NOTE: End date changed to 12/14/2018 per NSSC information (Ed., 10/1/18)

NOTE: End date changed to 5/2/2018 per NSSC information (Ed., 11/22/17)

NOTE: End date changed to 11/2/2017 per NSSC information (Ed., 1/23/17)

NOTE: End date changed to 11/2/2016 per NSSC information (Ed., 7/17/15)

NOTE: Gap Immune05 deleted per IRP Rev E (Ed., 3/24/14)

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

We successfully measured NK-cell killing potential against 4 separate tumor target cells in vitro and performed detailed immuophenotyping assays in whole blood samples identifying changes in the frequency of total T-cells, CD4+ T-cells, CD8+ T-cells, NK-cells, B-cells, granulocytes, monocytes, and their subtypes. In addition, interferon-gamma ELISpot assays were used to enumerate viral-specific T-cells against antigens derived from the herpesviruses cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella Zoster virus (VZV), and Herpes simplex virus-1 (HSV-1). We also measured multiple analytes in saliva including the stress biomarkers alpha-amylase, cortisol and DHEA, the anti-microbial proteins LL-37, HNP 1-3, lactoferrinandlysozyme, and the systemic inflammatory marker C-reactive protein. We also quantified viral DNA against the latent herpesviruses CMV, EBV, VZV, and HSV-1 using both saliva and urine samples collected from the crew and the ground-based controls and measured IgG antibody titers against each of these viruses in plasma. The major findings from this project have so far been presented in three separate manuscripts. First, the NK-cell phenotypic and functional data was recently published in the Journal of Applied Physiology. The American Physiological Society (APS) selected this paper for a prestigious APS Select Award. Second, a manuscript with the salivary stress and antimicrobial biomarkers was recently submitted to Journal of Applied Physiology and is currently in review (Ed. note January 2020--the paper is published now; see Bibliography section). Third, the B-cell immune phenotyping data was combined with Dr. Guillaume Spielmann’s omnibus project as we felt the story related to B-cell function would be better if it were combined with this data set. This manuscript was recently published in the Journal of Applied Physiology (PMID: 30496712).

[Ed. note: compiled from PI's final report received October 2019]

NOTE: End date changed to 12/14/2018 per NSSC information (Ed., 10/1/18)

NOTE: End date changed to 5/2/2018 per NSSC information (Ed., 11/22/17)

NOTE: End date changed to 11/2/2017 per NSSC information (Ed., 1/23/17)

NOTE: End date changed to 11/2/2016 per NSSC information (Ed., 7/17/15)

NOTE: Gap Immune05 deleted per IRP Rev E (Ed., 3/24/14)

