Immune Cell Response to Stimuli

The objective is to define the signaling pathways activated by lipopolysaccharide (LPS) and
other selected innate immunity stimuli, and the downstream inflammatory functional
consequences, in human leukocytes in vitro. Adult (greater than or equal to 18 - 65 years
old), nonpregnant, healthy volunteers will have 320 ml of whole blood collected by
venipuncture in a monitored setting no more frequently than once every 8 weeks. No further
interventions will be exercised upon the subjects. The whole blood will be fractionated into
neutrophil, red blood cell, mononuclear cell, and plasma fractions using plasma-Percoll
discontinuous centrifugation. Leukocytes will be subjected in vitro to inflammatory stimuli
(eg, LPS), and selected signaling outcomes (eg, mitogen-activated protein kinase activation,
Rho GTPase activation, protein-protein interactions) and functional measures (eg, chemotaxis,
superoxide anion and cytokine production) quantified in the absence and presence of relevant
chemical inhibitors (eg, SB203580, a p38 MAPK inhibitor). In each such experiment, cells from
the daily donor will be used as paired controls to the in vitro experimental intervention
(eg, SB203580 inhibitor vs. DMSO vehicle). Three or more repetitions (on different donors) of
each specific experimental outcome, as necessary, will be performed to establish statistical
significance of findings.

A specific focus of the studies planned will be to define the role of lipid raft membrane
microdomains in transduction of the LPS signal in human leukocytes. Lipid rafts are
cholesterol-rich microdomains in the plasma membrane, within which the LPS receptor,
Toll-like Receptor 4 (TLR4), has been described to reside. LPS signaling has been reported to
be sensitive to raft cholesterol content, presumably because the specific repertoire of
proteins in rafts is sensitive to raft cholesterol content. Rafts are thought to act as
dynamic signaling platforms for co-segregation of proximal adaptor proteins, kinases, and
other signaling proteins. Of interest, while LPS has been described to modulate the activity
of proteins that determine raft cholesterol content (eg, Liver X Receptor, ABCA1), virtually
no work has been done to clarify: 1) the mechanisms underlying LPS-induced intracellular
cholesterol redistribution, and, more importantly, 2) whether such intracellular
redistribution of cholesterol is causal to the signaling events triggered by LPS, or 3)
whether innate immunity signaling is dependent upon inter-subject variations in raft
cholesterol content.

Furthermore, we will investigate the role of the tumor suppressor gene p53 in the regulation
of inflammation. It is now widely accepted that inflammation and cancer development are
interconnected. Dr. Resnick is one of the international leaders in the study of the tumor
suppressor gene p53. His group has discovered that activation of p53 through exposure to
carcinogenic stimuli leads to differential expression of genes that have a direct effect on
the inflammatory response, such as several toll-like-receptor genes. Dr. Resnick will use
human leukocytes that will be isolated from whole blood. He will expose these cells to
stimuli that activate p53, such as doxorubicin (a chemotherapy agent) or radiation, and
examine the expression of toll-like-receptor genes as well as the response to LPS and other
inflammatory agents in vitro.

Finally, we will investigate the role of zinc-finger proteins in gene expression in
inflammatory cells. ZFP36 is an RNA binding zinc finger protein that is involved in the
turnover of mRNAs encoding several clinical important cytokines, including TNF(alpha).
Several missense, promoter and 3'-untranslated variants have been indentified in humans; in
some cases, these variants and their haplotypes have been associated with human inflammatory
disease. We will isolate peripheral blood macrophages and stimulate them with LPS and other
cytokines, then evaluate the behavior of cytokine mRNA and gene expression.

In addition to cell signaling experiments, we plan to test a novel detection system for the
presence of oxidized lipoprotein (LDL) in the blood. Inflammation in the body (like sepsis,
radiation injury, cancer) can induce the generation of reactive oxygen radicals (ROS) which
can react with proteins, DNA and other cell structures and alter their structure, therefore
causing cell damage. No reliable minimally invasive tests exist to detect biomarkers for
oxidative stress in humans. One such biomarker is N-formyl kynurenine (NFK) which can be
found on lipoproteins like LDL. We are developing polyclonal antiserum to NKF with the goal
of producing a simplified and high throughput method of detecting NFK via ELISA and Western
analyses. We propose to purify LDL from human serum by standard methods and use ELISA
analysis to determine if the samples contain KFK as detected using our anti-NFK polyclonal
serum. These experiments could lead to the development of a simple and reliable non-invasive
assay that detects a biomarker for oxidative stress in humans.

