User:Jssteil/sandbox/Illness due to altered immune response in space

Human immune function: Human immune function is altered in- and postflight, but it is unclear if this change leads to an increased susceptibility to disease. Reactivation of latent viruses has been documented in crewmembers, although this reactivation has not been directly correlated with the immune changes or with observed disease. Further research may elucidate whether microgravity exposure impairs the immune system, and whether this change represents a health risk to crews.

There is a large body of evidence associated with immune dysregulation and spaceflight; however, the current evidence base is insufficient to determine clinical risk related to immune dysregulation during exploration space missions. Evidence is lacking primarily because precious little of the current information is derived from in-flight studies with human subjects. The inflight studies that have been performed have used extremely small numbers of subjects or been limited to short-duration flight. There is more data derived from postflight testing of crew members, but these findings do not necessarily reflect the in-flight condition. Rather, landing-day observations may be skewed by the effects of re-entry and readaptation to gravity following deconditioning. Ground-analog testing of humans, while extremely useful for some applications (assay development, countermeasures validation, etc.), can never be said with certainty to mirror physiological changes during spaceflight. Each analog may simulate some aspect of flight, but no analog can cumulatively replicate all the aspects of flight.

In-flight testing of humans has revealed that latent herpes viruses reactivate to a high level during short-duration spaceflight, but it is currently unknown whether this phenomenon would persist or intensify during extended duration flight, or eventually resolve itself. During long duration flight, cell-mediated immunity has been demonstrated to be reduced in some subjects, and there may be a relationship between the observed in-flight immune changes and reactivation of latent viruses. Postflight human testing has revealed severely depressed T cell function following 6 months of flight, but unaltered function following short-duration flight. Altered cytokine production patterns and potentially a shift to the Th2 pattern have been observed following spaceflight. Natural killer (NK) cell, monocyte, and neutrophil function have all been found to be reduced following spaceflight. Stress hormone levels have been found to be elevated following flight, heavily dependent on mission duration. Various animal studies have demonstrated similar findings either during or after flight.

Human ground analog data have varied widely depending on the analog selected. To date, the 6-month Antarctic winter over appears to be the best terrestrial analog for long-duration spaceflight relevant to immunity. Reactivation of latent herpes viruses, immune dysregulation, and physiological stress that appear similar to spaceflight-associated immune dysregulation have all been observed in the Antarctic analog. Shorter-duration analogs such as the undersea NEEMO missions (off Key Largo, 2 weeks duration) and Arctic expeditions on Devon Island (Haughton-Mars Project, ~1.5 month’s duration) also appear to be promising analogs for short and intermediate duration spaceflight respectively. Recent pilot studies have found immune dysregulation similar to that associated with spaceflight during both of these analogs, and both are far easier to use, from a logistical perspective, than Antarctic winter-over. Bed rest has been reported by some investigators to induce immune changes, but the contrary has been reported by other investigators. While prolonged bed rest is an excellent analog for bone loss and muscle deterioration from lack of use, it does not simulate the primary suspected causes of spaceflight-associated immune dysregulation (including physiological stress, disrupted circadian rhythms, and microgravity).

Terrestrial clinical research has repeatedly demonstrated that altered immunity is associated with adverse conditions, and that immune “balance” is essential for good health. For example, depressed immunity may lead to increased incidence of infections, but elevated immunity may lead to allergies or autoimmunity. More specific alterations in Th1/Th2 cytokine balance may be associated with many conditions, including rheumatoid arthritis, multiple sclerosis, asthma, lupus, and allergies. If immune dysregulation were found to persist in the deep space environment, clinical risks could include hypersensitivities, allergies, autoimmunity, increased infection rates and even malignancies associated with impaired tumor surveillance. Rather than develop gradually, the risks related to altered immunity have the potential to suddenly impact a mission, likely in a manner very difficult to treat remotely. The reactivation of latent viruses, while not typically a clinical concern terrestrially, could pose a health risk if persisted for the duration of an exploration class mission.

Given the widely disparate nature of the immune evidence and the huge knowledge gap related to in-flight testing of human subjects, it cannot be determined at this time if significant immune dysregulation persists or resolves during extended duration spaceflight. This argues strongly for a comprehensive in-flight assessment of immunity, viral reactivation and physiological stress. It is intended that the new multi-laboratory Integrated Immune (SMO-015, SDBI-1900) flight study will provide sufficient in-flight long-duration data during the International Space Station (ISS) utilization phase to allow a determination of clinical risk (if any) related to immunity for exploration class missions. Other new studies, such as the European Space Agency’s Immuno experiment (ISS/Soyuz), will also add to the in-flight knowledge base.

