Mehta, Satish

Wilson, Brooke

NASA Technical Reports Server

Salivary biomarkers for stress and 
immune dysfunction 
Brooke Wilson
Cornell University, College of Veterinary Medicine
Satish Mehta
Microbiology
National Aeronautics and Space Administration
www.nasa.gov 
https://ntrs.nasa.gov/search.jsp?R=20140010672 2019-08-31T20:08:24+00:00Z
National Aeronautics and Space Administration Salivary biomarkers for stress and immune dysfunction 2
Introduction
• A little bit about me.....
National Aeronautics and Space Administration
Objectives of Internship
• Learn and have fun!
• Work to complete a project with the microbiology lab and assist 
in other areas as needed
Salivary biomarkers for stress and immune dysfunction 3
National Aeronautics and Space Administration
Project Objective
To measure an array of salivary hormones and cyotokines in 
healthy human adults to then correlate the levels of viral shedding 
and any changes in the microbiota of saliva which may have 
occurred due to stress.
Salivary biomarkers for stress and immune dysfunction 4
National Aeronautics and Space Administration
Background 
• Stress has been shown to lead to immune dysfunction 
• Stress can be measured in multiple ways
Blood
Urine
Saliva
• Spaceflight is a stressful environment!
Salivary biomarkers for stress and immune dysfunction 5
National Aeronautics and Space Administration
Background 
Salivary biomarkers for stress and immune dysfunction 6
• Immune dysfunction can lead to latent virus shedding and 
reactivation
Varicella zoster virus (can cause shingles) 
Epstein-Barr virus
etc.
• Potentially lead to major medical situations while on station
Various stressors associated with 
spaceflight acting through the 
hypothalamus-pituitary-adrenal 
(HPA)-axis is the basic mechanism 
involved. The HPA (brain and 
adrenal glands)-axis responding to 
stress increases the amount of 
stress hormones such as cortisol
from the adrenal glands, and the 
resulting reduction in cellular 
immunity allows the viruses to 
emerge from their latent state into 
an active infectious agent. 
7
National Aeronautics and Space Administration
Age groups: 20-35yr old 36-50yr old 51-70yr old
Female 3 0 3
Male 1 2 4
Total: 4 2 7
6am Noon 4pm
Day 1 13 12 12
Day 2 11 12 12
Day 3 12 11 12
Day 4 12 12 12
Total: 48 47 48
Project Design
Salivary biomarkers for stress and immune dysfunction 8
• Subjects- 13 total
• Time Points- 12 for each subject
National Aeronautics and Space Administration
Methods
Salivary biomarkers for stress and immune dysfunction 9
• Saliva was collect by passive drool method, aliquoted, and 
frozen at -80°C
• Multiple stress hormones were measured using Salimetrics 
assays
• Viral shedding was measured by real-time PCR in DNA 
extracted samples
• Cytokines were measured in collaboration with Dr. Brian 
Crucian's lab
• Microbiota analysis was performed using                           
illumina's MiSeq in collaboration                                                 
with Dr. Doug Botkin
National Aeronautics and Space Administration
Results: Cortisol
Salivary biomarkers for stress and immune dysfunction 10
Legend: Subjects (n=13) cortisol levels throughout four days of sample collection. Slight 
variation is seen between subjects but the overall trend is for cortisol levels to decrease 
throughout the day.
0.0000
0.2000
0.4000
0.6000
0.8000
1.0000
1.2000
1.4000
Day 1 AM Noon PM Day 2 AM Noon PM Day 3 AM Noon PM Day 4 AM Noon PM
C
o
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t
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(
u
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d
L
)
S1
S2
S3
S4
S5
S6
S7
S9
S10
S11
S12
S13
S14
National Aeronautics and Space Administration
Results: Cortisol
Salivary biomarkers for stress and immune dysfunction 11
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AM Noon PM
C
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(
u
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L
)
Day 1
Day 2
Day 3
Day 4
Legend: Subjects (n=13) cortisol levels averaged for each of the four days of sample 
collection. And the overall average (n=48) cortisol level throughout the day.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AM Noon PM
C
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(
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)
AVG
National Aeronautics and Space Administration
Results: Cortisol
Salivary biomarkers for stress and immune dysfunction 12
Legend: The overall average cortisol level throughout the day for females (n=24) and 
males (n=24). And for three different age groups, 20-35yr (n=16), 36-50yr (n=5), and 51-
70yr (n=24). No difference was seen in salivary cortisol levels due to age or sex.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AM Noon PM
C
o
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(
u
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/
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)
Gender
AVG (female)
AVG (male)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AM Noon PM
C
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(
u
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/
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)
Age
AVG (20-35)
AVG (36-50)
AVG (51-70)
National Aeronautics and Space Administration
Results: DHEA
Salivary biomarkers for stress and immune dysfunction 14
Legend: Subjects (n=13) DHEA levels averaged for each of the four days of sample 
collection. And the overall average (n=48) DHEA level throughout the day.
0
50
100
150
200
250
300
350
AM Noon PM
D
H
E
A
 