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

1. Complete all data collection procedures for 8 crewmembers (including 1y mission) and 7 ground-based control subjects.

2. Continue analysis of archived samples for all enrolled subjects.

3. Disseminate research findings to the scientific community.

Overview of Work Completed Study Progress: The study was initiated in September 2012 and data collection started in March 2013. We successfully enrolled the required number of subjects giving us a sample size of eight crewmembers and seven ground-based controls including an additional crewmember who completed a 1-year mission to the ISS. Baseline blood, urine, and saliva samples have been collected from all crewmembers and ground-based control subjects. All crewmembers and ground-based controls have completed all experimental procedures. We have obtained in flight samples and ambient blood returns from all enrolled crewmembers. All baseline and in flight ambient blood samples were processed and analyzed successfully. Frozen saliva, urine, and plasma samples have all been processed and analyzed. The IRB (Institutional Review Board) protocol was renewed in July 2018. During the last reporting period, the project team have been analyzing and interpreting data in preparation for dissemination to the scientific community. Data analysis and write up was somewhat delayed due to the Principal Investigator (PI) Dr. Simpson changing institutions (moving from the University of Houston to the University of Arizona) within the last reporting period. One scientific paper was published during the last reporting period, two others have been submitted for publication, and two others are currently in preparation (details below). One of the recent articles from this work “Long-duration spaceflight impairs NK-cell function” is currently under review with the Journal of Applied Physiology. Publications and Presentations: Our validation work for this project allowed us to assess NK-cell function in the context of latent cytomegalovirus infection, age, and exercise. This led to publications by Bigley et al. (2016) in the journals Cellular Immunology and Clinical and Experimental Immunology, Bigley et al. (2015) in Oxidative Medicine and Cellular Longevity and Brain, Behavior, and Immunity, and Bigley et al. (2018) in Clinical and Experimental Immunology. We also published a manuscript in European Journal of Applied Physiology, which stemmed from the validation work of our saliva assays for this project (Kunz et al., 2015). In addition, Spielmann et al. (2016) and Kunz et al. (2018) used the validation work from the viral T-cell quantification component of the “Salivary Markers” study for a publication in Scientific Reports and Physiology and Behavior, respectively. Furthermore, Graff et al. (2018), Agha et al. (2018), and LaVoy et al. (2017) used the validation work for cellular phenotyping of the “Salivary Markers” study in their recent publications in Brain, Behavior, and Immunity and Physiological Reports. The PI (Dr. Simpson) collaborated with NASA scientists and other space life scientists to generate two review articles pertaining to the effects of space travel on immune function (Crucian et al. 2018; Makedonas et al., 2018). Hematology data from this project was also used in the BMC Hematology publication by Kunz et al. (2017). NASA funding was acknowledged in all of these publications. The work supported by this research grant was presented at the International Society of Exercise and Immunology (ISEI) 2017 Symposium in July 2017, the Human Research Program Investigators' Workshop in January 2017 and February 2018, and the American College of Sports Medicine (ACSM) annual meetings in 2017 and 2018. Current and Future Work

All data has been collected and analyzed. The following papers are a direct result of this project and have been published (See also Cumulative Bibliography): Crucian BE, Chouker A, Simpson RJ, Mehta S, Marshall G, Smith SM, Zwart SR, Heer M, Ponomarev S, Whitmire A, Frippiat JP, Douglas GL, Lorenzi H, Buchheim JI, Makedonas G, Ginsburg GS, Ott CM, Pierson DL, Krieger SS, Baecker N & Sams C (2018). Immune System Dysregulation During Spaceflight: Potential Countermeasures for Deep Space Exploration Missions. Frontiers in Immunology, 9, 1437. Crucian, B., Simpson, R.J., Mehta, S., Stowe, R., Chouker, A., Hwang, S., Actor, J.K., Salam, A.P., Pierson, D. and C. Sams. (2014) Terrestrial stress analogs for spaceflight associated immune system dysregulation. Brain, Behavior and Immunity, 39, 23-32.

Kunz H, Quiriarte H, Simpson RJ, Ploutz-Snyder R, McMonigal K, Sams C & Crucian B. (2017). Alterations in hematologic indices during long-duration spaceflight. BMC Hematology, 8, 17-12. Makedonas G, Chouker A, Mehta S, Simpson RJ, Stowe R, Sams C, Pierson DL & Crucian BE (2018). Mechanistic Clues to Overcome Spaceflight-Induced Immune Dysregulation. Current Pathobiology Reports, 6, 185-192. Spielmann, G., Laughlin, M. S., Kunz, H., Crucian, B. E., Quiriarte, H. D., Mehta, S. K., Pierson, D. L. & Simpson, R. J. (2018). Latent viral reactivation is associated with changes in plasma antimicrobial protein concentrations during long-duration spaceflight. Acta Astronautica, 146, 111-116.

The following manuscripts have been submitted for publication or are in preparation:

Bigley, A.B., Agha, N.H., Kunz, H.E., Baker, F.L., Spielmann, G., Rooney, B.V., Mylabathula, P., Laughlin, M. S., Mehta, S. K., Pierson, D. L. Crucian, B. E., & Simpson, R. J. (2018). NK-cell function is impaired during long duration spaceflight. Journal of Applied Physiology (In Review).