Introduction
The assessment of immunity is a fast-developing, ever-changing area of science, made difficult by the complexity of the immune system. A number of distinct subpopulations of leukocytes (white blood cells) populate the blood, lymph nodes, and gut, and generally traffic around the entire body. The specific functions of these cell populations can vary widely, and in some cases they are counter regulatory. In addition, the emerging science of neuroimmunology illustrates the complexity with which the immune system interacts with other physiological systems via a network of communication involving hormones, cytokines, and cells. That altered immunity is directly related to the presence of disease is well-accepted and may be clinically related to increased incidence of infection and increased risk of tumor formation. For this report, immune dysregulation is defined as a deviation from “normal,” or from pre-flight baseline values. Dysregulation detected by individual immune assays may be either hyper-activity or hypo-activity. Clinically, hyper-immunity is associated with allergies and various auto-immune diseases, whereas hypo-immunity can be associated with increased incidence of infection and possibly tumor formation. In addition, the balance and bias of the immune system within itself (Th1/Th2) is correlated with risk and incidence of specific diseases.

To advance the study of immunity, a large number of assays have been developed. In the hospital laboratory, determination of the number of T cells positive for the surface protein CD4 (CD4+) and the titer of antibodies to viruses are well-established tests utilized in clinical practice. However, there are dozens of other direct measurements of specific immune parameters available for clinical research. Examples of immune assays are measurement of the level of immune cell subpopulations in the blood (phenotyping), isolation and stimulation of cultured immune cells followed by various functional assessments, determination of factors such as mRNA gene expression, secretion of cytokines, and expression of activation markers.

Published data strongly suggest that immune dysregulation is associated with spaceflight, regardless of duration, and several excellent reviews have been published recently regarding this subject (Borchers 2002, Sonnenfeld 2002). The specific single cause of immune dysregulation during flight is unknown, but likely associated with one or more of the following items: physiological stress, disrupted circadian rhythms, microgravity, isolation, altered environment, altered nutrition, and radiation. While the postflight status of the human immune system has been well characterized, the status of the immune system during flight (and particularly during longer duration flight) is incomplete. A general overview of spaceflight-associated immune dysregulation (potential causes, summary of observations to date, and potential clinical risk) is presented in figure 13-1. A current goal of NASA and the space life science community is to determine the specific clinical risks associated with all flight effects on human physiology, so that countermeasures may be developed prior to the initiation of exploration-class space missions. This need has been heightened by the impending NASA lunar/Mars program, which will be initiated within the next decade.

The published data regarding spaceflight and immunity may fall into numerous categories: during flight, after flight, ground analog, human, animal, and so on. For astronaut crewmembers, data are typically captured as follows: before flight to establish baseline values, during flight (when possible), and on landing day (R+0) to establish spaceflight associated changes, and after flight to monitor return to baseline. Due to the limits of practicality, it is impossible to review ALL the evidence. For this report, numerous representative articles have been summarized by category in Appendix 1. These articles reflect the primary scientific findings for the discipline. The narrative text of this report represents a summation of the data and likely clinical significance.



Evidence
A detailed presentation of representative published articles regarding various aspects of space-flight immunity is presented in Appendix 1. The narrative description of evidence represents a non-comprehensive summary of the published evidence.

In-flight human data
Due to the complexities associated with in-flight experiments, there have been comparatively few in-flight studies of human immune function. Those that have been performed have weakened by having a small number of subjects (n). Reactivation of latent herpes viruses has been observed repeatedly during short duration flights (Mehta/Pierson 2000, Payne 1999, Pierson 2005, Stowe 2001, Mehta 2004), and depressed cell mediated immunity (CMI), determined in vivo using the CMI skin test, has been observed during long duration flight (Cogoli 1993, Gmunder 1994).

Recently, the in-flight reactivation of latent herpes virus during a short-duration Soyuz taxi flight to the ISS was assessed (Mehta 2007). Three crewmembers who participated in a recent 14-day mission to the ISS were monitored for the reactivation of varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) using blood, urine, and saliva assays. During flight, only saliva was assayed; urine and blood were tested before and after flight. The data demonstrated EBV reactivation before, during and following flight. CMV was reactivated before and after flight, and VZV was reactivated during and after flight (Figure 13-2). No increases in titer of antibodies to these viruses were found, suggesting that an immune response may not be necessary for reactivation to occur. These data support the Shuttle findings, in that similar changes were found following short duration flight on different vehicles. No in-flight data exists regarding latent viral reactivation during long-duration flight.



Unfortunately, there has not yet been an integrated in-flight study assessing the various aspects of human immunity during long-duration space missions. The current European Space Agency (ESA) in-flight “Immuno” study (long duration, ISS/Soyuz, n=6) and the NASA inflight “Integrated Immune” study (long duration, ISS/Shuttle, n=17) will address this in-flight knowledge gap and allow a determination of clinical risk (if any) associated with immunity during prolonged exploration class flight.

