Download A Predictive Assay for Success Rates of Islet

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
Islet Cells
http://www.diabetesresearch.org
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 (HLAtyping) 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

Capture
antibody


Blocked
with protein
serum

Leukocytes or
splenocytes
are added
Cytokines
Released
Detection
Antibody

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-tocell ratio
Row
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
TNF Allo-Titration ELISPOT
TNF Allo-Titration ELISPOT
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
TNF Clinical
Patient ELISPOT
Positive
Controls


TNF Clinical
Patient ELISPOT
Positive
Controls

TNF Clinical
Patient ELISPOT

Negative
Controls


TNF Clinical
Patient ELISPOT

Successful transplant 
Failed Transplant

Failed Transplant

Successful Transplant
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