2. Study of cytokine gene polymorphism and graft outcome in live-donor kidney transplantation By Rashad Hassan MD Amgad El-Agroudy, Ahmad Hamdy, Amani Mostafa and Mohamed Sobh UNC

3. INTRODUCTION

4. Renal transplantation is the treatment of choice for patients afflicted with end stage renal disease. Patient as well as allograft survival rates exceed 90% at one year. However, acute rejection remains a formidable clinical challenge and is the most serious frequent complication in the first year of transplantation (Suthanthiran, 2000).

5. Acute rejection is also a risk factor for the subsequent development of chronic rejection. Indeed, it has been pointed out repeatedly that whereas one-year graft survival rates have improved substantially in the last decade, similar improvements have not been witnessed in the long-term outcome of renal allografts (Suthanthiran, 2000).

6. The unresolved issue then is why, with similar levels of HLA-incompatibility and managed with identical immuno-suppressive regimens, some patients suffer from rejection while others escape. For example, 20-30% of recipients of HLA-1 haplotype matched allografts experience acute rejection despite receiving the same immunosuppressive drugs that protected the other 70-80% of recipients (Suthanthiran, 2000).

7. An interesting and testable hypothesis for the clinical heterogeneity and differential responsiveness in allograft recipients is genetic variation.

8. Aim Of Work

9. Study the impact of IL-2 & IL-10 gene polymorphism on graft outcome

10. PATIENTS AND METHODS

11. The material of this work comprised fifty recipients of their first renal transplants who were transplanted in our center between May 2001 and February 2003. Exclusion criteria: 1-Patients who had prior transplantation. 2-Recipients below 18 years. 3-Pre-transplant chemistries demonstrating a total cholesterol greater than 300 mg/dl, triglycerides greater than 400 mg/dl, white blood cell count less than 4000/mm3 or platelets less than 150,000/mm3 {as hyperlipidemia and bone marrow suppression are well- known side effects of sirolimus}. 4-Change of the basal immunosuppressive protocol for whatever reason

12. The patients were randomized prior to transplantation into two groups: 1-Group (1) patients {38 patients} received oral prednisolone, sirolimus and Mycophenolate Mofetil. 2-Group (2) patients {12 patients} received oral prednisolone, sirolimus and Tacrolimus. All patients in both groups were given basiliximab (Simulect) 20 mg intravenously at surgery and on day 4 postoperative.

13. One year post transplantation, 43 patients were subjected to protocol biopsy Five years post transplantation, blood sampling and examination for IL-2 and IL-10 gene polymorphisms using RT-PCR technique. The patients were classified into low, intermediate or high producers of either cytokine.

14. Interpretation of results IL2 -330 genotypes: -TT----------Low producer -TG---------Intermediate producer -GG---------High producer (Reynard et al., 2000, McDaniel et al., 2003). IL10 -1082 genotypes : -AA----------Low producer -GA---------Intermediate producer -GG---------High producer (Perrey et al., 1998, Reynard et al., 2000, McDaniel et al., 2003).

15. RESULTS

16. Table (1) Demographic and clinical characteristics of the 50 renal allograft recipients at commencement of the study. ____________________________________________________________ 1. Recipient age 33.86 ± 9.79 (19-57) M ± SD (range), years ________________________________________________________ 2. Recipient gender 38/12 (76%) M/F (M %) ________________________________________________________ 3. Donor age, M ± SD 34.38+10.42 (21-65) (range), years ________________________________________________________ 4. Donor gender 24/26 (48%) M/F (M %) ________________________________________________________ 5. Donor relation (% of related) 41/9 (82%) R/U ________________________________________________________ 6. Duration of HD3 15.88+ 15.74 (0-48) M ± SD (range), months ________________________________________________________ 7. Number of pre-transplant blood 3.11+2.89 (1-10) M ± SD (range), units ------------------------------------------------------------------------------------- 8. Post-transplant follow up 72.48+6.28 (62-85) M ± SD (range), months

17. Table (2) Comparison between MMF group (group 1) and FK group (group 2) Group 1 (n=38) Group 2 (n=12) P value Number of acute rejection -zero -one -two * Proteinuria - No - >1gm/day for>6 months * Degree of proteinuria - 1-3 gm/day - > 3 gm/day * Onset of proteinuria (median), months *Protocol biopsy -Yes -No *Result of protocol biopsy -Normal -Chronic tubulo-interstitial fibrosis (mild degree) -Chronic tubulo-interstitial fibrosis (moderate degree) 3 4 1 29 9 (n=9) 7 2 8 31 7 9 22 0 10 2 0 11 1 (n=1) 1 0 4 12 0 2 8 2 0.735 0.246 0.598 0.537 0.109 0.058