- INCLUSION CRITERIA:

- Normal, healthy adult donors as judged by screening questionnaire

- Nonpregnant

- Weighing at least 110 lbs

- 18-65 years of age

- HIV negative (proof required every 6 months we will conduct test)*

- Hepatitis B surface antigen and hepatitis C serology negative (checked every 6 months
we will conduct test)*

- The rationale for HIV and hepatitis viral testing is that chronic viral infection
may alter and possibly invalidate our experimental results. HIV and hepatitis
results will be confidentially obtained. Testing will be contracted to an
external certified laboratory and will be paid for by the study group. Results
will be available only to the study doctor/PI (Fessler), the study coordinator,
the CRU Director (Garantziotis, LAI), and the donor, with the few caveats that
follow

All positive HIV, hepatitis B, and hepatitis C results will be promptly communicated to the
donor by the study doctor/PI or the CRU Director. The participant will be referred to their
physician and/or to the N.C. Department of Health for confirmatory testing and counseling.
As explained in detail in the attached Supplement describing N.C. State Department of
Health code will be followed. The state code mandates reporting of positive results along
with the participant s name and identifying information to the N.C. Department of Public
Health. Upon contracting with the testing laboratory, clarification will be obtained and
documented as to whether the contracted laboratory or the study MD will be responsible for
reporting positive results to the state to avoid duplication of reporting. Upon receipt of
the test results, the N.C. Department of Health will contact the participant to inform them
of the positive result, how to find care, how to avoid infecting others, how the newly
diagnosed HIV and/or hepatitis infection is reported, and the importance of informing their
partners at possible risk because of their HIV and/or hepatitis infection. If the HIV,
hepatitis B, and hepatitis C results are negative, the participant will be not be notified.
However, the participant may contact the research study nurse for their results.

HIV and hepatitis B/C test results, non-reactive and reactive, will be documented
confidentially by the PI or study coordinator in the subject s file, and kept in a locked
file cabinet in the CRU Medical Records Room.In order to document the reporting procedure
and the time associated with the reporting process, a document has been created and placed
in the study specific manual (Hepatitis B/C and HIV Notification Process for Reactive
Results Form)

EXCLUSION CRITERIA:

By questionnaire:

Feeling ill within the last 24 hours.

Alcohol consumption in the last 24 hours.

Visit to the dentist in the last 24 hours.

A doctor visit for illness or vaccination in the last 2 weeks.

Diarrhea in the last 2 weeks.

Recurrent fever (4 weeks).

Pregnant or suspected pregnancy in the last 6 weeks.

Blood or plasma donation that will cause the participant to exceed 550ml of blood in the
last 8 weeks.

Receiving a blood donation in the past 12 months.

Bleeding disorder.

Anemia.

Heart problems.

Insulin dependent diabetes.

Problems with blood donation.

Risk of or evidence of Creutzfeldt-Jacob Disease in the family.

HIV-positive status, Hepatitis B/C-positive status or other confirmed or suspected
immunosuppressive or immunodeficient conditions.

Use of immunosuppressants or other immune-modifying drugs.

Use of selected medications within the preceding 5 days unless the PI or AI receiving the
samples states otherwise (NSAIDS/aspirin/tylenol, antidepressants, antihistamines ,
corticosteroids, HMG CoA reductase inhibitors, and antihypertensives).

By exam:

Temperature over 99.5 F.

Blood pressure less than 90/50.

Blood pressure higher than 170/95 mm Hg.

Pulse rate less than 50 or greater than 100 beats/minute.

If blood donation exceeds 200ml:

- Hematocrit less than 34% for women or less than 36% for men, or greater than 56% for
either gender.

- Patients will be informed of disqualifying vital signs and hematocrit values and
advised by trained staff, as appropriate, to seek assistance from their physician.