Unpublished in-flight incidence rates
In addition to the experimental evidence, with regard to in-flight testing of human subjects there is also substantial anecdotal data regarding immunity during flight. This is almost entirely clinical and consists of crew reports regarding adverse medical events that occurred during various space missions. Such data are potentially very useful, if they reflect consistent flightrelated alterations in disease progression or wound healing. However, in accordance with Health Insurance Portability & Accountability Act of 1996 regulations, such clinical data are generally restricted and may be available for science purposes only in an un-attributable fashion. Recently, Dr. Kathy Johnson-Throop of the JSC Life Sciences Data Archive surveyed the Shuttle clinical data archive for in-flight incidence rates of infectious disease. The number of events that occurred in these very healthy, screened and essentially isolated individuals was remarkable, especially in consideration of the pre-flight quarantine of all Shuttle crewmembers (Table 13-1).

Postflight human data
Given the relative ease of performing postflight assessments of human immunity, a significant number of studies have been performed. Advantages of postflight evaluations include lower cost, readily available access to human participants, and minimal technical barriers. The primary disadvantage of a postflight assessment is the potential that data do not reflect the inflight condition and are skewed by the confounding physiological stress of re-entry and readaptation to unit gravity; however, as an important starting point, much has been learned about crewmember immune status immediately following both short- and long-duration spaceflights.

Specific immune system alterations that have been observed when testing was done immediately after spaceflight include dysregulation of the following: cytokine production patterns (Chapes 1994, Crucian 2000, Gould 1985, Gould 1987, Konstantinova 1995, Miller 1995, Sonnenfeld 1994, 1996, 1988, 1993); NK cell function (Buravkova 2004, Konstantinova 1995, Meshkov 1995); leukocyte distribution (Crucian 2000, Stowe 1999); monocyte function (Kaur 2005, Maine 1991); granulocyte function (Kaur 2004, Stowe 1999); T cell intracellular signaling (Cogoli 1997, 1993; Pippia 1996, Schwarzenberg 1999); neuroendocrine responses (Stowe 2003); and leukocyte proliferation following activation (Grove 1995, Nash 1992).

One may briefly summarize these observations as follows: there appears to be a generalized multi-faceted immuno-suppression that is detectable postflight. Production of various cytokines (intracellular, secreted, or otherwise) is reduced, and the functional capabilities of the various immunocytes (granulocytes, monocytes, NK cells, T cells) are reduced. Although some of these findings are almost certainly related to landing and readaptation, more recent findings indicate at least some postflight observations may reflect in-flight immune alterations. Specifically, when expression of EBV early, intermediate, and late genes was measured in infected peripheral B cells, expression of EBV late genes was elevated in samples collected immediately postflight (see unpublished postflight data: EBV gene expression).

Although latent herpesvirus reactivation has been found to occur to high levels during short duration space flight, some had speculated that the salivary measurements of viral DNA might not reflect infectious viral particles. To investigate this further, following a recent Space Shuttle mission, cultures were performed to determine if infectious virus was being shed. In 2 of 3 astronauts who participated in the study, following landing live VZV was found to be present in the saliva samples (Cohrs 2008).

Recently, a comprehensive post-flight immune assessment was performed on 17 short-duration Space Shuttle crewmembers and 8 long-duration International Space Station (ISS) crewmembers (Crucian 2008). The testing consisted of a comprehensive peripheral leukocyte subset analysis, determination of early T cell unctional capabilities, and intracellular/secreted cytokine profiles. For Shuttle crewmembers, the distribution of the peripheral leukocyte subsets was found to be altered post-flight. Early T cell function following culture stimulation was actually elevated post-flight; however, the percentage of T cell subsets capable of being stimulated to produce IL-2 and IFNg was decreased. The ratio of secreted IFNg:IL-10 following T cell stimulation declined after landing, indicating a Th2 shift. For the ISS crewmembers, some alterations in peripheral leukocyte distribution were also detected after landing. In contrast to Shuttle crewmembers, the ISS crewmembers demonstrated a statistically significant reduction in early T cell activation potential immediately post-flight (figure 13-3). The percentage of T cells capable of producing IL-2 was reduced, but IFNg percentages were unchanged. A reduction in the secreted IFNg:IL-10 ratio (Th2 shift) was also observed post-flight in the ISS crewmembers. These data indicate that consistent peripheral phenotype changes and altered cytokine production profiles occur following spaceflight of both short and long duration; however, functional immune dysregulation may vary related to mission duration. A more pronounced T cell dysfunction was observed in the ISS crewmembers, indicating that prolonged mission duration and deconditioning may affect immunity. The elevations in function in the Shuttle crewmembers were attributed to the acute stress of landing, in much less deconditioned subjects. Finally, the authors conclude that a Th2 cytokine shift appears to be associated with spaceflight. This potentially indicates specific classes of clinical risk (autoimmunity, hypersensitivities), should such shifts persist for exploration-class space missions. These data were derived from the Epstein Barr flight study (DSO-500, E129).



