a predictive assay for success rates of islet transplantation to treat type-1 diabetes tracy fuad...

A Predictive Assay for Success Rates

of Islet Transplantation to Treat Type-1 Diabetes

Tracy Fuad

2007

Diabetes Institute of Immunology and Transplantation

Objective• Engineer an assay to improve

success rates of islet transplantation in type-1 diabetics– As a tool to match patients to donors– As an in vitro monitoring tool

Type-1 Diabetes• An auto-immune

disease• Characterized by loss

of pancreatic islet cell function

Type-1 Diabetes• An auto-immune

disease• Characterized by loss

of pancreatic islet cell function

http://www.diabetesresearch.org

Islet Cells

Islet Transplantation

http://www.rsna.org/rsna/media/

Immune-Mediated Graft Rejection• Patient’s immune system recognizes

islets as foreign

• Immune system attacks and destroys transplanted tissues

HLA-Matching• Human leukocyte-antigen typing (HLA-

typing) matches tissues to minimize the immune response in a transplant

• Immune response can be also be measured by cytokine production

ELISPOT Assay

• Enzyme-linked immunospot assay

• Quantifies the cytokine production of lymphocytes (white blood cells)

• TNF is an inflammatory cytokine that is produced during transplantation and rejection

Goals• Optimize ELISPOT assay to test TNF

production in donor-stimulated patient cells

• Use the modified ELISPOT protocol to look for a correlation between failed transplants and elevated TNF production

Previous Studies

• Studies by Augustine et al. and Bellisola et al. have correlated heightened IFN production in an ELISPOT assay to increased rates of renal transplant rejection

Hypothesis

• Because IFN is an inflammatory cytokine often produced in conjunction with TNF, I hypothesized that heightened TNF production would correlate with failed islet transplants

Methods: ELISPOT Assay

• Uses a 96-well plate with nitrocellulose membranes

• Quantifies cytokines by capturing them locally and visualizing each cytokine

ELISPOT well

Capture antibody

Blocked with protein serum

Leukocytes or splenocytes are added

Cytokines Released

Detection Antibody

Strepdavidan-HRP,

a colored substrate

Precipitation reaction

Reading the ELISPOT Plate

http://www.biosciencetechnology.com/images

Reading the ELISPOT Plate

www.elispot.com/index.html?elispot_reader/software.html

ELISPOT Modification

• Increased the protein concentration of blocking buffers

• Reduced the number of cells per well

• Decreased secondary detection antibody

incubation time

• Added more washes between steps

Cell-to-Cell ELISPOT• Used to find the optimal donor-to-patient cell-to-

cell ratioRow Description, Columns 1-3 Description, Columns 4-6

A Media control Responder cells alone

B Responder cells (R) + PHA Responder cells (R) + Con-A

C Matched stimulator (S1) Mismatched stimulator (S2)

D 9:1 (S1) to (R) cell to cell ratio 9:1 (S2) to (R) cell to cell ratio

E 3:1 (S1) to (R) cell to cell ratio 3:1 (S2) to (R) cell to cell ratio

F 1:1 (S1) to (R) cell to cell ratio 1:1 (S2) to (R) cell to cell ratio

G 1:3 (S1) to (R) cell to cell ratio 1:3 (S2) to (R) cell to cell ratio

H 1:9 (S1) to (R) cell to cell ratio 1:9 (S2) to (R) cell to cell ratio

TNF Allo-Titration ELISPOT

Donor-to-Patient Induced Immune Response

• Islet transplant patient was selected

– First transplant failed

– Second transplant was successful

• Patient cells stimulated with cells from each donor

Donor-to-Patient Induced Immune Response

• Modified ELISPOT protocol used with the following plate map:

Row Description Cells/well

A Patient Cells Alone 90,000

B Patient cells with PHA 90,000

C Patient cells with Con-A 90,000

D Donor-Transplant 1 270,000

E Donor-Transplant 2 270,000

F Patient (P) + Donor 1 (D1) 90,000 (P) + 270,000 (D1)

G Patient (P) + Donor 2 (D2) 90,000 (P) + 270,000 (D2)

H Patient (P) + 3rd party (3P) 90,000 (P) + 270,000 (3P)

TNF Clinical Patient ELISPOT


PositiveControls


Negative Controls


Failed Transplant

Successful transplant

0

10

20

30

40

50

60

70

80

Patient+Donor 1 Patient+Donor 2 Patient+3rd Party

spots/well

0

10

20

30

40

50

60

70

80


spots/well

Failed Transplant

0

10

20

30

40

50

60

70

80


spots/well

Successful Transplant

0

10

20

30

40

50

60

70

80


spots/well

p=0.0000892

Conclusion

• TNF ELISPOT assay is an effective means to measure immune response in transplantation patients

• Heightened TNF production has a statistically significant correlation to failed transplantation

Applications• A tool to match patients to donors

• An in vitro monitoring assay–Identify immunosuppression needs of

individual patients

–Reduce the use of immunosuppresant medications

Future Studies• Test samples from additional islet

transplant patients

• Test patient samples from different points in transplantation timeline

Acknowledgements• The University of Minnesota and Dr.

Bernard Hering

• Dr. Pratima Pakala, Kelly Hire, Adam Nettles, and Olivia Thai

• Ms. Fruen and the Science Research Class

A Predictive Assay for Success Rates

of Islet Transplantation to Treat Type-1 Diabetes

Tracy Fuad

2007

a predictive assay for success rates of islet transplantation to treat type-1 diabetes tracy fuad...

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