1. Johnson Space Center 1 Salivary Markers Overview Human Research Facility The Effect of Long-Term Exposure to Microgravity on Salivary Markers of Innate Immunity Overview Training Instructors: Rickie Simpson713-743-9270rjsimpso@central.eh.edu.comrjsimpso@central.eh.edu.com Ali Schade281-218-3071alissa.zvada@lmco.comalissa.zvada@lmco.com

2. Johnson Space Center 2 Salivary Markers Overview Human Research Facility Session Objectives Identify the science objectives of the Salivary Markers experiment. Identify the time sequence for the pre-flight, on-orbit, and post-flight operations. Describe how to properly collect saliva and urine samples using the saliva collection hardware and Urine Collection Kit. Describe when samples are stowed in MELFI. Describe how to complete the Health Assessment.

3. Johnson Space Center 3 Salivary Markers Overview Human Research Facility The Salivary Markers Experiment will assess whether spaceflight induced immune system dysregulation increases infection susceptibility or poses a significant health risk to crewmembers onboard the International Space Station. Maintaining a robust immune system during prolonged spaceflight missions (i.e., to other planets or asteroids) will be important to ensure mission success. If any impairments in immunity are found due to spaceflight, this will allow us to develop appropriate countermeasures to help mitigate the risks. Science Overview

4. Johnson Space Center 4 Salivary Markers Overview Human Research Facility Spaceflight and the Immune System 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 Infectious episodes reported among crewmembers aboard the Mir station (i.e. conjunctivitis, acute respiratory and dental infections) Many microorganisms have been identified in space vehicles including Escherichia coli, Salmonella enterica, Bacillus subtilis, Staphylococci, Micrococci, and Coryneform Crewmembers have been found to shed live Varicella Zoster Virus (VZV) and other latent viruses both in-flight and on return to Earth Impaired immunity coupled with increased pathogen exposure could trigger a number of adverse events that could compromise crew safety and mission objectives.

5. Johnson Space Center 5 Salivary Markers Overview Human Research Facility POTENTIAL CONSEQUENCES OF PROLONGED IMMUNE DYSREGULATION DURING EXPLORATION- CLASS SPACEFLIGHT Increased incidence of infection Allergy/hypersensitivity reactions Viral reactivation Impaired tumor surveillance, increased incidence of malignancies Impaired wound healing Any adverse clinical event related to immune dysfunction would likely have an immediate mission impact.

6. Johnson Space Center 6 Salivary Markers Overview Human Research Facility Salivary Markers Specific Assays Salivary Antimicrobial Proteins/peptides Detailed leukocyte phenotype assays: -T-cells -NK-cells -Monocytes -B-cells -Dendritic cells Flow Cytometry Cellular Functional assays: -NK-cell cytotoxicity -Neutrophil degranulation -TLR ligand stimulations -Mitogenic T-cell stimulations Latent Viral Reactivation: -EBV (saliva) -VZV (saliva) -CMV (urine) Antibacterial capacity of saliva: -E. coli inhibition assay

7. Johnson Space Center 7 Salivary Markers Overview Human Research Facility The Salivary Markers study will collect blood, saliva, urine and a health assessment before, during and after a 6-month mission to the ISS. You will receive experiment unique blood training after you’ve completed several sessions of Generic Phlebotomy. Experiment Overview

8. Johnson Space Center 8 Salivary Markers Overview Human Research Facility Experiment Design Overview KEY: B: Single blood collection (29ml) collected S: Saliva sampling (3x oral swab) * On FD10, only 19ml of blood will be collected and frozen U: Single 24h urine collection. P: Health assessment

9. Johnson Space Center 9 Salivary Markers Overview Human Research Facility In-Flight 7 Day Saliva Collection 24 Hour Urine Collection Health Assessment Blood Collection FD-10FD-90* Soyuz Launch Soyuz Landing Handover Soyuz Undock R-1 Operations: Activities occur on or around FD10, FD90, and R-1 Saliva collection extends over a 7 day period Urine collection is collected over a 24hr period Blood collection and health assessment are each performed in a single session Total Collections: -3 blood collections (two are ambient return) -3 saliva collection sessions -3 urine collections -3 health assessments *The FD-90 window will be flexible to permit ambient blood to be returned on Soyuz.

10. Johnson Space Center 10 Salivary Markers Overview Human Research Facility Saliva Collection Saliva will be collected every day for 7 days. (21 samples per collection) The daily collection will include one timed sample and two untimed samples. Collections must occur at the beginning of crew day before eating, drinking, or brushing teeth. The timed sample will be collected prior to the untimed samples. Samples must be performed one at a time.

11. Johnson Space Center 11 Salivary Markers Overview Human Research Facility Saliva Collection Hardware Stored In HRF Supply Kits Timed Saliva Session Pack: Saliva Vial Salivette Saliva Session Pack: Saliva Bag Salivette Bag Salivette Session Packs can be located within crew quarters, stowed in Blood Collection Belt, for sample collection during post-sleep

12. Johnson Space Center 12 Salivary Markers Overview Human Research Facility Timed Saliva Collection Synthetic Salivette is placed under the tongue (sublingual) for at least 2 minutes but not longer than 3 minutes (Salivette must not become saturated) Do not touch Salivette with hands Do not bite down on or compress the Salivette. The duration, in seconds, that the Salivette was under the tongue is timed and recorded on Saliva Vial (measures flow rate) Salivette is placed into Saliva Vial after collection Saliva Vial is labeled, stowed in 3x5 Mesh Bag and inserted into MELFI

