might be due to training-induced alterations in parasympathetic and sympathetic nervous system activation.

The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis - B. cereus - B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains).

Spacecraft crewmembers live and work in a closed environment that is monitored to ensure health and safety. Lessons learned from previous spaceflight missions have been incorporated into the design and development of the International Space Station (ISS). The microbial control actions on the ISS include engineering designs, such as high efficiency particulate air filtering of the air, microbial monitoring of the air, surfaces, and water, as well as remediation procedures when needed. This chapter will describe an overview of microbial risks of spaceflight focusing on measures to prevent infectious disease. The information discussed in this chapter is focused on the microbial monitoring activities in United States Operating Segment (USOS) of the ISS and experimental data obtained on USOS crewmembers.

Objectives Since the routine use of polymerase chain reaction testing (PCR) in diagnosing herpes infections, varicella-zoster virus is increasingly recognized as a cause of varicella-zoster meningoencephalitis (VZV ME) among immunocompetent patients. We were interested to determine whether patients with VZV ME had VZV DNA in their saliva during the acute phase of the illness. Materials and methods Forty-five consecutive patients who underwent a lumbar puncture for diagnostic purposes were included in the study. The cerebrospinal fluid was examined for the presence of VZV DNA by PCR, and patients with positive findings were treated with acyclovir. The saliva was later analyzed in a blinded fashion for the presence of VZV DNA. Results VZV DNA was found in saliva in four of five (80%) patients with PCR confirmed VZV ME (sensitivity 0.8, specificity 0.84, and likelihood ratio 5). This was significantly more than in patients with non-zoster viral ME (0%, P = 0.009), parainfectious headache (12%, P = 0.03) and controls (9.5%, P = 0.007). In immunocompromised patients with systemic lymphoma and AIDS, VZV DNA was present at a similar rate (67%, P = 0.6). Conclusions We have found VZV DNA in saliva of patients with PCR confirmed VZV ME at a higher proportion than in controls and patients with non-VZV viral ME. This finding might be of clinical importance, especially in immunocompetent individuals with suspected VZV ME where the results of genetic and immunological testing are not conclusive.

Recent data indicates that dysregulation of the immune system occurs and persists during spaceflight. Impairment of immunity, especially in conjunction with elevated radiation exposure and limited clinical care, may increase certain health risks during exploration-class deep space missions (i.e. to an asteroid or Mars). Research must thoroughly characterize immune dysregulation in astronauts to enable development of a monitoring strategy and validate any necessary countermeasures. Although the International Space Station affords an excellent platform for on-orbit research, access may be constrained by technical, logistical vehicle or funding limitations. Therefore, terrestrial spaceflight analogs will continue to serve as lower cost, easier access platforms to enable basic human physiology studies. Analog work can triage potential in-flight experiments and thus result in more focused on-orbit studies, enhancing overall research efficiency. Terrestrial space analogs generally replicate some of the physiological or psychological stress responses associated with spaceflight. These include the use of human test subjects in a laboratory setting (i.e. exercise, bed rest, confinement, circadian misalignment) and human remote deployment analogs (Antarctica winterover, undersea, etc.) that incorporate confinement, isolation, extreme environment, physiological mission stress and disrupted circadian rhythms. While bed rest has been used to examine the effects of physical deconditioning, radiation and microgravity may only be simulated in animal or microgravity cell culture (clinorotation) analogs. This article will characterize the array of terrestrial analogs for spaceflight immune dysregulation, the current evidence base for each, and interpret the analog catalog in the context of acute and chronic stress.