Unpublished postflight data: EBV immunity
EBV-specific T cell immunity, responsible for controlling viral reactivation, is altered following spaceflight. Representative crewmember data are presented in Figure 13-4. Levels of EBV-specific T cells (measured by the MHC tetramer method) rise following flight, likely indicating an attempt to control latent virus reactivation. However, EBV- specific T cell function (IFNg cytokine expression following EBV peptide stimulation in culture) was dramatically reduced following spaceflight. These data (derived from the Epstein Barr flight study [DSO-500, E129]) indicated that the immune defect that allows viral reactivation to occur is altered T cell function, not a loss of specific T cells responsible for control of virus. Prolonged similarly reduced function of the entire T cell population could result in immunosuppression and disease susceptibility.



Unpublished postflight data: EBV gene expression
The level of EBV mRNA gene expression in infected peripheral B cells was found to be altered following spaceflight compared to both the pre-flight baseline and normal healthy controls. Several EBV genes were measured by quantitative polymerase chain reaction (PCR); they were subdivided into genes expressed during EBV latency, genes expressed intermediateearly or early (IE/E), and genes expressed late in the active replicative phase of infection and reactivation. For this assay, actin mRNA was measured as a positive control and the EBV EBER gene was measured as a control for the presence of infected B cells. Transcripts of the EBER gene are expressed to varying degrees in all infected B cells, regardless of the viral latency state.

EBV gene expression in peripheral blood from healthy adults is highly restricted. Five of 24 samples were positive for latent gene expression, while only 3 of 24 samples were positive for IE/E expression; none of the 24 control subjects were positive for any of the late replication EBV genes. For Shuttle crewmembers, 5 of 12 samples showed evidence of latent gene expression, while nine of 12 samples had IE/E gene expression. Notably, some of the samples were positive for multiple viral gene transcripts. None of the pre- or postflight samples were positive for late gene transcripts.

For ISS crewmembers, nine of 12 samples were positive for latent gene transcripts, while 11 of 12 were positive for IE/E gene transcripts. In addition, a much higher percentage of samples were positive for multiple viral gene transcripts. Importantly, for the first time, evidence of late (i.e., replicate) gene transcription was found, postflight only: four of the six landing day samples were positive for one or more of the late genes. None of the samples from healthy controls or the Shuttle postflight samples demonstrated any late EBV gene expression. Note that for the astronaut crewmembers, all subjects demonstrated EBER expression; thus the actin control is not shown. These data (derived from the “Epstein Barr flight study [DSO-500, E129]) suggest that reactivation of latent EBV is to some degree observed at the L-10 postflight timepoint. However, it is known that for some stress measurements, L-10 may be too close to launch to serve as a fair baseline as crewmember stress is present by this point in the countdown. Also, the data show that EBV reactivation is affected by mission duration. This may be due to poorer cellular immune control over viral replication during prolonged space missions. Alternatively, spaceflight may have a more direct effect on viral reactivation and replication. Persistent reactivation of latent herpes viruses during long duration space missions, in conjunction with dysregulated immunity, high energy radiation, and other factors, may represent a significant clinical health risk to crewmembers participating in  exploration-class deep space missions. (Publication anticipated 2009)

Inflight animal data
Unfortunately, the majority of spaceflight immunity studies with animal subjects have involved postflight testing of animals flown in space. Those studies are listed under “postflight animal data.” Some animal studies have assessed immunity during spaceflight; these primarily involved dissection of animals during flight and return of samples to earth. On the Shuttle SLS-2 mission, rats were flown and samples returned for analysis. Spleen T cell and NK cell function were reduced during and after flight; however, marrow NK cells appeared to be unaltered. Altered cytokine production patterns were also reported (Lesnyak 1996).

Postflight animal data
A number of animal studies during spaceflight have provided biosamples available for postflight testing. In particular, immune splenocytes and thymocytes have been available from rats and mice flown in space on several SLS Shuttle missions. On several flights of Russian COSMOS satellites, live rats were flown and recovered. Several variables in these studies cannot be controlled, such as mission duration, differing launch vehicles, and different animal species. These additional variables make comparison of data from different studies difficult; however, actual flight data remains extremely valuable, regardless of vehicle.

The postflight animal data include observations of altered leukocyte distribution (Sonnenfeld 1990, 1992) and altered cytokine production (Gould 1987, Grove 1995, Miller 1995, Sonnenfeld 1996). One study indicated that postflight mitogenic and proliferative responses of lymph node lymphocytes, as well as IL-2 production, were unaltered in space-flown rats (Nash 1992). In general, the animal data are similar to the postflight human data: immune dysregulation is observed post spaceflight. Changes include dysregulated cell function (proliferation, cytokine production, and other functions). Interestingly, Nash et al. suggested that microgravity has a tissue-specific effect on lymphocyte function, a finding that is impossible to evaluate in human subjects and highlights the greater utility of animal studies for in-flight immune investigations.

