Immune System Responses Related to Environmental

Uranium Exposure – DiNEH Project Results

E. Erdei & J. LewisUniversity of New Mexico, Health Sciences Center,

College of Pharmacy, Community Environmental Health Program

Navajo Nation Human Research Review Board Conference

Window Rock, AZ

November 16, 2011

Environmental Legacy Exposures Increase the Likelihood of Several Diseases: Autoimmune

DiseaseFigures below show similar increases in risks for autoimmune disease (self-reported) based on an increase from 1 to 2 activities

DiNEH Survey Responses

35.9%

25.1%

17.1%

6.2% 5.4% 5.1%3.5% 3.1% 3.1% 2.5%

45.4%

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

Prevalence of Self-Reported Health Conditions Among 1,304 DiNEH Survey Participants

(*Cancer prevalence based on 1,011 participants surveyed)

Goal of this portion of the study

• Direct response to community members’ requests for research on immune system function during the capacity building and environmental risk evaluation work

• Address possible pathways within the human body in association with environmental uranium and other heavy metal exposures

• Find early indicators of

health effects

DiNEH biological sample collection

• DiNEH project participants from 20 chapters

• Samples collected from 267 individuals

• A subset has been analyzed for immune biomarkers (N=65)

• Early markers, showing alterations

in immune cell distribution and activity

Flow cytometry measurements

• Lymphocyte subpopulations from whole blood samples.

• Becton Dickinson Simultest IMK Plus lymphocyte kit was used.

• 6 cell populations were measured:– T cells (CD3+), T helpers (CD4+), T suppressors

(CD8+); – B cells (CD19+); – HLA-DR+ cell activation in T cells and– B cells and other cell types; NK cells

(CD3-/CD16+/CD56+).

UNM HSC Core Facility

• Director: Dr. Bruce Edwards

• Flow cytometry machine

• FACScan 2 lasers, 2-color

simultaneous detection of lymphocytes

• Membrane markers, CD coding

Flow cytometry results I.CD3+ T cells (in %) among DiNEH participants

y = 25.601x + 44.298

R2 = 0.0446

0

10

20

30

40

50

60

70

80

90

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Exposure score

Perc

en

tag

e o

f cell

s

CD19+ B cells (in %) among DiNEH participants

y = 16.366x + 0.8651

R2 = 0.1538

0

5

10

15

20

25

30

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Exposure Score

Per

cen

tag

e o

f ce

lls

Flow cytometry results II.

• Increased percentage of activated T cells

• Decreased percentage of activated B cells

• Decoupling of T cell and B cell activities suggest altered immune response among this subset of participants

• Can lead to lower production of protective antibodies

• Preliminary interpretation of data; more complex modeling incorporating other variables pending

Serum cytokines

• Cytokines are small molecular weight proteins produced by immune cells and other cells through the human body

• Their role is to promote communication, activation processes in the immune system

• Cytokine productions show immune status and disease developmental pathways

Serum cytokine measurements

• Applied xMAP multiplexing technology

• UNM HSC Core Facility- Luminex 100™ detection (96-well format)

• Detection of 10 human serum cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, INF-γ, TNF-α, and

GM-CSF) high sensitivity assay

• Uses only 50 μl of serum sample/ participant

Serum cytokine concentrations related to environmental uranium waste exposures

IFN-g cytokine and Exposure Score among DiNEH project participants

y = 0.0832x + 0.0385

R2 = 0.0265

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Exp. score

IFN

-g [

pg

/ml)

IL-4 (hypereactivity) cytokine production and Exposure Score among DiNEH participants

y = 0.2936x + 0.1276

R2 = 0.1202

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Exp. score

IL-4

cc.

[pg

/ml]

Interleukin-8 (IL-8; mucosal protection) cytokine and Exposure Score among DiNEH project participants

y = 3.8629x + 2.2598

R2 = 0.00660

5

10

15

20

25

30

35

40

45

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Exp. Score

cyto

kin

e cc

. [p

g/m

l]

Serum cytokine concentrations (pg/ml) related to environmental uranium

waste exposures (N=47)• Percent of variance by exposure for IL-4 and IL-1ß suggests a

potential inflammatory process

ConclusionsBased on our preliminary analysis• DiNEH participants with increased exposure to

uranium waste had increased number of activated T cells and decreased activity of B cells and other antigen-producing cells

• If consistent w/ other modeling results (e.g. water source) – indication of the decoupling of the immune response

• Alterations in cytokine production indicative of the presence of an inflammatory response

Future directions

• These results are based on our preliminary analyses – work in progress

• To continue to complete immune assays for entire sample set

• Further modeling works will allow us more detailed evaluation of suggestive inflammatory response

• In connection with other pathway analyses –cardiovascular process

DiNEH Acknowledgements

• NIEHS, EPA and UNM for financial support• Community Advisory Board

– Ed Carlisle, Jay DeGroat, Herbert Enrico, Thomas Manning,Sr., Lynnea Smith, Jean Whitehorse,

• UNM-HSC Community Environmental Health Program & Clinical and Translational Science Center

– Johnnye L. Lewis, PhD; Miranda Cajero, BCH; Matthew Campen, PhD; Jeremy DeGroat; Mallery Downs, RN; Eszter Erdei, PhD; Molly Harmon; Gabriel Huerta, PhD; Curtis Miller; Bernadette Pacheco; Glenn Stark; Mary Woodruff; research nursing support

• Crownpoint Service Unit, I H S– Virgil Davis

• Navajo Area IHS– Lisa Allee, CNM; John Hubbard; Ryan

Johnson, MD; Doug Peter, MD• UT-Houston Nephrology

– Donald Molony, MD• Southwest Research Information Center

– Chris Shuey, MPH, Sarah Henio-Adeky, Teddy Nez, Sandy Ramone

• Students– Jamie deLemos, PhD – Tufts Univ.– Christine George – Stanford Univ.– Tommy Rock, MA, UNM Health Policy

Student– Christine Samuel-Nakamura, PhD

Candidate, UCLA• Dartmouth

– Ben Bostick, PhD• University of Arizona Cancer Center &

Northern Arizona University, NACRP– Jani Ingram, PhD, Margaret Briehl, PhD

• USEPA Region IX– Harry Allen, Rich Bauer, Clancy Tenley

• State of New Mexico Diagnostic Laboratory• Navajo Nation EPA Air Quality Division, Public

Water Supply Supervision Program, Superfund Program

• Navajo Nation Division of Health• Former Contributors:

– Bess Seschillie, Bernice Norton, Jerry Elwood, Harrison Gorman, Harris Arthur (in memoriam), Alta McCabe, Margaret Menache, PhD, Alexis Kaminsky, PhD; Eastern Navajo Health Board

• Thanks to the many others who’ve contributed

Project funding support

• DiNEH project – supported through the following grants

• NIEHS, RO1 ES014565; R25  ES013208; P30 ES-012072;

• USEPA/ERRG pass through contract; with support from DHHS/NIH/NCRR #1UL1RR031977-01 

Questions?

THANK YOU!