The ubiquitous nature of microbiology is reflected in the diversity of microbiological research and operational efforts at NASA. For example, the impact of microorganisms on other planets and the protection of Earth from the potential of microbial life elsewhere is the responsibility of the Office of Planetary Protection (http://planetaryprotection.nasa.gov/ about). While the Office of Planetary Protection does not include forward or back contamination to or from a low Earth orbit, research platforms, such as the ISS, are being used to better understand the survival of microorganisms and corresponding contamination control in the extreme conditions of space (2, 4, 10). Another example is the NASA Center for Astrobiology, which focuses on the origin, evolution, distribution, and future of life in the universe (https:// astrobiology.nasa.gov/). The large focus on microbiology is within its human exploration operations. NASA has historically set microbiological requirements, including stringent monitoring regimes, to mitigate risks to the health and performance of astronauts. Microorganisms can have both positive and negative impacts on many aspects of human spaceflight, including the risk and prevention of infectious diseases, performance of Environmental Control and Life Support Systems (ECLSS), spaceflight foods, and vehicle design and integrity. Even though a great amount of information has been obtained, several key questions regarding the impact of microorganisms on human spaceflight still remain. Research into the uncertainties of risks that may affect crew health are the responsibility of the NASA Human Research Program (http://www.nasa.gov/exploration/human research/). The research that addresses our fundamental understanding of space life science and its translation for benefits to the general public on Earth is the responsibility of NASA Space Biology.

High-efficiency particulate air (HEPA) filtration was first developed by the U.S. Atomic Energy Commission during the World War II era to capture microscopic radioactive particles that existing fil...

Epstein-Barr virus (EBV) is the causative agent of mononucleosis and is also associated with several malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, among others. EBV reactivates during spaceflight, with EBV shedding in saliva increasing to levels ten times those observed pre-and post-flight. Although stress has been shown to increase reactivation of EBV, other factors such as radiation and microgravity have been hypothesized to contribute to reactivation in space. We used a modeled spaceflight environment to evaluate the influence of radiation and microgravity on EBV reactivation. BJAB (EBV-negative) and Raji (EBV-positive) cell lines were assessed for viability/apoptosis, viral antigen and reactive oxygen species expression, and DNA damage and repair. EBV-infected cells did not experience decreased viability and increased apoptosis due to modeled spaceflight, whereas an EBV-negative cell line did, suggesting that EBV infection provided protection against apoptosis and cell death. Radiation was the major contributor to EBV ZEBRA upregulation. Combining modeled microgravity and radiation increased DNA damage and reactive oxygen species while modeled microgravity alone decreased DNA repair in Raji cells. Additionally, EBV-infected cells had increased DNA damage compared to EBV-negative cells. Since EBV-infected cells do not undergo apoptosis as readily as uninfected cells, it is possible that virus-infected cells in EBV seropositive individuals may have an increased risk to accumulate DNA damage during spaceflight. More studies are warranted to investigate this possibility.

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts.

Microorganisms impact spaceflight in a variety of ways. They play a positive role in biological systems, such as waste water treatment but can be problematic through buildups of biofilms that can affect advanced life support. Of special concern is the possibility that during extended missions, the microgravity environment will provide positive selection for undesirable genomic changes. Such changes could affect microbial antibiotic sensitivity and possibly pathogenicity. To evaluate this possibility, Escherichia coli (lac plus) cells were grown for over 1000 generations on Luria Broth medium under low-shear modeled microgravity conditions in a high aspect rotating vessel. This is the first study of its kind to grow bacteria for multiple generations over an extended period under low-shear modeled microgravity. Comparisons were made to a non-adaptive control strain using growth competitions. After 1000 generations, the final low-shear modeled microgravity-adapted strain readily outcompeted the unadapted lac minus strain. A portion of this advantage was maintained when the low-shear modeled microgravity strain was first grown in a shake flask environment for 10, 20, or 30 generations of growth. Genomic sequencing of the 1000 generation strain revealed 16 mutations. Of the five changes affecting codons, none were neutral. It is not clear how significant these mutations are as individual changes or as a group. It is concluded that part of the long-term adaptation to low-shear modeled microgravity is likely genomic. The strain was monitored for acquisition of antibiotic resistance by VITEK analysis throughout the adaptation period. Despite the evidence of genomic adaptation, resistance to a variety of antibiotics was never observed., Evolution: Bacteria gain advantageous mutations under simulated microgravity Bacteria grown for an extended period of time under simulated microgravity adopt growth advantages. George Fox and colleagues from the University of Houston, Texas, USA, cultured Escherichia coli bacteria for 1000 generations in a high aspect rotating vessel to simulate the low fluid shear microgravity environment encountered during spaceflight. They then performed growth competition assays and found that the 1000-generation adapted bacteria outcompeted control bacteria grown without simulated microgravity. Genomic sequencing of the adapted bacteria revealed 16 mutations, five of which altered protein sequences. These DNA changes likely explain the growth advantage of the bacteria grown for multiple generations in simulated microgravity. Similar adaptations during prolonged space missions could result in nastier pathogens that might threaten the health of astronauts. Fortunately, the microbes did not appear to acquire antibiotic resistance over the 1000 generation in the modeled microgravity culture.