Recently, mice flown on the STS-118 Space Shuttle mission were available for immunological studies. For this study, spleens and thymuses from flown mice were evaluated in comparison to similarly held ground controls. Samples were collected 3-6 hours following Shuttle landing. In general, the observations were similar to those obtained from human subjects. Alterations in the distribution of the lymphocyte subsets, a reduction in blastogenesis following mitogenic stimulation, and shifted cytokine profiles. Specifically, IL-2 production was decreased, whereas IL10 and IFNg were increased. In addition, alterations in the expression of 30 cancer-related genes was reported (Gridley 2009). During the same mission, innate immune function was investigated, by determining responses to LPS stimulation. Secretion of IL-6 and IL-10, but not that of TNFa was increased in the flown mice as compared to ground control mice (Baqai 2009). Also, the genes responsible for scavenging ROS were upregulated.

In-flight cell culture data
Several studies involving in-flight culture of immune cells have been performed. These data should be interpreted with caution, as it is unknown if in-flight culture observations accurately reflect altered (or unaltered) in vivo human immune function. Specific findings include: unaltered NK function (Buravkova 2004), altered cytokine production profiles (Chapes 1994), and various observations of altered progression of cellular activation following mitogenic stimulation (Hughes-Fulford 2001 and 2003, Cogoli 1993 and 1997, Hashemi 1999).

Ground-analog human data
To evaluate the effects of mission-associated factors on human physiology, ground-based “spaceflight analogs” may be used (Schmitt 1993). A variety of analogs are available, each unique and exerting some influence on human physiology that is similar to one or more aspects of spaceflight. Examples of the most well-known human ground-based spaceflight analogs are presented in Table 13-2.

In addition, analogs may be augmented. For example, a recent study at Brooks Air Force Base by Dr. Raymond Stowe added high-G human centrifugation prior to and after a 16-day prolonged head-down tilt bed rest, to simulate launch and landing and thus better replicate the physiological aspects of a shuttle mission (Stowe 2008). Another recent study by Dr. Bill Paloski added a daily 1-hour 2.5xG human centrifugation to a 16-day bed rest study, to evaluate artificial gravity as a possible countermeasure. For ground-based studies, it is very important to choose the analog that is most appropriate for the physiological system of interest. For example, bed rest may be an excellent analog for muscle loss, whereas NEEMO or Antarctic winter over would not, since the prime causal factor (microgravity) is not replicated.

Since analog use is comparatively cost-effective, the immunology discipline at NASA is pursuing validation of an appropriate ground-based spaceflight analog for spaceflight-associated immune dysregulation. This pursuit is based on suggestions made at the 2006 Immunology Program Review and on consensus direction statements provided during the 2007 NASA Human Research Program Workshop. Validation of a ground-based analog would be extremely useful for basic science as well as countermeasures validation, in the event that spaceflight-associated immune dysregulation is found to persist during long-duration flight and countermeasures development is warranted. An excellent ground-based flight analog for immune studies would simulate mission-associated stress, isolation, and disrupted circadian rhythms. An overview of immune data collected during various analog studies follows.

Antarctic Winter-over Analog. During Antarctic winter-over (AWO), subjects experience prolonged isolation in a harsh extreme environment, and several comprehensive immune studies have been conducted during these expeditions. It is likely that AWO represents the closest analog to long-duration or exploration class spaceflight available on Earth. This is because the mission length, extreme environment, extreme isolation, mission associated activities, disrupted circadian rhythms, and other factors are similar to those of long-duration spaceflight. During AWO, participants are completely isolated, as no aircraft are capable of reaching the various Antarctic outposts during this time. Immune studies performed during winter-over missions have shown decreased cell mediated immune responses (Muller 1995a, 1995b, Mehta/Lugg 2000), reduced T cell function (Tingate 1997), altered cytokine production profiles (Shearer 2002, Tingate 1997) and latent herpesvirus reactivation (Mehta/Lugg 2000, Tingate 1997). A study of antibody production following immunization during AWO found no mid-mission alterations (Shearer/Reuben 2001), potentially indicating that humoral immunity is unchanged in the presence of altered cellular immunity. These data support the Antarctic analog as the most successful to date in simulating long duration spaceflight-associated immune dysregulation. The only serious limiting factor regarding physiology studies during AWO, is the logistical access during a mission. Ironically, this is directly related to the very factors that make AWO such a good analog, thus truly making it “flight-like.” Studies that require simple collection and freezing of samples (blood, saliva) are obviously very compatible with mid-mission AWO studies. However, as recent data have indicated, it is immune function that appears to be compromised following (and during?) spaceflight. Assessments of immune function typically require live cell culture and more immediate processing and analysis of samples. This difficulty was addressed during preparation for the Haughton-Mars 2001 immune pilot study (Crucian 2007).