Spielmann, G., Simpson, R. J. Laughlin, M. S., Kunz, H., Crucian, B. E., Mehta, S. K. & Campbell, J.C. (2018). B-cell homeostasis is maintained during long-duration spaceflight. Journal of Applied Physiology (In Review).

Agha, N.H., Kunz, H.E., Baker, F.L., Spielmann, G., Rooney, B.V., Mylabathula, P., Bigley, A.B., Laughlin, M. S., Mehta, S. K., Pierson, D. L. Crucian, B. E., & Simpson, R. J. (2018). Long-duration spaceflight alters antimicrobial proteins in astronaut saliva (In preparation).

Agha, N.H., Spielmann, G., Kunz, H.E., Baker, F.L., Rooney, B.V., Mylabathula, P., Bigley, A.B., Laughlin, M. S., Mehta, S. K., Pierson, D. L. Crucian, B. E., & Simpson, R. J. (2018). T-cell and NK-cell responses to latent viral reactivation onboard the International Space Station (In preparation).

NOTE: End date changed to 5/2/2018 per NSSC information (Ed., 11/22/17)

NOTE: End date changed to 11/2/2017 per NSSC information (Ed., 1/23/17)

NOTE: End date changed to 11/2/2016 per NSSC information (Ed., 7/17/15)

NOTE: Gap Immune05 deleted per IRP Rev E (Ed., 3/24/14)

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

Publications and Presentations: Our validation work for this project allowed us to assess NK-cell function in the context of latent cytomegalovirus infection, age, and exercise. This led to publications by Bigley et al. (2016) in the journals Cellular Immunology and Clinical and Experimental Immunology, and Bigley et al. (2015) in Oxidative Medicine and Cellular Longevity and Brain, Behavior, and Immunity. We also published a manuscript in European Journal of Applied Physiology, which stemmed from the validation work of our saliva assays for this project (Kunz et al., 2015). In addition, Spielmann et al. (2016) used the validation work from the viral T-cell quantification component of the ‘Salivary Markers’ study for a publication in the Nature affiliated journal Scientific Reports. NASA funding was acknowledged in all of these publications. The work supported by this research grant was presented at the National Space Biomedical Research Institute (NSBRI) Summer Bioastronautics Institute and American College of Sports Medicine annual meeting in July 2016 and the Human Research Program Investigator’s Workshop in February 2016.

Current and Future Work: The study protocol is ongoing. By the end of year 5, we expect to have all data processed and analyzed for all crewmembers and ground-based controls. The remainder of FY17 will be spent analyzing frozen biological samples, interpreting data, and producing scientific papers.

Applications and Acquisition of Funding: Work on the ‘Salivary Markers’ project has allowed us to apply for further research funding. We have received additional research funding for space life science specific research projects but also from the National Institutes of Health (NIH). The latter project requires us to assess the impact of latent CMV infection on NK-cell phenotype and cytotoxicity in multiple myeloma patients following autologous stem cell transplantation. Our ideas for this project and preliminary data stemmed from the ‘Salivary Markers’ project. We also received a student research grant from the American College of Sports Medicine to analyze archived data related to astronaut fitness levels and latent viral reactivation.

Bibliography

The following are some of the published papers supported by this work (see also Task Book Bibliography below):

Bigley AB, Rezvani K, Shah N, Sekine T, Spielmann G, Pistillo M, Agha N, Kunz H, LaVoy ECP, Bollard CM & R.J. Simpson (2016). Latent CMV infection enhances anti-tumor cytotoxicity through accumulation of NKG2C+ NK-cells in healthy humans. Clinical and Experimental Immunology, 185, 239-251.

Bigley AB, Spielmann G, Agha N, O’Connor, D.P. & R.J. Simpson (2016). Dichotomous effects of latent CMV infection on the phenotype and functional properties of CD8+ T-cells and NK-cells. Cellular Immunology, 300, 26-32.

Bigley, A.B., Spielmann, G., Agha, N., & R.J. Simpson (2015). The effects of age and latent cytomegalovirus infection on NK-cell phenotype and exercise responsiveness in man. Oxidative Medicine & Cellular Longevity, 2015, 979645.

