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While astronauts are returning from long duration spaceflight with multiple ocular signs that mimic those seen in terrestrial patients with elevated intracranial pressure (ICP), evidence has yet to prove a clinically significant increase in ICP during space.1 Preliminary research evidence may even suggest that ICP decreases in microgravity. Idiopathic intracranial hypertension (IIH) has long been considered the ideal terrestrial analogue to Spaceflight Associated Neuro-ocular Syndrome (SANS).1 However, there are several critical features of SANS that do not complement any reported case of IIH on Earth. These findings mandate a closer look at the accuracy of IIH as a terrestrial SANS analog.

The proposed technology uses advanced image analysis techniques to maximize the resolution and utility of medical imaging methods being used during spaceflight. We utilize COTS technology for medical imaging, but our applications require higher resolution assessment of the medical images than is routinely applied with nominal system software. By leveraging advanced data reduction and multidimensional imaging techniques utilized in analysis of Planetary Sciences and Cell Biology imaging, it is possible to significantly increase the information extracted from the onboard biomedical imaging systems. Year 1 focused on application of these techniques to the ocular images collected on ground test subjects and ISS crewmembers. Focus was on the choroidal vasculature and the structure of the optic disc. Methods allowed for increased resolution and quantitation of structural changes enabling detailed assessment of progression over time. These techniques enhance the monitoring and evaluation of crew vision issues during space flight.

Determining the effect of space travel on the human immune system has proven to be extremely challenging. Limited opportunities for in-flight studies, varying mission durations, technical and logistical obstacles, small subject numbers, and a broad range of potential assays have contributed to this problem. Additionally, the inherent complexity of the immune system, with its vast array of cell populations, sub-populations, diverse regulatory molecules, and broad interactions with other physiological systems, makes determining precise variables to measure very difficult. There is also the challenge of determining the clinical significance of any observed immune alterations. Will such a change lead to disease, or is it a transient subclinical observation related to short-term stress? The effect of this problem may be observed by scanning publications associated with immunity and spaceflight, which began to appear during the 1970s. Although individually they are each valid studies, the comprehensive literature to date suffers from widely varying sampling methods and assay techniques, low subject counts, and sometimes a disparate focus on narrow aspects of immunity. The most clinically relevant data are derived from in-flight human studies, which have demonstrated altered cell-mediated immunity and reactivation of latent herpes viruses. Much more data are available from post-flight testing of humans, with clear evidence of altered cytokine production patterns, altered leukocyte distribution, continued latent viral reactivation, and evidence of dramatically altered virus-specific immunity. It is unknown if post-flight assessments relate to the in-flight condition or are a response to landing stress and readaptation. In-flight culture of cells has clearly demonstrated that immune cells are gravity-sensitive and display altered functional characteristics. It is unknown if these data are related to in vivo immune cell function or are an artifact of microgravity culture. Ground analog testing of humans and animals, as well as microgravity-analog cell culture, has demonstrated utility. However, in all cases, it is not known with certainty if these data would reflect similar testing during space travel. Given their ready availability, ground analogs may be extremely useful for assay development and the evaluation of potential countermeasures. In general, the evidence base suffers from widely disparate studies on small numbers of subjects that do not directly correlate well with each other or spaceflight itself. Also lacking are investigations of the effect of gender on adaption to spaceflight. This results in significant knowledge 'gaps' that must be filled by future studies to completely determine any clinical risk related to immunity for human exploration-class space missions. These gaps include a significant lack of in-flight data, particularly during long-duration space missions. The International Space Station represents an excellent science platform with which to address this knowledge gap. Other knowledge gaps include lack of a single validated ground analog for the phenomenon and a lack of flight-compatible laboratory equipment capable of monitoring astronauts (for either clinical or research purposes). However, enough significant data exist, as described in this manuscript, to warrant addressing this phenomenon during the utilization phase of the ISS. A recent Space Shuttle investigation has confirmed the 31 in-flight nature of immune dysregulation, demonstrating that it is not merely a post-flight phenomenon. Several current studies are ongoing onboard the ISS that should thoroughly characterize the phenomenon. NASA recognizes that if spaceflight-associated immune dysregulation persists during exploration flights in conjunction with other dangers, such as high-energy radiation, the result may be a significant clinical risk. This emphasizes the need for a continued integrated comprehensive approach to determining the effect of prolonged spaceflight, separated from transient launch and landing stresses, on human immunity. After such studies, the phenomenon will be understood, and, hopefully, a monitoring strategy will have been developed that could be used to monitor the effectiveness of countermeasure

