In-vivo Effects of E-cigarette Aerosol on Innate Lung Host Defense

Investigators will evaluate the acute effect of CA-flavored e-cigs on MCC and IS immune cells
in up to 22 healthy, young adults who are current e-cig users with a total of less than 10
pack-years cigarette smoking history. MCC will be measured by gamma scintigraphy at baseline
and following controlled vaping of e-liquids with and without cinnamon flavoring. Two
different e-liquids (one completely devoid and one containing at least 30mM CA similar to
"Hot Cinnamon Candies" which is commercially available) will be used for two separate
randomized vaping sessions.

The randomization scheme for the two different e-liquids (e-liquids with and without CA) will
be generated by using the Web site Randomization.com (http://www.randomization.com), assigned
treatment Regimen A and B by an assigned study team member, and provided to the study team.
This individual will also be responsible for loading the e-cigarette with the appropriate
solution for that session prior to the vaping sessions.

Participants will undergo baseline testing during the screening visit, which will occur 2-3
weeks prior to the first controlled vaping session. Investigators will also recruit
non-vaping control subjects (n=22), who will only undergo the baseline testing and thus serve
as a non-exposed/non-vaping control group. will aim to recruit similar numbers of males and
females in both cohorts. While investigators cannot guarantee age-matching and sex-matching
in these cohorts, based on our previous studies, investigators do not expect to find
significant age and sex differences in the two cohort. In addition, potential confounders,
such as age, sex, and BMI will be included as covariates in our multivariate analysis.

Observations obtained from the non-vaping control group will provide necessary information on
potential baseline differences in the two cohorts (i.e. current vapers versus non-vaping
controls). These data from the non-vaping control group are important to provide a reference
for any potential CA-induced changes in the vaping group. Hence, there are two stages of the
study:

Stage 1. A cross sectional observational cohort comparison of baseline MCC and IS immune
cells in a reference cohort of n=22 non-vaping control subjects and E-cig cohort of n=22
currently vaping subjects (confounding based on other variables such as BMI, sex, age is
possible for this stage).

Stage 2. A randomized comparison of changes in MCC and IS immune cells after Regimen A (e-cig
us without CA) and Regimen B (e-cig use with CA). The cohort of e-cig users will undergo a
randomized 2-treatment, 2-period, 2-sequence crossover study of CA exposure.

For stage 1, baseline measurements of Tc99m-SC clearance will be used to measure each
subject's normal baseline MCC and IS immune cell characteristics. For both stages, subjects
will be asked to complete a vaping diary to record information on the device and e-liquids
(name/vendor/e-liquids/puffs/device settings) used during their normal vaping sessions for
the entire duration of the study. In addition, for stage 2, participants will be asked to
maintain their current habits for the duration of the study, not to significantly increase or
decrease their vaping patterns, including the nicotine concentrations of their e-liquids.

For stage 2, for each e-cig vaping session (Training and MCC Test Days), subjects will be
asked to follow a laboratory-based protocol involving 6, 5-minute paced vaping segments (1
puff/minute) over a 1 hour time period, vaping the e-liquid with and without CA provided by
us. On each Test Day, participants will undergo the vaping protocol immediately prior to
inhalation of the Tc99m-SC (10 min between end of vaping and inhalation of Tc99m-SC). An
initial deposition scan of Tc99m-SC will then be obtained followed by dynamic imaging of the
lung with subjects seated in front of the gamma camera to determine potential changes in MCC
induced by acute exposure to CA-flavored e- cigarettes. Induced sputum samples will be
collected at baseline, and after each MCC scan.

24 hours after completion of the MCC scans. The two randomized vaping sessions will be
separated by 2-3 weeks. While there are no data providing specific information on the
duration needed to washout the effects of CA on MCC, previous studies examining changes in
MCC following inhalation of other aerosols have shown that this washout period is sufficient
to prevent potential carryover between the two treatments.

Inclusion Criteria:

- An equal number of participants who currently use a vaping device and those who do not
use a vaping device

- Age 18-40

- Must have a Forced Vital Capacity (FVC) and Forced Expiratory Volume in 1 second
(FEV₁) of at least 80% of predicted. Participants who fall out of the normal range
will be offered a copy of the test to share with their personal physician.

Exclusion Criteria:

- Any pre-existing lung disease (asthma, cystic fibrosis, etc.)

- Any significant chronic illness, such as, but not limited to, heart disease,
uncontrolled hypertension, diabetes, auto-immune disease

- Any use of tobacco products (other than e-cig) in the past 3 months, or a greater than
10 pack year history of smoking cigarettes

- Pregnant or nursing women

- Participants with a history of radiation exposure in the past year which exceeds
annual safe limits.