Bigley, A.B. Rezvani, K., Pistillo, M., Reed, J., Kunz, H., Agha, N., Bollard, C.M. & R.J. Simpson (2015). Acute exercise preferentially mobilizes NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells. Part II: Impact of latent cytomegalovirus infection and catecholamine sensitivity. Brain, Behavior and Immunity, 49, 59-65.

Kunz, H. Bishop, N.C., Spielmann, G., Pistillo, M., Reed, J., Ograjsek, T., Park, Y., Mehta, S., Pierson, D.L. & R.J. Simpson (2015) Fitness level impacts salivary antimicrobial responses to a single bout of cycling exercise. European Journal of Applied Physiology, 115, 1015-27.

Spielmann, G., Bollard, C.M., Kunz, H., Hanley, P.J. & R.J. Simpson (2016). A single exercise bout enhances the ex vivo manufacture of viral-specific T-cells from healthy donors: implications for allogeneic adoptive transfer immunotherapy. Scientific Reports, 16; 6:25852.

The following presentations were delivered and supported by this work:

Simpson, R.J., Bigley, A.B., Spielmann, G., Kunz, H.E., Agha, N., Baker, F., Rooney, B., Mylabathula, P.L., Graff, R.M., Crucian, B.E., Laughlin, M., Mehta, S.K., & D.L. Pierson. Long duration spaceflight impairs NK-cell function in astronauts. ACSM Annual Meeting, Boston, MA, USA, May 31st – June 4th, 2016.

Bigley, A.B. The role of microgravity and stress-related humoral factors in dysregulated NK-cell function during spaceflight. NSBRI Summer Bioastronautics Institute, Houston, TX, USA, May 31st – June 3rd, 2016.

Bigley, A.B. The effects of exercise and spaceflight on NK-cells. Invited Presentation at University of Calgary, Human Performance Laboratory Seminar, Sports Medicine Centre, Calgary, AB, Canada, March 30th – March 31st, 2016.

Mylabathula, P.L., Bigley, A.B., & R.J. Simpson. Simulated microgravity ‘disarms’ human NK-cells and inhibits cytotoxicity. HRP Investigators’ Workshop, Galveston, TX, USA, February 8th – February 11th, 2016.

Simpson, R.J. Immune responses to prolonged spaceflight: the ‘Salivary Markers’ study. NASA Human Research Program Investigator’s Workshop, Galveston, TX, USA, Feb 8th – Feb 11th 2016.

Interviews/Press Releases:

Simpson, R.J. interviewed for: Want a stronger immune system? Try hitting the gym. NBC Nightly News with Lester Holt: http://www.nbcnews.com/nightly-news/video/want-a-stronger-immune-system--try-hitting-the-gym-592155715731

Simpson, R.J. interviewed for: After record 340 days in space, Scott Kelly is coming down to Earth by Kim McGuire. Houston Chronicle, February 27, 2016: http://www.houstonchronicle.com/news/houston-texas/houston/article/After-record-340-days-in-space-Scott-Kelly-is-6858657.php

Simpson, R.J. Article in the Houston Chronicle regarding Dr Simpson’s NASA-funded research: http://www.houstonchronicle.com/local/history/city-of-possibilities/article/Trying-to-make-space-exploration-a-healthier-10415337.php?t=cd985e8469438d9cbb&cmpid=email-premium#photo-11641970

NOTE: End date changed to 11/2/2017 per NSSC information (Ed., 1/23/17)

NOTE: End date changed to 11/2/2016 per NSSC information (Ed., 7/17/15)

NOTE: Gap Immune05 deleted per IRP Rev E (Ed., 3/24/14)