The Risk of Crew Adverse Health Event Due to Altered Immune Response is identified by the National Aeronautics and Space Administration (NASA) Human Research Program (HRP) as a recognized risk to human health and performance in space. The HRP Program Requirements Document (PRD) defines these risks. This Evidence Report provides a summary of the evidence that has been used to identify and characterize this risk. It is known that human immune function is altered in‐ and post‐flight, but it is unclear at present if such alterations lead to increased susceptibility to disease. Reactivation of latent viruses has been documented in crewmembers, although this reactivation has not been directly correlated with immune changes or with observed diseases. As described in this report, further research is required to better characterize the relationships between altered immune response and susceptibility to disease during and after spaceflight. This is particularly important for future deep‐space exploration missions.

Background: Aspects of immune system dysregulation associated with long‐duration spaceflight have yet to be fully characterized, and may represent a clinical risk to crewmembers during deep space missions. Plasma cytokine concentration may serve as an indicator of in vivo physiological changes or immune system mobilization. Methods: The plasma concentrations of 22 cytokines were monitored in 28 astronauts during long‐duration spaceflight onboard the International Space Station. Blood samples were collected three times before flight, 3‐5 times during flight (depending on mission duration), at landing and 30 days post‐landing. Analysis was performed by bead array immunoassay. Results: With few exceptions, minimal detectable mean plasma levels (<10 pg/ml) were observed at baseline (launch minus 180) for innate inflammatory cytokines or adaptive regulatory cytokines, however IL‐1ra and several chemokines were constitutively present. An increase in the plasma concentration IL‐8, IL‐1ra, Tpo, CCL4, CXCL5, TNF(alpha), GM‐CSF and VEGF was observed associated with spaceflight. Significant post‐flight increases were observed for IL‐6 and CCL2. No significant alterations were observed during or following spaceflight for adaptive/T‐regulatory cytokines (IL‐2, IFN(gamma), IL‐17, IL4, IL‐5, IL‐10). Conclusions: This pattern of cytokine dysregulation suggests multiple physiological adaptations persist during flight, including inflammation, leukocyte recruitment, angiogenesis and thrombocyte regulation.

Background: Concordia Station is located inside Antarctica about 1000km from the coast at an altitude of 3200m (Dome C). Hence, individuals living in this harsh environment are exposed to two major conditions: 1.) hypobaric hypoxia and 2.) confinement and extreme isolation. Both hypoxia and confinement can affect human immunity and health, and are likely to be present during exploration class space missions. This study focused on immune alterations measured by a new global immunity test assay, similar to the phased out delayed type hypersensitivity (DTH) skin test. Methods: After informed written consent 14 healthy male subjects were included to the CHOICE-study (Consequences-of-longterm-Confinement-and-Hypobaric-HypOxia-on-Immunity-in-the Antarctic-Concordia-Environment). Data collection occurred during two winter-over periods lasting each one year. During the first campaign 6 healthy male were enrolled followed by a second campaign with 8 healthy males. Blood was drawn monthly and incubated for 48h with various bacterial, viral and fungal antigens followed by an analysis of plasma cytokine levels (TNF-alpha, IL2, IFN-gamma, IL10). As a control, blood was incubated without stimulation ("resting condition"). Goals: The scope of this study was to assess the consequences of hypoxia and confinement on cellular immunity as assessed by a new in vitro DTH-like test. Results: Initial results indicate that under resting conditions the in vitro DTH-like test showed low cytokine levels which remained almost unchanged during the entire observation period. However, cytokine responses to viral, bacterial and fungal antigens were remarkably reduced at the first month after arrival at Concordia when compared to levels measured in Europe prior to departure for Antarctica. With incrementing months of confinement this depressed DTH-like response tended to reverse, and in fact to show an "overshooting" immune reaction after stimulation. Conclusion: The reduced in vitro DTH-like test response in the early phase of Antarctic wintering over con rms distinct immune suppressive effects seen after (sub-)acute hypobaric hypoxia. The reversal and overshooting reaction of cellular immune responses upon stimulation, but not the resting state, indicate either a) priming of immune answers and/or b) an uncoupled or disregulated control of cellular immune answers by auto-, para- and endocrine pathways. Further analyses and correlations are warranted. Acknowledgement: Supported by the European Space Agency (ESA), the French (IPEV) and Italian (PNRA) polar institutes, the German National Space Program (DLR, 50WB0719/WB0919), by BELSPO/PROEDEX/ESA (C90-380/-391), NASA and by the Concordia crews who have participated with great enthusiasm.

Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on Earth by shaking in glass vials, it cannot readily be performed this way in outer space without entraining air bubbles. The present device can be preloaded with the powder and diluent(s) in separate compartments. The powder and diluent( s) can be mixed, without introducing air bubbles, by removing the clip(s), then shaking. This use of the device could also be advantageous in terrestrial applications because it maintains the isolation of the constituents until the time of use.

Determine the in-flight status of immunity, physiological stress, viral immunity/reactivation. Specific measurements include leukocyte distribution, T cell function, cytokine production profiles (mRNA, intracellular, secreted, plasma), virus-specific T cell number/function, latent herpesvirus reactivation, stress hormone levels. Determine the clinical risk related to immune dysregulation for exploration class spaceflight, as well as an appropriate monitoring strategy for spaceflight-associated immune dysfunction, that could be used for the evaluation of countermeasures. Specific Study Objectives: Determine the nutritional status of astronauts before, during, and after spaceflight ensure adequate intake of energy, protein, and vitamins during missions. The Clinical Nutritional Status Assessment measures dietary intake, body composition, protein, bone, iron, mineral, vitamin, and antioxidant status (60 total analytes). Currently, it is a medical requirement for U.S. crewmembers on-board the ISS. The results of data analysis are used both to understand the connections between nutrition and human health during space flight, and to develop effective dietary strategies to reduce adverse health impacts (including bone loss, loss of important vitamins and minerals, and increased genetic damage from radiation).

Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of: (1) peripheral leukocyte distribution, (2) plasma cytokine levels and (3) cytokine production profiles following whole blood mitogenic stimulation

NASA is concerned with the health risks to astronauts, particularly those risks related to radiation exposure. Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of (A) peripheral leukocyte distribution; (B) plasma cytokine levels; (C) cytokine production profiles following whole blood stimulation of either T cells or monocytes.

Immune dysregulation has been demonstrated following spaceflight of varying durations and limited in-flight studies indicate this phenomenon may persist during spaceflight. Causes may include microgravity, physiological stress, isolation, confinement and disrupted circadian rhythms. To further investigate the mechanisms associated with flight-associated immune changes, murine splenocytes immune parameters were assessed following 14 day space flight on Space Shuttle mission STS-135.

Space flight is known to affect immune responses of astronauts and animals, decreasing lymphocytic responses to mitogenic stimuli, delayed typed hypersensitivity reactions, and T-cell activation. Despite changes in immune suppression, there are no reports of consistent adverse clinical events post flight. To further investigate the spectrum of affected immune responses, murine splenocytes were stimulated immediately post-shuttle flight (14 days on STS-135) with T-cell stimulators or toll-like receptor agonists. Comparisons were made to ground control splenocytes from age-matched mice. Cell phenotypes were assessed, as well as activation markers and associated cytokine production. The CD4+ population decreased with no concurrent decrease in CD8+ cells from shuttle mice post flight compared to ground controls. Regarding antigen presenting cell populations, the number of CD11c+ cells were slightly elevated post flight, compared to ground controls, with increased MHC Class I expression (I-A(sup b)) and no change in Class II expression (H-2K(sup b)). CD86+ populations were also significantly diminished. However, the decreased markers did not correlate with activity. Stimulation of splenocytes post flight showed significant increase in bead uptake, increased Class I expression, increased TNF-alpha and IL-6 production in response to TLR-2 (zymosan) and TLR-4 (LPS) agonists. While most activated (ConA or anti-CD3/anti-CD28) CD4+ cells showed markedly diminished responses (reduced IL-2 production), non-specific T cell responses to superantigen (SEA/SEB) increased post flight as determined by expression of early activation markers. Production of additional cytokines was also dysregulated postflight. Overall, persistent immune changes during space flight could represent unique clinical risks for exploration class missions. The consequences of pathogenic encounter remain an important concern that should be addressed.

