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A Predictive Assay for Success Rates of Islet Transplantation to Treat Type-1 Diabetes

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

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A Predictive Assay for Success Rates of Islet Transplantation to Treat Type-1 Diabetes

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

  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. Capture antibody

  17. Blocked with protein serum

  18. Leukocytes or splenocytes are added

  19. Cytokines Released

  20. Detection Antibody

  21. Detection Antibody

  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

  28. TNF Allo-Titration ELISPOT

  29. TNF Allo-Titration ELISPOT

  30. TNF Allo-Titration ELISPOT

  31. TNF Allo-Titration ELISPOT

  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:

  34. TNF Clinical Patient ELISPOT

  35. TNF Clinical Patient ELISPOT  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

  39. Failed Transplant

  40. Successful Transplant

  41. p=0.0000892

  42. 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

  43. 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

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

  45. 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

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

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