(
p
g
/
m
L
)
Day 1
Day 2
Day 3
Day 4
0
50
100
150
200
250
300
350
AM Noon PM
D
H
E
A
 
(
p
g
/
m
L
)
AVG
National Aeronautics and Space Administration
Results: DHEA
Salivary biomarkers for stress and immune dysfunction 15
Legend: The overall average DHEA level throughout the day for females (n=24) 
and males (n=24). And for three different age groups, 20-35yr (n=16), 36-50yr 
(n=5), and 51-70yr (n=24). No difference is seen in DHEA due to sex; however, 
DHEA levels peak in the 36-50yr old group before drastically dropping after the 
age of 50. 
0
50
100
150
200
250
300
350
AM Noon PM
D
H
E
A
 
(
p
g
/
m
L
)
Age
AVG (20-35)
AVG (36-50)
AVG (51-70)
0
50
100
150
200
250
300
350
AM Noon PM
D
H
E
A
 
(
p
g
/
m
L
)
Gender
AVG (female)
AVG (male)
National Aeronautics and Space Administration
Results: Cortisol:DHEA ratio
Salivary biomarkers for stress and immune dysfunction 17
Legend: Subjects (n=13) cortisol:DHEA ratio averaged for each of the four days of 
sample collection. And the overall average (n=48) of cortisol:DHEA ratio throughout the 
day.
0
50
100
150
200
250
300
AM Noon PM
C
o
r
t
i
s
o
l
:
D
H
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A
Day 1
Day 2
Day 3
Day 4
0
50
100
150
200
250
300
AM Noon PM
C
o
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t
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s
o
l
:
D
H
E
A
AVG
National Aeronautics and Space Administration
Results: Cortisol:DHEA ratio
Salivary biomarkers for stress and immune dysfunction 18
Legend: The overall average cortisol:DHEA ratio throughout the day for females 
(n=24) and males (n=24). And for three different age groups, 20-35yr (n=16), 
36-50yr (n=5), and 51-70yr (n=24). No difference is seen in cortisol:DHEA ratio 
due to sex; however, the ratio appears to be higher in subjects over the age of 
50. 
0
50
100
150
200
250
300
AM Noon PM
C
o
r
t
i
s
o
l
:
D
H
E
A
Gender
AVG (female)
AVG (male)
0
50
100
150
200
250
300
AM Noon PM
C
o
r
t
i
s
o
l
:
D
H
E
A
Age
AVG (20-35)
AVG (36-50)
AVG (51-70)
National Aeronautics and Space Administration
Results: α-amylase
Salivary biomarkers for stress and immune dysfunction 20
Legend: Subjects (n=13) α-amylase concentrations averaged for each of the four 
days of sample collection. And the overall average (n=48) of α-amylase
concentration throughout the day.
0
50
100
150
200
250
300
350
400
450
500
AM Noon PM
a
l
p
h
a
-
A
m
y
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(
U
/
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)
Day 1
Day 2
Day 3
Day 4
0
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1 2 3
a
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p
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a
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m
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(
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AVG
National Aeronautics and Space Administration
Conclusion
Salivary biomarkers for stress and immune dysfunction 22
• Salivary cortisol levels begin high in the morning and decrease 
throughout the day; appear to be unaffected by age or gender
• Salivary DHEA levels have a similar diurnal pattern as cortisol; 
levels peak in middle aged subjects and then significantly 
decline after the age of 50
• The Cortisol:DHEA ratio has a more gradual decline throughout 
the day; ratio increases in subjects that are older than 50
• Salivary α-amylase levels gradually increase through the day; 
levels become more consistent throughout the day in subjects 
that are over 50 years old
National Aeronautics and Space Administration
Conclusion
Salivary biomarkers for stress and immune dysfunction 23
• Data has been collected but not analyzed for salivary cytokines 
• EBV expression has been measured but not analyzed; VZV and 
CMV will also be measured
• Data on the microbiota of each saliva sample is currently 
underway
• Further samples and data collection on healthy                     
adults are required to obtain a enough                             
information to validate the preliminary findings
• This information will serve as a baseline of                                
data for normal values that can be used to                         
compare against samples obtained from                          
astronauts
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Acknowledgments
• Lauren Merkle and Judy Hayes
• Missy Matthias and Diego Rodriguez
• The microbiology and immunology labs, Brian Crucian, Doug Botkin, and 
Cherie Oubre
• Duane Pierson, Satish Mehta, and Ashlie Renner
Salivary biomarkers for stress and immune dysfunction 24
National Aeronautics and Space Administration Your Title Here 25


Salivary Biomarkers for Stress and Immune Dysfunction

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