Haughton-Mars Project Analog (Canadian Arctic). Another potentially useful analog for spaceflight associated immune dysregulation is the NASA Haughton-Mars Project (HMP). The HMP is an international field research project centered on the scientific study of the Haughton meteorite impact structure and surrounding terrain on Devon Island, Nunavut Territory, Canadian High Arctic. It is viewed as an analog for planetary exploration, in particular for exploration of the Moon and Mars. It is particularly well suited for exploration-related human physiology studies because field personnel are subject to actual and relevant nvironmental stressors, although they are clearly not as extreme as those encountered in space. In addition, personnel are engaged in field exploration tasks that in many cases are direct analogs of those anticipated for the Moon and Mars. The following factors encountered by HMP field participants are particularly relevant to spaceflight or planetary exploration:


 * Long travel to and from Devon Island (several days of travel followed by weeks of stay)
 * Relatively harsh polar desert environment
 * Disrupted circadian rhythms (24 hours of daylight during the summer field season)
 * Relative isolation from the outside world (with limited exception)
 * Limited local infrastructure (HMP Research Station is analogous to early lunar or Martian outpost)
 * Activities relevant to those that crewmembers on lunar and Mars missions would be expected to perform, including exploration, field work, EVA
 * Reliance on remote telemedicine and communications equipment.

These factors and mission duration make the HMP potentially a good analog for spaceflight-associated immune dysfunction studies. The 30- to 45-day mission duration seems to make HMP a potentially useful analog for ISS Flight Engineer-2 subjects, who rotate on successive Shuttle flights and have mission durations longer than those of Shuttle-only crewmembers but shorter than those of ISS-Soyuz (6-month) crewmembers. In 2002 a NASA pilot study was performed with the following objectives:
 * 1) develop and field-evaluate methods for processing biological samples to support immune function testing in remote locations, and
 * 2) use the protocol to assess mission-associated immune changes during an HMP mission.

The data demonstrated that in the HMP participants, changes in immune function and physiological stress occurred that were in some ways similar to those observed in astronauts following spaceflight (Crucian 2007). Specifically, phenotypic alterations, reductions in intracellular cytokine levels, humoral data that suggested EBV reactivation, and altered stress hormone levels were all observed during this intermediate length HMP mission.

NEEMO Undersea Analog. A third likely relevant analog for immune changes observed during short-duration spaceflight is the NASA Extreme Environment Mission Operations (NEEMO) project. The NEEMO project was developed by NASA to use extended undersea missions based in Aquarius (the world’s only permanent undersea station) as a high-fidelity ground-based spaceflight analog. Aquarius was constructed and is operated by a partnership of the National Oceanic and Atmospheric Administration, the University of North Carolina at Wilmington, and the National Undersea Research Center, and is utilized routinely for undersea oceanic research. It is located 7 miles offshore of Key Largo, Florida at a depth of approximately 65 feet. During research missions, which typically last 7-14 days, crewmembers (“aquanauts”) use saturation diving. In this dive protocol, easy return to the surface is not possible and nominal resurfacing requires approximately 18 hours of decompression. NEEMO missions are timelined and executed in such a way that the spaceflight analog conditions are the best possible:


 * Confinement to the station lasts the duration of the mission.
 * EVA activities are performed, linked to Mission Control Center in Houston for support.
 * A variety of Shuttle and ISS experiments are performed.
 * Telemedicine is used to communicate with NASA flight surgeons.
 * For high fidelity, only Shuttle or ISS food may be consumed (NEEMO-5).

It is important to note that although the NEEMO missions simulate high-fidelity actual spaceflight conditions, they are actual missions in their own right with real health risks and are not necessarily only simulations. Given the short mission duration and high-fidelity similarity to a Shuttle mission, NEEMO may represent a useful analog for the spaceflight-associated immune dysregulation that has been observed during short duration spaceflight. Also, NEEMO is extremely easy to use logistically, making it an attractive test bed for hardware and initial countermeasures development. Pilot data generated during the NEEMO 4, 5, 12, and 13 missions have indicated that immune dysregulation and viral reactivation similar to those observed during or following spaceflight occur during NEEMO missions.