Varicella zoster virus (VZV) in saliva from 6 herpes zoster patients and 1 chickenpox patient was found to be exclusively associated with epithelial cells by confocal microscopy. VZV localization with antibody specific to the VZV glycoprotein E was detected primarily on the membrane but was also inside the cell. Epithelial cells with VZV were still present in saliva in 1 out of two tested zoster patients after 10 months of recovery. Saliva from healthy controls (non-shingles patients, n = 5) did not show any sign of VZV by polymerase chain reaction or by confocal microscopy. No VZV was found in the liquid fraction of saliva. Further work is required to understand the movement of VZV in the saliva cells of infected patients. This article is protected by copyright. All rights reserved

How microorganisms function in the microgravity environment associated with spaceflight had been an open question for 50 years. A variety of unique microbial responses have been observed in experiments throughout the spaceflight program; however, the impact of these responses on medical operations was never thoroughly investigated. Answers were slow coming for several reasons, including a low priority for microbial studies because of the perception that microbes have relatively small impact on short duration spaceflights on the Space Shuttle. However, as the goals of the National Aeronautics and Space Administration (NASA) have shifted to much longer exploration missions, the potential impact of microbes to human health and ultimately long term habitation in space became evident. Longer stays in space resulted in the realization of emerging microbial-based problems associated with human occupation of the relatively small, closed environments of spacecraft/habitats.

Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight.

Post-flight data suggests immunity is dysregulated immediately following spaceflight, however this data may be influenced by the stress effects of high-G entry and readaptation to terrestrial gravity. It is unknown if immunity is altered during spaceflight. Blood samples were collected from 19 US Astronauts onboard the Space Shuttle ~24 h prior to landing and returned for terrestrial analysis. Assays consisted of leukocyte distribution, T cell blastogenesis and cytokine production profiles. Most bulk leukocyte subsets (WBC, differential, lymphocyte subsets) were unaltered during spaceflight, but were altered following landing. CD8+ T cell subsets, including cytotoxic, central memory and senescent were altered during spaceflight. T cell early blastogenesis varied by culture mitogen. Functional responses to staphylococcal enterotoxin were reduced during and following spaceflight, whereas response to anti-CD3/28 antibodies was elevated post-flight. The level of virus specific T cells were generally unaltered, however virus specific T cell function was depressed both during and following flight. Plasma levels of IFNα, IFNγ, IL-1β, IL-4, IL-10, IL-12, and TNFα were significantly elevated in-flight, while IL-6 was significantly elevated at R + 0. Cytokine production profiles following mitogenic stimulation were significantly altered both during, and following spaceflight. Specifically, production of IFNγ, IL-17 and IL-10 were reduced, but production of TNFα and IL-8 were elevated during spaceflight. This study indicates that specific parameters among leukocyte distribution, T cell function and cytokine production profiles are altered during flight. These findings distinguish in-flight dysregulation from stress-related alterations observed immediately following landing.

In a typical microbiological or biomedical laboratory, potentially harmful biological hazards are contained by laboratory practices and techniques, safety equipment (primary barriers and personal protective equipment), and facility design. Despite the unique and restrictive environment in space, sound biosafety principles and practices that are commonly used on the ground, such as adequate medical surveillance, thorough risk assessment, sufficient level of containment, effective remedial controls, and the use of proper personal protective equipment, can still be applied in space to mitigate the potential risks that biohazardous materials present to the astronauts and to the internal environment of the International Space Station (ISS).