Reduced T cell, granulocyte, NK and monocyte function have all been reported following both long and short duration spaceflight, however these data indicate crews are generally not experiencing inflammatory or adaptive immune activation during spaceflight. There appear to be varied individual crew responses, and specific relationships between cytokines and markers of iron status and muscle turnover that warrant further evaluation. Increases in growth factors and chemokines may indicate other types of adaptation occurring during spaceflight, such as attempts to overcome diminished immunocyte function.

This viewgraph presentation reviews the lessons learned from manned space flight on the International Space Station. The contents include: 1) Overview of space flight effects on crewmembers; 2) General overview of immune system; 3) How does space flight alter immune system? 4) What factors associated with space flight inteact with crewmember immune function and impact health risks? 5) What is the current understanding of space flight effects on the immune system? and 6) Why should NASA be interested in immunology? Why is it significant?

This viewgraph presentation describes the various flight investigations performed on the International Space Station as part of the NASA Human Research Program (HRP). The evaluations include: 1) Stability; 2) Periodic Fitness Evaluation with Oxygen Uptake Measurement; 3) Nutrition; 4) CCISS; 5) Sleep; 6) Braslet; 7) Integrated Immune; 8) Epstein Barr; 9) Biophosphonates; 10) Integrated cardiovascular; and 11) VO2 max.

NASA's Vision for Space Exploration centers on exploration class missions including the goals of returning to the moon and landing on Mars. One of NASA's objectives is to focus research on astronaut health and the development of countermeasures that will protect crewmembers during long duration voyages. Exposure to microgravity affects human physiology and results in changes in the urinary chemical composition favoring urinary supersaturation and an increased risk of stone formation. Nephrolithiasis is a multifactorial disease and development of a renal stone is significantly influenced by both dietary and environmental factors. Previous results from long duration Mir and short duration Shuttle missions have shown decreased urine volume, pH, and citrate levels and increased calcium. Citrate, an important inhibitor of calcium-containing stones, binds with urinary calcium reducing the amount of calcium available to form stones. Citrate inhibits renal stone recurrence by preventing crystal growth, aggregation, and nucleation and is one of the most common therapeutic agents used to prevent stone formation. Methods: Thirty long duration crewmembers (29 male, 1 female) participated in this study. 24-hour urines were collected and dietary monitoring was performed pre-, in-, and postflight. Crewmembers in the treatment group received two potassium citrate (KCIT) pills, 10 mEq/pill, ingested daily beginning 3 days before launch, all in-flight days and through 14 days postflight. Urinary biochemical and dietary analyses were completed. Results: KCIT treated subjects exhibited decreased urinary calcium excretion and maintained the levels of calcium oxalate supersaturation risk at their preflight levels. The increased urinary pH levels in these subjects reduced the risk of uric acid stones. Discussion: The current study investigated the use of potassium citrate as a countermeasure to minimize the risk of stone formation during ISS missions. Results suggest that supplementation with potassium citrate decreases the risk of stone formation during and immediately after spaceflight.

This viewgraph presentation describes the risks of renal stone formation in manned space flight. The contents include: 1) Risk; 2) Evidence; 3) Nephrolithiasis -A Multifactorial Disease; 4) Symptoms/signs; 5) Urolithiasis and Stone Passage; 6) Study Objectives; 7) Subjects; 8) Methods; 9) Investigation Results; 10) Potassium Citrate; 11) Calcium Balance; 12) Case Study; 13) Significant Findings; 14) Risk Mitigation Strategies and Recommended Actions; and 15) Future Potential.