To investigate if NEEMO induces in-flight immune alterations similar to those observed during Shuttle missions, and to evaluate assays developed for SMO-015, a pilot study was performed on NEEMO 12 and 13, during 2007. Assays were performed that assessed immune status, physiological stress, and latent viral reactivation. Blood and saliva samples were collected at pre-, mid-, and post-mission timepoints. The data revealed minimal changes in peripheral leukocyte subsets, as would be expected from healthy subjects in an adverse environment in the absence of actual illness. Also revealed, however, were dramatic alterations in T cell function. Intracellular cytokine profiles within T cell subsets were altered, and generalized T cell function was diminished during the missions, in a fashion similar to that observed postflight in ISS crewmembers (Figure 13-5). Serological evidence of EBV reactivation was observed in 50% of the subjects. As evidence of latent VZV reactivation, salivary VZV DNA was detected in 2 of the 4 NEEMO-12 subjects. Plasma cortisol was elevated in some of the NEEMO subjects, and salivary concentrations of cortisol were greater during the mission than before or after it. Taken together, the pilot study data seem to validate the NEEMO analog as being appropriate to induce some of the aspects of spaceflight-associated immune dysregulation that are observed during short duration Shuttle flights. In addition, the ease of utility and high-fidelity of the analog make it attractive for rapid investigations. However, to investigate immune dysregulation associated with prolonged missions (a key element to determining clinical risk for exploration class missions), another analog would be required.



Head Down Tilt Extended Bed Rest Analog. The use of long duration head-down tilt bed rest has also been investigated to determine if this analog is appropriate for spaceflight associated immune dysregulation. The most obvious relevance for bed rest is for studying muscle loss and bone demineralization, but some investigators believe that the fluid shifts replicated during bed rest may be relevant to in vivo immune alterations. Pieces of the evidence conflict regarding validation of the bed rest analog as a replicate for spaceflight-associated immune dysregulation. Schmitt, Uchakin and others have published data indicating that some immune changes, including decreases in T cell activation potential and altered cytokine patterns, are associated with this analog (Schmitt 1996, Uchakin 2007). However, these data were induced by exogenous delivery of stress hormones to the participants. Data generated during a general immune assessment as part of the recent NASA Flight Analogs Project bed rest campaigns did not show altered leukocyte distribution, altered cytokine production patterns, reduced T cell function, or significant viral reactivation during the campaigns (Crucian 2009). Given the absence of the most likely causes of spaceflight-associated immune dysregulation (disrupted circadian rhythms, mission associated stress, isolation, etc.) bed rest most likely does not represent the best analog for exploration class spaceflight-associated immune dysregulation.

Ground-analog animal data
Ground analog animal data are essentially confined to hind limb elevation, suspension, unloading or restraint. Altered cytokine production patterns and reduced ability to fight infection have been observed using such analogs (Berry 1991, Sonnenfeld 1988). Although these results are extremely interesting, their direct correlation with human astronaut clinical risk from prolonged spaceflight-associated immune dysregulation is obviously debatable. There are obvious differences between humans and mice, and the animal suspension/restraint analogs are clearly different from prolonged spaceflight.

Ground-analog cell culture data
Ground cell culture analogs for modeled microgravity such as clinorotation, bioreactor, High Aspect Ratio Vessel, all essentially subject cultured cells to a continuous free fall which has been shown to replicate some cellular effects of microgravity exposure. A variety of cellular effects, including altered actin polymerization, reduced lymphocyte locomotion, disrupted transmission of intracellular signals, and altered gene expression are all commonly observed that are believed to be similar to spaceflight observations (Pellis 1997, Licato 1999, Hughes Fulford 2005, Ward 2006). Sundaresan et al. identified the intracellular defect responsible for altered locomotion in modeled microgravity at the level of PKC or upstream, with lymphocyte calcium signaling pathways found to be functional (Sundaresan 2002). Hughes Fulford et al. found that alterations in 10 key genes were associated with simulated microgravity culture, indicating that the intracellular protein kinase A pathway was a key pathway altered during microgravity conditions and likely responsible for some of the observed spaceflight-associated immune dysregulation effects in humans (Hughes Fulford 2005). Ground cell-culture analogs may have significant utility for mechanistic studies that will determine the root causes of cell-specific microgravity induced immune system changes. However, any conclusions regarding clinical risk for exploration-class missions will require human subject studies, as variables such as stress and isolation cannot be replicated by these cellular analogs.

Microbial environment and virulence during spaceflight
The incidence of infectious disease associated with early spaceflight missions was commonplace and observed on Apollo 7 (upper respiratory infection), Apollo 8 (preflight viral gastroenteritis), Apollo 9 (rhinitis and pharyngitis), and Apollo 13 (urinary tract infection). After Apollo 13, the implementation of the Health Stabilization Plan greatly decreased the frequency of reported disease (Billica1994) though its continued occurrence is well documented (see Table 13-1).