Introduction Immune system dysregulation has been demonstrated to occur during and immediately following spaceflight. As the initial bias and magnitude for an immune response is heavily influenced by monocyte/macrophage secreted cytokines, this study investigated monocyte phenotype and cytokine production patterns following short-duration spaceflight. Methods Secreted cytokine profiles were examined by cytometric bead array analysis of culture supernatants following whole blood culture activation with LPS or PMA+ionomycin. Nine short-duration Space Shuttle crewmembers participated in this study. Results Peripheral monocyte percentages were unaltered postflight. Constitutive monocyte expression of both CD62L and HLA-DR was reduced following spaceflight in a mission-specific fashion. Loss of either molecule indicates a functional disability of monocytes, either by inhibition of adhesion and tissue migration (CD62L) or by impaired antigen presentation (HLA-DR). Following LPS stimulation of monocytes, postflight expression of IL-6, TNFalpha, and IL-10 were significantly reduced (by 43%, 44%, and 41%, respectively) and expression of IL-1b was elevated (65%). IL-8 production was either elevated or reduced in a mission-specific fashion. Following PMA+ionomycin stimulation of all leukocyte populations, only expression of IL-6 was significantly reduced postflight. Discussion These data indicate that changes in monocyte constitutive phenotype and inflammatory cytokine production occur following short-duration spaceflight, which may impact overall crewmember immunocompetence. Also, monocyte/macrophage function may be highly sensitive to mission specific parameters.

INTRODUCTION: Short-term spaceflight is associated with significant but reversible immunological alterations. However, little information exists on the effects of long-duration spaceflight on neuroimmune responses. METHODS: We collected multiple pre- and postflight samples from Shuttle and International Space Station (ISS) crewmembers in order to compare adrenocortical and immune responses between short- (approximately 11 d) and long-duration (approximately 180 d) spaceflight. RESULTS: In Shuttle crewmembers, increased stress hormone levels and altered leukocyte subsets were observed prior to launch and at landing. Additionally, typical stress-induced shifts in leukocyte and lymphocyte subsets, as well as the percentage of T-cells capable of producing intracellular IFN-gamma were also decreased just before launch and immediately after landing. Plasma IL-10 levels were increased before launch but not postflight. No preflight changes occurred in ISS crewmembers, but long-duration crewmembers exhibited significantly greater spikes in both plasma and urinary cortisol at landing as compared to Shuttle crewmembers. The percentage of T-cells capable of producing intracellular IFN-gamma was decreased in ISS crewmembers. Plasma IL-10 was increased postflight. Unexpectedly, stress-induced shifts in lymphocyte subpopulations were absent after long-duration flights despite significantly increased stress hormones at landing. CONCLUSION: Our results demonstrate significant differences in neuroimmune responses between astronauts flying on short-duration Shuttle missions versus long-duration ISS missions, and they agree with prior studies demonstrating the importance of mission duration in the magnitude of these changes.

Maintaining vitamin D status without sunlight exposure is difficult without supplementation. This study was designed to better understand interrelationships between periodic vitamin D supplementation and immune function in Antarctic workers. The effect of 2 oral dosing regimens of vitamin D supplementation on vitamin D status and markers of immune function was evaluated in people in Antarctica with no UV light exposure for 6 mo. Participants were given a 2000-IU (50 μg) daily (n = 15) or 10,000-IU (250 μg) weekly (n = 14) vitamin D supplement for 6 mo during a winter in Antarctica. Biological samples were collected at baseline and at 3 and 6 mo. Vitamin D intake, markers of vitamin D and bone metabolism, and latent virus reactivation were determined. After 6 mo, the serum 25-hydroxyvitamin D concentration (mean ± SD) increased from 56 ± 17 to 79 ± 16 nmol/L and from 52 ± 10 to 69 ± 9 nmol/L in the 2000-IU/d and 10,000-IU/wk groups, respectively (main effect over time, P < 0.001). Participants with a greater BMI (participant BMI range = 19–43 g/m2) had a smaller increase in 25-hydroxyvitamin D after 6-mo supplementation (P < 0.05). Participants with high serum cortisol and higher serum 25-hydroxyvitamin D were less likely to shed Epstein-Barr virus in saliva (P < 0.05). The doses given raised vitamin D status in participants not exposed to sunlight for 6 mo, and the efficacy was influenced by baseline vitamin D status and BMI. The data also provide evidence that vitamin D, interacting with stress, can reduce risk of latent virus reactivation during the winter in Antarctica.