A viewgraph presentation on biomedical data from International Space Station (ISS) Expeditions 1-12 is shown. The topics include: 1) ISS Expeditions 1-12; 2) Biomedical Data; 3) Physiological Assessments; 4) Bone Mineral Density; 5) Bone Mineral Density Recovery; 6) Orthostatic Tolerance; 7) Postural Stability Set of Sensory Organ Test 6; 8) Performance Assessment; 9) Aerobic Capacity of the Astronaut Corps; 10) Pre-flight Aerobic Fitness of ISS Astronauts; 11) In-flight and Post-flight Aerobic Capacity of the Astronaut Corps; and 12) ISS Functional Fitness Expeditions 1-12.

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-gamma) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS+CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

As logistical access for in-flight space research becomes more limited, the use of ground based spaceflight analogs for life science studies will increase. These studies are particularly important as NASA progresses towards the Lunar and eventually Mars missions outlined in the 2005 Vision for Space Exploration. Countermeasures must be developed to mitigate the clinical risks associated with exploration class space missions. In an effort to coordinate studies across multiple disciplines, NASA has selected 90-day bed rest as the analog of choice, and initiated the Flight Analogs Project to implement research studies with or without the evaluation of countermeasures. Although bed rest is not the analog of choice to evaluate spaceflight-associated immune dysfunction, a standard Immune Assessment was developed for subjects participating in the 90-day bed best studies. The Immune Assessment consists of: leukocyte subset distribution, T cell functional responses, intracellular cytokine production profiles, latent viral reactivation, virus specific T cell levels, virus specific T cell function, stress hormone levels and a behavioral assessment using stress questionnaires. The purpose of the assessment during the initial studies (without countermeasure) is to establish control data against which future studies (with countermeasure) will be evaluated. It is believed that some of the countermeasures planned to be evaluated in future studies, such as exercise, pharmacologic intervention or nutritional supplementation, have the ability to impact immune function. Therefore immunity will likely be monitored during those studies. The data generated during the first three control studies showed that the subjects in general did not display altered peripheral leukocyte subsets, constitutive immune activation, significant latent viral reactivation (EBV, VZV) or altered T cell function. Interestingly, for some subjects the level of constitutively activated T cells (CD8+/CD69+) and virus-specific T cells (CMV and EBV) both decreased during the studies. This likely reflects the isolation of the subjects (from an immunological perspective) and absence of everyday subclinical challenges to the immune system. Cortisol levels (plasma and saliva) did not vary significantly during the studies. This probably reflects a lack of physiological stress during the study and the stress of readaptation to the 1xG environment at R+1. These data demonstrate the absence of significant immune alteration during 90-day bed rest, and establish control data against which future studies (including countermeasures) may be compared.

Immune system dysregulation has been demonstrated to occur during spaceflight and has the potential to cause serious health risks to crewmembers participating in exploration-class missions. A comprehensive immune assessment was recently performed on 13 short duration Space Shuttle crewmembers and 8 long duration International Space Station (ISS) crewmembers. Statistically significant post-flight phenotype alterations (as compared to pre-flight baseline) for the Shuttle crewmembers included: granulocytosis, increased percentage of B cells, reduced percentage of NK cells, elevated CD4/CD8 ratio, elevated levels of memory CD4+ T cells, and a CD8+ T cell shift to a less differentiated state. For the Shuttle crewmembers, T cell function was surprisingly elevated post-flight, among both the CD4+ and CD8+ subsets. This is likely an acute stress response in less-deconditioned crewmembers. The percentage of CD4+/IL-2+, CD4+/IFNg+ and CD8+/IFNg+ T cells were all decreased at landing. Culture secreted IFNg production was significantly decreased at landing, whereas production of Th2 cytokines was largely unchanged. It was found that the IFNg:IL-10 ratio was obviously declined in the Shuttle crewmembers immediately post-flight. A similar pattern of alterations were observed for the long duration ISS crewmembers. In contrast to Shuttle crewmembers, the ISS crewmembers demonstrated a dramatic reduction in T cell function immediately post-flight. This may be related to the effect of acute landing stress in conjunction with prolonged deconditioning associated with extended flight. The reduction in IFNg:IL-10 ratio (Th2 shift) was also observed post-flight in the ISS crewmembers to a much higher degree. These data indicate consistent peripheral phenotype changes and altered cytokine production profiles occur following space travel of both short and long duration.

Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

Exposure to the microgravity environment results in many metabolic and physiological changes to humans. Body fluid volumes, electrolyte levels, and bone and muscle undergo changes as the human body adapts to the weightless environment. Changes in the urinary biochemistry occur as early as flight day 3-4 in the short duration Shuttle crewmembers. Significant decreases were observed both in fluid intake and urinary output. Other significant changes were observed in the urinary pH, calcium, potassium and uric acid levels. During Shuttle missions, the risk of calcium oxalate stone formation increased early in the flight, continued at elevated levels throughout the flight and remained in the increased risk range on landing day. The calcium phosphate risk was significantly increased early in-flight and remained significantly elevated throughout the remainder of the mission. Results from the long duration Shuttle-Mir missions followed a similar trend. Most long duration crewmembers demonstrated increased urinary calcium levels despite lower dietary calcium intake. Fluid intake and urine volumes were significantly lower during the flight than during the preflight. The calcium oxalate risk was increased relative to the preflight levels during the early in-flight period and continued in the elevated risk range for the remainder of the space flight and through two weeks postflight. Calcium phosphate risk for these long duration crewmembers increased during flight and remained in the increased risk range throughout the flight and following landing. The complexity, expense and visibility of the human space program require that every effort be made to protect the health of the crewmembers and ensure the success of the mission. Results from our early investigations clearly indicate that exposure to the microgravity environment of space significantly increases the risk of renal stone formation. The early studies have indicated specific avenues for development of countermeasures for the increased renal stone risk observed during and following space flight. Increased hydration and implementation of pharmacological countermeasures are being tested for their efficacy in mitigating the in-flight risk of renal stones. Maintaining the health and well-being of crewmembers during space flight requires a means of minimizing potential detrimental health effects of microgravity. The formation of a renal stone during flight obviously has severe consequences for the affected crewmember as well as the success of the mission.

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent functionally different monocyte subsets with distinct functions. Whole blood culture eliminates the need to purify cell populations prior to culture and may have significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. It is likely that the altered cytokine production observed following whole blood culture more accurately represents the in-vivo immune balance.

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent distinct monocyte subsets with unique functions. CONCLUSIONS: Whole blood culture eliminates the need to purify cell populations prior to culture and may have Significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. In this study, alterations in cytokine production are demonstrated between whole blood and PBMC activation. It is likely that whole blood activation more accurately represents the in-vivo immune balance than PBMC activation.

The present studies were designed to determine effects of a microgravity model system upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF-alpha) activity and indices of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1726+/-1 17,150 u IEU) from Wistar Furth rats were treated as: 1) HARV (High Aspect Ratio Vessel cell culture) , 2) HARV plus LPS, 3) static culture, 4) static culture plus LPS. TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (p<0.05). A decrease in insulin concentration was demonstrated in the LPS stimulated HARV culture (p<0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. While nitrogenous compound analysis indicated a ubiquitous reliance upon glutamine in all experimental groups, arginine was converted to ornithine at a two-fold greater rate in the islets cultured in the HARV microgravity model system (p<0.05). These studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV. These alterations in fuel homeostasis may be promulgated by gravity averaged cell culture methods or by three dimensional cell assembly.

