a predictive assay for success rates of islet transplantation to treat type-1 diabetes tracy fuad...
TRANSCRIPT
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A Predictive Assay for Success Rates
of Islet Transplantation to Treat Type-1 Diabetes
Tracy Fuad
2007
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Diabetes Institute of Immunology and Transplantation
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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
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Type-1 Diabetes• An auto-immune
disease• Characterized by loss
of pancreatic islet cell function
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Type-1 Diabetes• An auto-immune
disease• Characterized by loss
of pancreatic islet cell function
http://www.diabetesresearch.org
Islet Cells
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Islet Transplantation
http://www.rsna.org/rsna/media/
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Immune-Mediated Graft Rejection• Patient’s immune system recognizes
islets as foreign
• Immune system attacks and destroys transplanted tissues
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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
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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
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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
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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
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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
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Methods: ELISPOT Assay
• Uses a 96-well plate with nitrocellulose membranes
• Quantifies cytokines by capturing them locally and visualizing each cytokine
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ELISPOT well
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Capture antibody
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Capture antibody
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Blocked with protein serum
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Leukocytes or splenocytes are added
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Cytokines Released
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Detection Antibody
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Detection Antibody
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Strepdavidan-HRP,
a colored substrate
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Precipitation reaction
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Reading the ELISPOT Plate
http://www.biosciencetechnology.com/images
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Reading the ELISPOT Plate
www.elispot.com/index.html?elispot_reader/software.html
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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
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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
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TNF Allo-Titration ELISPOT
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TNF Allo-Titration ELISPOT
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TNF Allo-Titration ELISPOT
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TNF Allo-Titration ELISPOT
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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
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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)
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TNF Clinical Patient ELISPOT
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TNF Clinical Patient ELISPOT
PositiveControls
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TNF Clinical Patient ELISPOT
PositiveControls
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TNF Clinical Patient ELISPOT
Negative Controls
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TNF Clinical Patient ELISPOT
Failed Transplant
Successful transplant
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0
10
20
30
40
50
60
70
80
Patient+Donor 1 Patient+Donor 2 Patient+3rd Party
spots/well
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0
10
20
30
40
50
60
70
80
Patient+Donor 1 Patient+Donor 2 Patient+3rd Party
spots/well
Failed Transplant
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0
10
20
30
40
50
60
70
80
Patient+Donor 1 Patient+Donor 2 Patient+3rd Party
spots/well
Successful Transplant
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0
10
20
30
40
50
60
70
80
Patient+Donor 1 Patient+Donor 2 Patient+3rd Party
spots/well
p=0.0000892
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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
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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
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Future Studies• Test samples from additional islet
transplant patients
• Test patient samples from different points in transplantation timeline
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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
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A Predictive Assay for Success Rates
of Islet Transplantation to Treat Type-1 Diabetes
Tracy Fuad
2007