Influence of Lifestyle Factors on Neutrophil Migration Pilot Study

This study will investigate how external factors implicated in immunity such as exercise,
caffeine ingestion and ethanol consumption influence neutrophil migration.

Neutrophils are the most prominent immune cell in human blood and are involved in a complex
equilibrium of immune protection and autoimmune damage. Their recruitment to an inflammatory
or wounding site is controlled by the sensing and directed migration to a concentration
gradient of attractant molecules, a process called chemotaxis. Immune cells are also
implicated in many diseases including cancer. The ability to measure the amplitude of a
response over time for a specific patient, and the variation of this response when the
patient engages in certain activities or consumes certain substances will improve
understanding of how certain lifestyle factors impact the immune response. Traditional assays
require large volumes of blood and a long purification process, which may affect neutrophil
function and strictly limits the number of draws possible from a single patient. The novel
microfluidic assay proposed limits these drawbacks as it has the capability to purify
neutrophils from a 3 µL drop of blood in less than 5 minutes and measure their chemotaxis in
a gradient of chemokines. Critically, the proposed studies will begin to fill a gap in
current understanding of immune response as previous studies focused on single endpoints
likely missing early events in the response to external stimulus. Understanding this temporal
response may have implications in the development of new treatments as well as improvements
in diagnosis of improper immune response.

The KOALA (Kit On A Lid Assay) approach was developed in Professor Dave Beebe's lab and has
been validated in a mouse model and in human asthmatic patients. In a collaboration with Dr.
Anna Huttenlocher, it has been shown that, in contrast to traditional neutrophil
purification, KOALA can be performed with small volumes of blood, and a much quicker
purification time.

Due to its unique qualities, KOALA allows for repeated evaluation of neutrophil adhesion and
chemotaxis properties, thus making it an attractive method for studying dynamic neutrophil
changes that may occur as a result of an external factor.

Using traditional macrobiology tools, researchers have identified several factors that may
play important roles in reducing neutrophil responsiveness and migration ability. Lifestyle
and diet factors, amongst others, have been shown to impact neutrophil count, migration and
biochemical function. For example, sleep deprivation has been linked with higher neutrophil
count, despite a known immuno-depressive effect. Physical exercise results in increased
neutrophil counts and increased neutrophil degranulation. Dietary factors, such as caffeine
and ethanol have been shown to impact immune function.

To build on this research, the investigators intend to probe the role of these lifestyle
factors on neutrophil migration over much shorter time scales than has been previously
studied. The investigators propose to examine the effects of exercise, ethanol ingestion and
caffeine consumption on neutrophil behavior.

Whole blood obtained from subjects by finger stick will be used in KOALA to isolate
neutrophils. Neutrophil function will be assessed by measuring absolute migration speed,
chemotactic index and chemotaxis velocity (directional velocity toward the formation of the
gradient of chemoattractant).

The primary outcome of this study will be to determine whether exercise, caffeine consumption
and ethanol ingestion affect neutrophil chemotactic velocity. The secondary outcomes are to
determine whether exercise, caffeine consumption and ethanol ingestion affect the absolute
speed and chemotactic index of neutrophils.

Inclusion Criteria:

- Capacity to provide informed consent and ability to speak and read English.

- Age 21-40 years (participants in the ethanol study will be required to present ID to
verify their age)

- Male or female with no chronic or acute health concerns that might affect subject
safety during the study or interfere with the study results

- No intake of medication that the researchers believe will significantly influence
immune function in the 48 hours proceeding the lancet puncture (examples given in the
section entitled "Exclusion Criteria")

- In good physical health

- Regularly exercise at least 30 minutes 3 times per week (exercise cohort)

Exclusion Criteria:

- Currently participating in another clinical trial

- History of significant systemic disease (eg. cancer, infection, hematological, renal,
hepatic, coronary artery disease or other cardiovascular disease, endocrinologic,
neurologic, rheumatologic, or gastrointestinal disease)

- Use of beta blockers or corticosteriods

- Currently taking medications that are not recommended to be taken in conjunction with
alcohol

- Acute illness or evidence of clinically significant active infection

- Currently receiving immunotherapy

- Pregnant women

- Ingested medication (e.g. systemic corticosteroids) within 48 hours preceding the draw
that the researchers believe may have an effect on immune response or the immune
system

- Performed any activity that conflicts (eg. drinking any alcohol prior to the study),
in the judgment of the investigator, with the external factor to be tested in the
study (if any)

- Alcoholic or other health conditions for which alcohol consumption is contraindicated

- Consume more than 7 drinks per week (women alcohol cohort)

- Consume more than 14 drinks per week (men alcohol cohort)

- Consume more than three (8 oz.) servings of coffee, caffeinated soft drinks/tea (12
oz.) per day (caffeine cohort)