An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

Space flight has been shown to induce changes in the physiology of crewmembers which can adversely affect their performance. The objectives of Countermeasures Evaluation and Validation Project (CEVP) are to develop and validate potential countermeasures to the deleterious effects of space flight for operational implementation and use. In order to accomplish this objective, a group of standardized tests, the Integrated Testing Regimen or ITR, will be utilized for the evaluation of candidate countermeasures. Additionally, the ITR will illuminate intersystem effects of the potential countermeasures and provide normative values for the physiological responses. Due to the small number of crewmembers available as test subjects, statistically rigorous validation of countermeasures presents a challenging problem. Strategies for reliable evaluation of small N clinical studies will be utilized for these activities. These approaches will permit effective analysis of promising countermeasure to support human space flights of increasing duration.

The present studies were designed to determine effects of microgravity upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF - alpha) activity and indices of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1726+/-117,150 u IEU) from Wistar Furth rats were treated as: 1) HARV (High Aspect Ratio Vessel cell culture) , 2) HARV plus LPS 3) static culture, 4) static culture plus LPS TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (p<0.05). A decrease in insulin concentration was demonstrated in the LPS stimulated HARV culture (p<0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. While nitrogenous compound analysis indicated a ubiquitous reliance upon glutamine in all experimental groups, arginine was converted to ornithine at a two-fold greater rate in the islets cultured in the HARV microgravity paradigm (p<0.05). These studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV paradigm. These alterations in fuel homeostasis may be promulgated by gravity averaged cell culture methods or by three dimensional cell assembly.

The changes in immune function which occur during space flight potentially expose the crews to an increased risk for development of illness. Decreased cellular immune function has been repeatedly documented after space flight and confirmed during flight by in vivo delayed-type hypersensitivity testing. However, correlation of immune changes with a clinically significant risk factor has not yet been performed. Our hypothesis is that space flight induces a decrease in cell-mediated immune function accompanied by a shift from a type 1 cytokine pattern (favoring cell-mediated immunity) to a type 2 cytokine pattern (favoring humoral immunity). We further hypothesize that reactivation of latent viruses will occur during space flight in association with the decreased cellular immunity. To test these hypotheses, we will determine the effects of space flight on cell-mediated immunity and viral reactivation. We will utilize delayed-type hypersensitivity testing as an in vivo measure of integrated cell-mediated immune function. The production of cytokines and immunoregulatory factors by lymphocytes and monocytes will be measured to determine whether changes in cytokine patterns are associated with the space flight-induced immune dysregulation. Correlation of antigen-specific immune changes with reactivation of latent herpes viruses will be determined by measuring peripheral levels of viral (CMV, VZV, EBV) antigen-specific T cells and comparing to the levels of EBV-infected B-cells by fluorescence in situ hybridization and flow cytometry. A comparison of cell-mediated immune function, cytokine regulation and viral reactivation will provide new insights into crew member health risks during flight.

Exposure to microgravity during space flight results in profound physiologic changes. Numerous studies have shown changes in circulating populations of peripheral blood immune cells immediately after space flight. It is currently unknown if these changes result from exposure to microgravity or are caused by the stress of reentry and readaptation to gravity. We have developed the whole blood staining device as a system for the staining of whole blood collected during space flight for subsequent flow cytometric analysis, This device contains all liquids to address safety issues concerned with space flight and also moves the cells through the staining, lyse/fixation and dilution steps.

