1. A Predictive Assay for Success Rates of Islet Transplantation to Treat Type-1 Diabetes Tracy Fuad 2007

2. Diabetes Institute of Immunology and Transplantation

3. 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

4. Type-1 Diabetes An auto-immune disease Characterized by loss of pancreatic islet cell function

5. Type-1 Diabetes An auto-immune disease Characterized by loss of pancreatic islet cell function http://www.diabetesresearch.org Islet Cells

6. Islet Transplantation http://www.rsna.org/rsna/media/

7. Immune-Mediated Graft Rejection Patient’s immune system recognizes islets as foreign Immune system attacks and destroys transplanted tissues

8. 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

9. 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

10. 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

11. 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

12. 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

13. Methods: ELISPOT Assay Uses a 96-well plate with nitrocellulose membranes Quantifies cytokines by capturing them locally and visualizing each cytokine

14.  ELISPOT well

15. Capture antibody 

16. 

17. Blocked with protein serum 

18. Leukocytes or splenocytes are added 

19. Cytokines Released

20.  Detection Antibody

21. 

22.  Strepdavidan-HRP, a colored substrate 

23.  Precipitation reaction

24. Reading the ELISPOT Plate  http://www.biosciencetechnology.com/images

25. Reading the ELISPOT Plate www.elispot.com/index.html?elispot_reader/software.html

26. 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

27. Cell-to-Cell ELISPOT Used to find the optimal donor-to-patient cell-to- cell ratio RowDescription, Columns 1-3Description, Columns 4-6 AMedia controlResponder cells alone BResponder cells (R) + PHAResponder cells (R) + Con-A CMatched stimulator (S1)Mismatched stimulator (S2) D9:1 (S1) to (R) cell to cell ratio9:1 (S2) to (R) cell to cell ratio E3:1 (S1) to (R) cell to cell ratio3:1 (S2) to (R) cell to cell ratio F1:1 (S1) to (R) cell to cell ratio1:1 (S2) to (R) cell to cell ratio G1:3 (S1) to (R) cell to cell ratio1:3 (S2) to (R) cell to cell ratio H1:9 (S1) to (R) cell to cell ratio1:9 (S2) to (R) cell to cell ratio

28. TNF  Allo-Titration ELISPOT

31. 

32. 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

33. Donor-to-Patient Induced Immune Response Modified ELISPOT protocol used with the following plate map: RowDescriptionCells/well APatient Cells Alone90,000 BPatient cells with PHA90,000 CPatient cells with Con-A90,000 DDonor-Transplant 1270,000 EDonor-Transplant 2270,000 FPatient (P) + Donor 1 (D1)90,000 (P) + 270,000 (D1) GPatient (P) + Donor 2 (D2)90,000 (P) + 270,000 (D2) HPatient (P) + 3 rd party (3P)90,000 (P) + 270,000 (3P)

34. TNF  Clinical Patient ELISPOT

35. Positive Controls  

36. TNF  Clinical Patient ELISPOT Positive Controls 

37. TNF  Clinical Patient ELISPOT    Negative Controls

38. TNF  Clinical Patient ELISPOT   Failed Transplant Successful transplant

40.  Failed Transplant

41.  Successful Transplant

42. p=0.0000892

43. 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

44. 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

45. Future Studies Test samples from additional islet transplant patients Test patient samples from different points in transplantation timeline

46. 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

47. A Predictive Assay for Success Rates of Islet Transplantation to Treat Type-1 Diabetes Tracy Fuad 2007