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

Immune system dysregulation has been documented during and after spaceflight, but it is not known if these changes increase infection susceptibility or pose a significant health risk to crewmembers. Inherent problems with current in-flight research are small sample sizes and the difficulty to control for the many confounding factors that impact on the immune system. As such, it is not known if changes in immunity are due to the microgravity environment per se, or to the stressors associated with landing and re-adaptation to the 1G environment. The present Flight Definition investigation will utilize a longitudinal repeated measures design to determine the effects of long-term exposure to microgravity on a host of salivary antimicrobial proteins (AMPs), latent viral reactivation, antibacterial properties of saliva, and blood markers associated with innate host immune defense, whilst also considering the impact of other acute stressors such as Soyuz landing. Saliva, urine, and blood samples will be collected from crewmembers selected for ISS mission and ground-based controls pre-flight (L-180-L-45), At “early”, “mid,” and “late” phases during the 6-month period on the ISS, and up to R+63 on return to Earth. Saliva sampling was selected as the primary source because it is an excellent biological fluid with which to detect broad-spectrum biomarkers of front-line host immune defense and is suitable for the spaceflight environment. Attempts will also be made to establish relationships between salivary and cellular immune markers, viral reactivation and other stressors associated with spaceflight (i.e. mood state disturbances, circadian desynchronization, sleep loss/disruption, stress biomarkers) using serial data. Blood samples will be used for monocyte, NK-cell, and neutrophil phenotype and functional assays. This project will help to establish if spaceflight alters innate immune function, which is important to determine if altered immunity poses a significant risk of an adverse health event among crewmembers. Moreover, these data will serve as a foundation for future countermeasure developments and technological advances to detect real time changes in immune function during subsequent lunar or Mars missions.

Task Objective/Description

Year 3 objectives for this project were:

1. Continue test subject recruitment and complete all data collection procedures for 1 crewmember and 1 ground-based control subject; 2. Continue experimental procedures for all enrolled subjects; 3. Disseminate research findings to the scientific community.

Overview of Work Completed in Year 3

Study Progress: The study was initiated in September 2012 and data collection started in March 2013. As of October 2014, we have enrolled the required number of subjects giving us a sample size of six crewmembers and six ground-based controls. We also recruited a crewmember on the 1-year ISS mission and will continue to obtain samples from this subject into FY16. Baseline blood, urine, and saliva samples have been collected from all crewmembers and ground-based control subjects. One crewmember has completed all experimental procedures and a second crewmember has one remaining sample to provide before completing the entire protocol (this is currently scheduled for November 2014). We have obtained in flight samples and ambient blood returns from a total of three crewmembers to date. All baseline and in flight ambient blood samples were processed and analyzed successfully. Saliva, urine, and blood plasma samples have been stored at -80°C until analysis. Frozen samples will be analyzed on completion of the study protocol. The IRB protocol was renewed in July 2014.

Publications and Presentations:

Dr. Crucian and Dr. Simpson took the lead on compiling a review article focused on terrestrial analogs of spaceflight-induced immune system dysregulation. The manuscript entitled: “Terrestrial Stress Analogs for Spaceflight Associated Immune System Dysregulation” was published in Brain, Behavior and Immunity in July 2014 (Crucian, Simpson et al. 2014). The paper by Bigley et al. in which we developed the NK-cell functional assay for use in this study, was also published in Brain, Behavior and Immunity in July 2014 (Bigley, Rezvani et al. 2014). Our validation work for this project also allowed us to assess NK-cell function in the context of latent cytomegalovirus infection. This led to a submitted manuscript that is currently under review with the Journal of Immunology. We also submitted a manuscript to European Journal of Applied Physiology, which stemmed from the validation work of our saliva assays for this project (Kunz et al). This paper has been accepted subject to minor revisions. NASA funding was acknowledged in all of these publications. The work supported by this research grant was presented at the Human Research Program Investigator’s Workshop in February 2014 and the Psychoneuroimmunology Research Society Symposium in July 2014.