Space flight produces a number of metabolic and physiological changes in the crewmembers exposed to microgravity. Following launch, body fluid volumes, electrolyte levels, and bone and muscle undergo changes as the human body adapts to the weightless environment. Changes in the urinary chemical composition may lead to the potentially serious consequences of renal stone formation. Previous data collected immediately after space flight indicate changes in the urine chemistry favoring an increased risk of calcium oxalate and uric acid stone formation (n = 323). During short term Shuttle space flights, the changes observed include increased urinary calcium and decreased urine volume, pH and citrate resulting in a greater risk for calcium oxalate and brushite stone formation (n = 6). Results from long duration Shuttle/Mir missions (n = 9) followed a similar trend and demonstrated decreased fluid intake and urine volume and increased urinary calcium resulting in a urinary environment saturated with the calcium stone-forming salts. The increased risk occurs rapidly upon exposure to microgravity, continues throughout the space flight and following landing. Dietary factors, especially fluid intake, or pharmacologic intervention can significantly influence the urinary chemical composition. Increasing fluid intake to produce a daily urine output of 2 liters/day may allow the excess salts in the urine to remain in solution, crystals formation will not occur and a renal stone will not develop. Results from long duration crewmembers (n = 2) who had urine volumes greater than 2.5 L/day minimized their risk of renal stone formation. Also, comparisons of stone-forming risk in short duration crewmembers clearly identified greater risk in those who produced less than 2 liters of urine/day. However, hydration and increased urine output does not correct the underlying calcium excretion due to bone loss and only treats the symptoms and not the cause of the increased urinary salts. Dietary modification and promising pharmacologic treatments may also be used to reduce the potential risk for renal stone formation. Potassium citrate is being used clinically to increase the urinary inhibitor levels to minimize the development of crystals and the growth of renal stones. Bisphosphonates are a class of drugs recently shown to help in patients with osteoporosis by inhibiting the loss of bones in elderly patients. This drug could potentially prevent the bone loss observed in astronauts and thereby minimize the increase in urinary calcium and reduce the risk for renal stone development. Results of NASA's renal stone risk assessment program clearly indicate that exposure to microgravity changes the urinary chemical environment such that there is an increased risk for supersaturation of stone-forming salts, including calcium oxalaie and brushite. These studies have indicated specific avenues for development of countermeasures for the increased renal stone risk observed during and following space flight. Increased hydration and implementation of pharmacologic countermeasures should largely mitigate the in-flight risk of renal stones.

In this study, we have attempted to combine standard immunological assays with the cellular resolving power of the flow cytometer to positively identify the specific cell types involved in spaceflight-induced immune alterations. We have obtained whole blood samples from 27 astronauts collected at three timepoints (L-10, R+0 and R+3) surrounding four recent space shuttle missions. The duration of these missions ranged from 10 to 18 days. Assays performed included serum/urine cortisol, comprehensive subset phenotyping, assessment of cellular activation markers and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following spaceflight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated trends towards a decreased percentage of T cells and an increased percentage of B cells. Nearly all of the astronauts exhibited an increased CD4:CD8 ratio, which was dramatic in some individuals. Assessment of memory (CD45RA+) vs. naive (CD45RO+) CD4+ T cell subsets was more ambiguous, with subjects tending to group more as a flight crew. All subjects from one mission demonstrated an increased CD45RA:CD45RO ratio, while all subjects from another Mission demonstrated a decreased ratio. While no significant trend was seen in the monocyte population as defined by scatter, a decreased percentage of the CD14+ CD16+ monocyte subset was seen following spaceflight in all subjects tested. In general, most of the cellular changes described above which were assessed at R+O and compared to L-10 trended to pre-flight levels by R+3. Although no significant differences were seen in the expression of the cellular activation markers CD69 and CD25 following exposure to microgravity, significant alterations were seen in cytokine production in response to mitogenic activation for specific subsets. T cell (CD3+) production of IL-2 was significantly decreased after at R+O as was IL-2 production by both CD4+ and CD8+ T cell subsets for most subjects. Production of IFN(sub gamma) did not appear to be affected by microgravity exposure in either T cells in general or in the CD8+ T cell subset. There was a spaceflight-induced decrease in IFN(sub gamma) production in the CD4+ T cell subset, however it did not reach statistical significance. Serum and urine stress-hormone analysis indicated significant physiologic stresses in astronauts following spaceflight. In summary, these results demonstrate alterations in the peripheral immune system of astronauts immediately after spaceflight of 10 to 18 days duration and support continued research regarding microgravity and immunology (including in-flight sampling) prior to routine long-term spaceflight for astronauts.

An improved bio-reactor vessel and system useful for carrying out mammalian cell growth in suspension in a culture media are presented. The main goal of the invention is to grow and maintain cells under a homogeneous distribution under acceptable biochemical environment of gas partial pressures and nutrient levels without introducing direct agitation mechanisms or associated disruptive mechanical forces. The culture chamber rotates to maintain an even distribution of cells in suspension and minimizes the length of a gas diffusion path. The culture chamber design is presented and discussed.