Among the many stressors astronauts are exposed to during spaceflight, cosmic radiation may lead to various serious health effects. Specifically, space radiation may contribute to decreased immunity, which has been documented in astronauts during short- and long-duration missions, as evidenced by several changes in cellular immunity and plasma cytokine levels. Reactivation of latent herpes viruses, either directly from radiation of latently infected cells and/or from perturbation of the immune system, may result in disease in astronauts. Epstein‒Barr virus (EBV) is one of the eight human herpes viruses known to infect more than 90% of human adults and persists for the life of the host without normally causing adverse effects. Reactivation of several latent viruses in astronauts is well documented, although the mechanism of reactivation is not well understood. We studied the effect of four different types of radiation, (1) 137Cs gamma rays, (2) 150-MeV protons, (3) 600 MeV/n carbon ions, and (4) 600 MeV/n iron ions on the activation of lytic gene transcription and of reactivation of EBV in a latently infected cell line (Akata) at doses of 0.1, 0.5, 1.0, and 2.0 Gy. The data showed that for all doses used in this study, lytic gene transcription was induced and median viral loads were significantly higher for all types of radiation than in corresponding control samples, with the increases detected as early as four days post-exposure and generally tapering off at later time points. The viability and size of EBV-infected Akata cells were highly variable and exhibited approximately the same trend in time for all radiation types at 0.1, 0.5, 1.0, and 2.0 Gy. This work shows that reactivation of viruses can occur due to the effect of different types of radiation on latently infected cells in the absence of changes or cytokines produced in the immune system. In general, gamma rays are more effective than protons, carbon ions, and iron ions in inducing latent virus reactivation, though these high-energy particles did induce more sustained and later reactivation of EBV lytic gene transcription. These findings also challenge the common relative biological effectiveness concept that is often used in radiobiology for other end points.

All neurological and ocular complications of varicella zoster virus (VZV) reactivation can occur without rash. Virological verification requires detection of VZV DNA or anti-VZV IgG antibody in cerebrospinal fluid (CSF), or anti-VZV IgM antibody in serum or CSF. If VZV were readily detected in other tissue in patients with neurological disease without rash and found to correlate with tests listed above, more invasive tests such as lumbar puncture might be obviated. Saliva is a potential source of VZV DNA. To study the potential diagnostic value of detecting VZV DNA in saliva from patients with neurological disease, saliva of healthy adults searched for VZV DNA. A single saliva sample obtained by passive drool was centrifuged at 16,000 × g for 20 mins. DNA was extracted from the supernatant and cell pellet and examined in triplicate for VZV DNA by real time PCR. A single random saliva sample from 80 healthy men and women aged 20–59 years revealed no VZV DNA (Table 1), but was uniformly positive for cell (GAPdH) DNA. Because VZV DNA was not found in a random saliva sample from 80 individuals 20–59 years-old, a VZV-positive sample during neurologic disease may have potential significance. Further studies will determine whether VZV DNA in saliva correlates with VZV DNA or anti-VZV antibody in CSF in patients with neurological disease. TABLE 1 Absence of VZV DNA in saliva of healthy individuals aged 20–59 years Keywords: VZV, saliva, diagnosis, PCR INTRODUCTION Varicella zoster virus (VZV) is a ubiquitous neurotropic alphaherpesvirus. Primary infection usually produces varicella (chickenpox), after which virus becomes latent in ganglionic neurons along the entire neuraxis [Gilden et al., 1983; Kennedy et al., 1998; 1999]. Decades later, reactivation produces zoster (shingles) which may be further complicated by meningoencephalitis, myelopathy, vasculopathy and multiple ocular disorders. Importantly, these neurological and ocular complications of VZV reactivation can occur without rash [Gilden et al., 2010]. In such instances as well as in zoster sine herpete (radicular pain in the absence of rash), virological verification relies on detection of VZV DNA or anti-VZV IgG antibody in cerebrospinal fluid (CSF), or less often, the presence of VZV DNA in blood mononuclear cells or anti-VZV IgM antibody in serum or CSF. If VZV were readily detected in other tissue samples (e.g., saliva or tears) in patients with neurological disease in the absence of rash and shown to correlate with the standard tests listed above, more invasive tests such as lumbar puncture might be obviated. Saliva is a potential source of VZV DNA in such patients [Gershon 2011]. First, VZV DNA was present not only in saliva in all of 54 patients with acute herpes zoster involving the face, trunk, upper and lower extremities [Mehta et al., 2008], but also in saliva of patients with acute peripheral facial palsy without rash [Furuta et al., 2000; 2001; 2005], indicative of geniculate zoster (Ramsay Hunt syndrome) sine herpete. VZV DNA was also present in saliva up to 28 days after zoster immunization of people aged >60 years with no history of rash [Pierson et al., 2011]. Finally, the frequency of VZV DNA detection in saliva samples from individuals aged >60 years was significantly higher in all patients with a history of zoster than in age-matched subjects with no zoster history [Nagel et al., 2011].