Current and Future Work

The study protocol is ongoing. By the end of year 4 we expect to have 4 crewmembers complete the entire experimental protocol. The current Soyuz schedule for our enrolled subjects means we will have to extend the project into a fifth year. This is required not only to complete the experimental procedures for the ‘Salivary Markers’ subjects, but also for the subject on the 1-year ISS mission. The 1-year mission subject is currently scheduled to return in March 2016 and our protocol requirements include the collection of biological samples up to 66 days after landing. Based on the current ‘Salivary Markers’ crewmember enrollment and Soyuz schedule, we expect to collect our final samples by May 2016 (45S). The remainder of FY16 will be spent analyzing frozen biological samples, interpreting data and producing scientific papers.

Bibliography

The following published papers (and papers in revision) were supported by this work:

Bigley, A. B., K. Rezvani, C. Chew, T. Sekine, M. Pistillo, B. Crucian, C. M. Bollard and R. J. Simpson (2014). "Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells." Brain Behav Immun 39: 160-171.

Bigley A.B., Rezvani K., Shah N., Sekine T., Spielmann G., Pistillo M., Agha N., Kunz H., LaVoy E.C.P., Bollard C.M., Simpson R.J. "Latent CMV infection enhances anti-tumor cytotoxicity through accumulation of NKG2C+ NK-cells in healthy humans". J Immunol (In Review, submitted September 2014).

Crucian, B., R. J. Simpson, S. Mehta, R. Stowe, A. Chouker, S. A. Hwang, J. K. Actor, A. P. Salam, D. Pierson and C. Sams (2014). "Terrestrial stress analogs for spaceflight associated immune system dysregulation." Brain Behav Immun 39: 23-32.

Kunz, H. Bishop, N.C., Spielmann, G., Pistillo, M., Reed, J., Ograjsek, T., Park, Y., Mehta, S., Pierson, D.L. and R.J. Simpson. “Fitness level impacts salivary antimicrobial responses to a single bout of cycling exercise”. Eur J Appl Physiol (In Revision, submitted July 2014)

The following presentations were delivered and supported by this work:

Bigley, A.B., G. Spielmann, E. C. LaVoy, M. Pistillo, H. Kunz, N. Agha, R. J. Simpson. “Latent cytomegalovirus infection enhances baseline anti-tumor cytotoxicity but impairs NK-cell responses to acute exercise through preferential expansion of NKG2C+ NK-cells in healthy humans”. Psychoneuroimmunology Research Society Annual Meeting, Philadelphia, PA, USA, May 28th -31st 2014.

Kunz, H., G. Spielmann, M. Pistillo, J. Reed, T. Ograjsek, R. J. Simpson. “The impact of workload and training status on salivary antimicrobial proteins following acute exercise”. Psychoneuroimmunology Research Society Annual Meeting, Philadelphia, PA, USA, May 28th -31st 2014.

Spielmann, G., A. B. Bigley, B. E. Crucian, S. Mehta, D. Pierson, H. Kunz, N. Agha, E. C. LaVoy and R. J. Simpson. “Immune cell phenotypes and NK-cell function in astronauts and controls 5 months before a 6-month mission to the International Space Station”. Psychoneuroimmunology Research Society Annual Meeting, Philadelphia, PA, USA, May 28th -31st 2014.

Spielmann, G. B. E. Crucian, S. Mehta, H. Kunz, D. Pierson, and R. J. Simpson. “The impact of long-duration spaceflight on plasma antimicrobial proteins”. NASA Human Research Program Investigator’s Workshop, Galveston, TX, USA, Feb 12th - 13th 2013.

NOTE: Gap Immune05 deleted per IRP Rev E (Ed., 3/24/14)

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

CPHS approval for the study was renewed in July 2013.

To date, 4 crewmembers and 2 backup crewmembers have agreed to participate in the study and have provided informed consent. Baseline blood, urine, and saliva samples have been collected from 4 crewmembers and 4 ground-based control subjects. All cellular blood products have been processed and analyzed. Saliva, urine, and blood plasma samples have been stored at -80°C until analysis.