13. Johnson Space Center 13 Salivary Markers Overview Human Research Facility Untimed Saliva Collection First untimed Salivette is placed in mouth and rolled over the tongue from cheek to cheek for 3-4 minutes until saturated. Salivette is placed into Saliva bag Second untimed salivette is placed under tongue (sublingual) for 4-5 minutes until saturated and placed into Saliva bag Do not touch Salivette with hands Do not bite down on or compress the Salivette. Saliva Bags are labeled, stowed in 3x5 Mesh bags and inserted into MELFI

14. Johnson Space Center 14 Salivary Markers Overview Human Research Facility Saliva Collection Review Samples placed in Saliva Vial or Saliva Bag must be labeled with crewmember ID and GMT. Timed collection must include number of seconds Salivette was under tongue. For untimed collections, a check must be placed in either “Sublingual” or “Rolled” box Barcodes must be called down, placed in crew notes or scanned with the BCR/RFID Scanner MELFI insertion must occur within 30 minutes of collection

15. Johnson Space Center 15 Salivary Markers Overview Human Research Facility 24 Hour Urine Collection 24 hour collection will be shared with other experiments when possible Begins with first urine void after waking After each void, two Frozen Syringes are filled and stowed in MELFI Frozen Syringes must be labeled with Crew ID (your initials) and GMT to the minute Frozen Syringes are placed in a 3x5 Mesh Bag prior to MELFI insertion within 30 minutes of collection Frozen Syringe barcodes may be called down, placed in crew notes or scanned with the BCR/RFID Scanner

16. Johnson Space Center 16 Salivary Markers Overview Human Research Facility 24 Hour Urine Collection Urine Collection Kit: Urine Collection Device Urine Tube Kit: Frozen Syringes Urine Collection Device (male catheter held at left)

17. Johnson Space Center 17 Salivary Markers Overview Human Research Facility 24 Hour Urine Collection Urine Collection Kit: Urine Collection Device (UCD) Designed with adaptors for males or females at one end A blue sample port is situated at the other end Frozen Syringe tips are inserted into blue port to remove samples Contains a Lithium Chloride (LiCl) volume marker Urine voids must be mixed thoroughly with LiCl before sample removal Male adaptor (urine entry point) Sample port Urine Collection Device: One UCD will be used for every void.

18. Johnson Space Center 18 Salivary Markers Overview Human Research Facility Urine Tube Kit: Frozen Syringes Two Frozen Syringes will be filled per void. (The tips are capped in this picture.) Frozen Syringe: tip at left (cap is removed) is inserted into UCD sample port. Plunger at left is extended to draw sample into tube.

19. Johnson Space Center 19 Salivary Markers Overview Human Research Facility Urine Collection Hardware: Urine Containment Bag (UCB) After Frozen Syringes are filled from a Urine Collection Device, the Urine Collection Device should be returned to a sealed ziplock bag and disposed of in the Urine Containment Bag (below)

20. Johnson Space Center 20 Salivary Markers Overview Human Research Facility Urine Collection Summary: Void into Urine Collection Device and mix. Insert Frozen Syringe into blue port of Urine Collection Device. Draw sample (7 ml), by pulling the plunger until the black sharpie mark is visible. Place Frozen Syringes in 3 x 5 Mesh Bag then insert into MELFI. Reseal used UCD in its ziplock bag and dispose in Urine Containment Bag. Label Frozen Syringes with Crew ID and GMT day and time to the minute.

21. Johnson Space Center 21 Salivary Markers Overview Human Research Facility Health Assessment Done through the NASA Data Collection Tool (DCT) on the SSC Station Support Computer (SSC) Health Assessment

22. Johnson Space Center 22 Salivary Markers Overview Human Research Facility Critical Points for Successful Science Return For Saliva Collection Do NOT touch the Salivette with anything other than your mouth Perform saliva collection at crew wake before eating, drinking and brushing teeth Saliva Bags/Vials must be labeled and barcode called down, placed in crew notes or scanned via the BCR/RFID scanner Salivettes must be placed in MELFI within 30 minutes of collection For Urine Collection Thoroughly mix urine void in collection device before removing samples Do not draw more than 7ml into each Frozen Syringe (black sharpie mark on the plunger is your guide) to allow for freezer expansion Frozen Syringes must be labeled with time of void and Crew ID. After each sample collection, barcodes may be reported by calling down, placing them in crew notes or using the BCR/RFID Scanner Collection extends over a full 24-hours Frozen Syringes must be placed in MELFI within 30 minutes of collection

23. Johnson Space Center 23 Salivary Markers Overview Human Research Facility BCRBarcode Reader BDCBaseline Data Collection CMVCytomegalovirus DCTData Collection Tool EBVEpstein-Barr Virus FDFlight day GMTGreenwich Mean Time Hrhour HRFHuman Research Facility LLaunch LiClLithium Chloride MELFIMinus Eighty Laboratory Freezer for ISS METMission Elapsed Time NK CellNatural Killer Cell OprOperator OSTPVObnoard Short Term Plan Viewer OvOverview PAOPublic Affairs Office PDLPayload Development Lab PIPrincipal Investigator P/LPayload RReturn RFIDRadio Frequency Identification TLRToll-Like Receptor UCBUrine Containment Bag UCDUrine Collection Device UCKUrine Collection Kit VZVVaricella Zoster Virus Acronyms and Abbreviations