To minimize crew exposure to medically significant organisms, spacecraft air, surfaces, potable water, and food are evaluated prior to flight. In general, air, surface, and water samples reflect low concentrations of normal flora commonly seen in the environment (Castro, 2004, Pierson, 2001). Staphylococcus, Micrococcus, and Bacillus are the most common bacterial genera recovered from the air. The genera of bacteria most commonly cultured from spacecraft surfaces are Staphylococcus, Micrococcus, Corynebacterium, and Bacillus. Aspergillus, Penicillium, and Cladosporium are the most frequently cultured fungi from air or surfaces. Spacecraft potable water is maintained at very low microbial levels and contains common environmental microorganisms, such as Burkholderia cepacia, Sphingomonas paucimobilis, Ralstonia, eutropha, and Methylobacterium species (Castro, 2004; Pierson, 2001). The species associated with routine air, surface, and water monitoring generally do not contain medically significant organisms beyond opportunistic pathogens, such as Staphylococcus aureus. However, isolated incidences of contamination have been documented which suggest the potential of crew exposure to medically significant microorganisms during a mission (Ott, 2004). During the NASA Mir Program, an evaluation of multiple samples of water condensate from behind panels on the Mir Space Station indicated the presence of microorganisms not commonly isolated from routine environmental sampling, including Escherichia coli, Serratia marcescens and a Legionella species. In addition, microscopic analysis indicated the presence of protozoa, dust mites, and spirochetes (Ott, 2004). In addition to the environment, food is also monitored prior to flight. While the incidence of contamination is low, preflight analyses of food samples have indicated the presence of organisms such as Salmonella typhimurium, Staphylococcus aureus, Enterobacter cloacae and Enterobacter sakazakii (unpublished data). Contaminated lots are immediately removed before shipment for flight; however, these findings suggest a potential route of infection to the crew.

Determination of infectious disease risk is further complicated by a great deal of evidence that suggests genotypic and phenotypic changes, including virulence, occur in microorganisms in response to spaceflight (Nickerson, 2004). For over 40 years, multiple spaceflight experiments have indicated changes in phenotypic characteristics such as microbial growth, morphology, metabolism, genetic transfer, and antibiotic susceptibility (Nickerson, 2004). Recent groundbased analogs, using bioreactors designed to simulate aspects of the spaceflight environment, have supported the in-flight observations. These ground-based experiments also suggested changes in gene expression and an increase in virulence during flight (Nickerson, 2004). This evidence was supported by a flight experiment in 2006 aboard STS-115 in which Salmonella typhimurium was grown during flight and compared to identically cultured ground controls (Wilson, 2007). The cultures were either placed in an RNA fixative during flight or returned as live cultures for virulence testing. The cultures grown aboard the Space Shuttle displayed an extracellular matrix that was not seen in the ground controls. Evaluation of the gene expression indicated 167 genes were differentially regulated compared to ground controls, with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. In addition, cultures grown during flight displayed a 2.7 fold lower LD50 in a murine model when compared to inoculation with ground control cultures (Wilson 2007).

In a follow up study designed to address potential mechanisms for such bacterial changes, the same authors have reported that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model (Wilson 2008). Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, they identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested.

These data are suggestive that the microbial environment may be altered during spaceflight, with a potential for increased virulence. For exploration-class spaceflight, in conjunction with dysregulated immunity, this may represent an additional health risk to crewmembers.

Computer-based simulation information
We know of no clinically relevant computer-based simulations for the human immune system.

Risk in context of exploration mission operational scenarios
The likelihood of an adverse clinical event (allergy, hypersensitivity, infection, malignancy) related to immunology is difficult to calculate due to the paucity of in-flight data and lack of understanding of the in-flight condition. Even when sufficient data from low Earth orbit missions of any duration are obtained, the situations experienced by crewmembers on such missions will likely be vastly different from the ones experienced by crewmembers on lunar long-duration or Mars missions. Current prediction of clinical risk in the context of different operational scenarios (vehicle, destination, duration, etc.) is best summarized as presented in Figure 13-6.

In general, the Discipline Team feels that low-Earth orbital flight of up to 6 months duration does NOT pose a significant health risk related to immunology. This is because of several factors unique to orbital flight. These include protection from certain kinds of deep-space high-energy radiation only beyond the Van Allen belt, as well as a readily-available return option. Also, orbital flight is likely a vastly different experience with regard to physiological stress than will be encountered during exploration class flight. It is expected that exploration class flight, with up to six-fold increases in mission duration, planetary exploration, and exposure to higher energy radiation, will increase the clinical risk. Radiation is a factor in assessing clinical risk related to immunology, due to the direct link between immunity, radiation, and cancer. Immune precursor cells residing in the marrow are particularly sensitive to radiation. Also, if immune dysregulation is found to persist during longer missions, the clinical risk related to tumor surveillance and development of malignancies may become significant.



Conclusion
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. Also, 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 extremely complicated. There is also the challenge to determine the clinical significance of any observed immune alteration. 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 dealing 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 the in-flight human studies, which have demonstrated altered cell mediated immunity and reactivation of latent herpes viruses. Much more data are available from postflight 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 postflight 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, and microgravity-analog cell culture have all 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. 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 providing a monitoring of astronauts (for either clinical or research purposes).

However, enough significant data exists, as described in this manuscript, to warrant addressing this phenomenon during the utilization phase of the ISS. NASA has recognized 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 an 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 countermeasures.