Archived plasma samples from 20 crewmembers that participated in the ‘Integrated Immune’ study were analyzed for a range of antimicrobial proteins. A scientific manuscript is currently being prepared with these data. We expect to submit the paper to a scientific journal by the end of the 2013 calendar year. Dr. Guillaume Spielmann presented the work at the International Society of Exercise Immunology (ISEI) meeting in Newcastle, Australia in poster form. For this work he was awarded the New Investigator prize for best presentation. He also presented work on the effects of latent viral infections, age, and acute stress on immune cell distribution. This work was supported by our validation experiments and Dr. Spielmann was also awarded the New Investigator prize for best oral presentation. It was first time in ISEI history that a single investigator was awarded both prizes.

Finally, blood samples will be collected before and after the mission to determine the impact of spaceflight on cellular aspects of innate immunity. Given the potential of salivary AMPs to serve as an indicator of weakened immunity during spaceflight, this project will serve as a foundation for future countermeasure developments and technological advances to detect real time changes during subsequent lunar or Mars missions.

1. Determine the salivary antimicrobial proteins that can be accurately measured in saliva obtained using collection devices suitable for spaceflight and the microgravity environment

2. Determine the stability of all immune system biomarkers measured in both blood and saliva for up to 72h post collection

Funding for this project began on November 3rd 2011. This allowed us to start the hiring process for a postdoctoral research fellow (Dr Guillaume Spielmann) who was appointed in March 2012.

Objective #1 Summary

Our goal was to identify a saliva collection method that would be accurate for detecting a number of key project biomarkers and would also be suitable for implementation on the International Space Station (ISS). Although whole saliva would be considered the gold standard, our attempts to collect whole saliva samples in a microgravity environment using the saliva collection aid (SCA) and a C9 parabolic flight were not successful. We therefore attempted to validate our assays with saliva collected using the Salivette and salimetrics oral swab (SOS) technologies. It became apparent that the Salivette method (which uses a cotton roll to collect saliva) would not be suitable as analyte recovery was extremely low and was not related to pair-wise concentrations determined in whole saliva. On the other hand, the SOS technology, although not resulting in complete analyte recovery, was significantly associated with concentrations determined in whole saliva and was sensitive enough to detect within-subject changes following a bout of acute physical stress. Moreover, the relative change to acute stress for all saliva biomarkers measured was not different between the passive drool and SOS collection methods. These data indicate that the SOS method, in addition to its suitability to be implemented on the ISS, is capable of accurately measuring a wide range of salivary biomarkers to detect changes within subjects over time. Dr. Pierson and Dr. Mehta’s laboratory also attempted to recover viral DNA from SOS versus salivette and found that 10x more DNA could be recovered from the SOS.

Objective #2 Summary

Our goal was to determine the stability of the salivary and blood biomarkers and assays for up to 72h post thaw/collection to meet mission operational constraints for returning samples to Earth. Proportions of lymphocyte and monocyte subtypes appear to be stable in whole blood samples up to 48h post draw in either EDTA or ACD treated blood tubes. T-cell populations also appear to be stable up to 72h post draw. NK-cell function can be measured in both EDTA and ACD treated blood tubes and is stable for up to 48h post draw in ACD tubes. However, 48h stability was demonstrated in only 2 out of 4 samples so there is a need to increase the sample size here to determine the likelihood of NK-cell function being assessed on blood samples collected in-flight. The saliva biomarkers measured appear to be stable for up to 72h post thaw and are impervious to multiple repeat freeze-thaw cycles.

Current and Future Work

We are currently assessing the suitability of the SOS technology to measure additional biomarkers in saliva, including histatins, ß-defensins, lactoperoxidase, and C-reactive protein. We recently completed the analysis of 5 antimicrobial proteins in plasma samples obtained from 12 ISS astronauts from the previous “Integrated Immune” study. Analyses of these data are still ongoing. We are also in the process of optimizing assays to determine the antimicrobial capacity of saliva and neutrophil/monocyte oxidative burst and degranulation. Our first informed consent briefing to ISS crewmembers will take place in September 2012, with the first test subjects being recruited in March 2013. We anticipate that all feasibility work will be completed by January 31st 2013.