Maintaining immune system integrity is of paramount importance for astronauts embarking on long-duration spaceflight missions. We have shown that Natural killer (NK) cell function is impaired during spaceflight; however, the mechanisms responsible for this effect are unknown. We exposed primary human blood NK cells to 12 h of simulated microgravity (SMG) using a rotating wall vessel zero gravity cell culture analog. Compared to static and vertical axis rotational controls, SMG (horizontal axis rotation) was found to decrease subsequent NK-cell cytotoxic function in 1G against allogeneic target cell lines of leukemia (K562), multiple myeloma (U266), B-lymphoma (721.221) and HLA-E transfected lymphoma (221.AEH) origin. Flow cytometric analysis revealed that SMG had little effect on the surface expression of a wide-range of NK-cell activating and inhibitory receptors, but did decrease intracellular perforin expression. Using cytometry by time of flight (CyTOF), SMG was found to lower NK-cell degranulation as determined by CD107a expression following co-incubation with K562 target cells in 1G. Moreover, SMG reduced NK-cell expression of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in response to K562 co-culture at 1G. These results indicate that microgravity may be involved in spaceflight-associated reductions in NK cell function. Specifically, pre-exposure to SMG appears to disrupt NK-cell killing by ‘disarming’ them of cytolytic granules and impairing their ability to secrete effector cytokines when confronted with tumor target cells in the 1G environment.

NF-κB is a transcriptional activator of many genes, including some that lead to muscle atrophy and bone resorption—significant concerns for astronauts. NF-κB activation is inhibited by eicosapentaenoic acid (EPA), but the influence of this omega-3 fatty acid on the effects of weightlessness are unknown. We report here cellular, ground analogue, and spaceflight findings. We investigated the effects of EPA on differentiation of RAW264.7 monocyte/macrophage cells induced by receptor activator of NF-κB ligand (RANKL) and on activation of NF-κB by tumor necrosis factor α (TNF-α) or exposure to modeled weightlessness. EPA (50 µM for 24 hours) inhibited RANKL-induced differentiation and decreased activation of NF-κB induced by 0.2 µg/mL of TNF-α for 30 minutes or by modeled weightlessness for 24 hours (p

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. These results reveal that many genes play previously unrecognized roles in multiple DNA repair responses, all of which are required for successful repair of IR-induced damage.

Astronauts live and work in relatively crowded, confined environments on the Space Shuttle and the International Space Station. They experience a unique set of stressors that contribute to a diminishment of many immune responses. This study investigated the ability of the shuttle crew members' monocytes to respond to gram-negative endotoxin that they could encounter during infections. Blood specimens were collected from 20 crew members and 15 control subjects 10 days before launch, 3 to 4 h after landing, and 15 days after landing and from crew members during their annual medical examination at 6 to 12 months after landing. When challenged with gram-negative endotoxin, the crew member's monocytes collected at all three time points produced lower levels of interleukin-6 (IL-6) and IL-1β and higher levels of IL-1ra and IL-8 compared to those of control subjects. Cytokines were assessed by measuring the number of cells positive for intracellular cytokines. These values returned to normal 6 to 12 months after landing, except for IL-1ra, which was still higher (five- to sixfold) than in controls. This phenomenon was accompanied by an increased expression of Toll-like receptor 4 and decreased expression of CD14 on the crew members' monocytes at all time points. There were also increased levels of the lipopolysaccharide binding protein in the plasma of the crew members 3 to 4 h and 15 days after landing. This study shows that spaceflight-associated factors (in-flight and preflight) modulate the response of monocytes to gram-negative endotoxins.