18. I Analysis of the data in relation to IL-2 production

19. Table (3) Relation between number, degree & management in acute rejection episodes; incidence of chronic allograft nephropathy and IL-2 production IL-2 production P value Low (n=28) Intermediate (n=12) High (n=10) *Number of acute rejection episodes Zero - one - two *Degree of 1st acute rejection -ACR grade 1 -BLR *Management of 1st acute rejection -steroids -other therapy *Degree of 2nd acute rejection -ACR grade 1 -BLR *Management of 2nd acute rejection -steroids -other therapy *Chronic allograft nephropathy -No -Yes 24 4 0 1 3 4 -- 0 -- 0 -- 24 4 11 1 0 1 -- 0 -- 0 -- 12 0 8 1 0 2 -- 1 -- 1 -- 10 0 0.357 0.646 0.181

20. Table (4) Relation between results, Banff classification of the graft biopsies; results of protocol biopsies and IL-2 production IL-2 production P value Low (n=28) Intermediate (n=12) High (n=10) *Result of 1st biopsy -Insufficient -Normal -ACR1 -Acute FK toxicity *Banff classification of 1st biopsy -BLR2 -ACR1grade 1 *Result of 2nd biopsy -ACR1 -Acute FK toxicity *Banff classification of 2nd biopsy -BLR -ACR1 grade 1 *Result of protocol biopsy -Normal -Chronic tubulo-interstitial fibrosis (mild degree) -Chronic tubulo-interstitial fibrosis (moderate degree) 0 3 1 2 1 0 3 1 2 1 (n=25) 6 17 2 1 0 1 0 (n=9) 3 6 0 1 2 0 2 0 1 0 1 (n=9) 2 7 0 0.620 0.567 0.248 0.768

21. Table (5) Relation between graft function at serial follow up intervals and IL-2 production IL-2 production P value Low (n=28) Intermediate (n=12) High (n=10) Creatinine at 14 days M+SD (range), mg/dl Creatinine at 1 month M+SD (range), mg/dl * Creatinine at 3 months M+SD (range), mg/dl * Creatinine at 6 months M+SD (range), mg/dl * Creatinine at 12 months M+SD (range), mg/dl * Creatinine at 24 months M+SD (range), mg/dl * Creatinine at 36 months M+SD (range), mg/dl * Creatinine at 48 months M+SD (range), mg/dl * Creatinine at 60 months M+SD (range), mg/dl * Creatinine at 72 months M+SD (range), mg/dl * Creatinine at last follow up M+SD (range), mg/dl 1.09±0.19 1.17±0.21 1.17±0.22 1.18±0.28 1.15±0.29 1.20±0.32 1.22±0.27 1.31±0.32 1.31±0.31 1.33±0.37 1.32±0.37 1.09±0.25 1.08±0.23 1.09±0.26 1.13±0.30 1.13±0.26 1.14±0.31 1.17±0.31 1.17±0.22 1.15±0.24 1.23±0.28 1.23±0.33 1.18±0.34 1.13±0.26 1.20±0.33 1.20±0.32 1.23±0.27 1.28±0.26 1.36±0.28 1.3±0.22 1.33±0.25 1.38±0.29 1.40±0.31 0.591 0.485 0.563 0.822 0.679 0.585 0.292 0.335 0.208 0.532 0.540

22. Table (6) Relation between graft & patient survival and IL-2 production IL-2 production P value Low (n=28) Intermediate (n=12) High (n=10) *Period of functioning graft M+SD (range), months *Condition at last follow up -alive with functioning graft -alive with graft failure -died with functioning graft -died with graft failure 73.32±6.45 28 -- 71.17±5.54 12 -- 71.70±6.88 10 -- 0.564

23. II Analysis of the data in relation to IL-10 production

24. Table (7) Relation between number, degree & management in acute rejection episodes; incidence of chronic allograft nephropathy and IL-10 production IL-10 production P value Low (n=22) Intermediate (n=15) High (n=13) *Number of acute rejection episodes Zero - one - two *Degree of 1st acute rejection -ACR grade 1 -BLR *Management of 1st acute rejection -steroids -other therapy *Degree of 2nd acute rejection -ACR grade 1 -BLR *Management of 2nd acute rejection -steroids -other therapy *Chronic allograft nephropathy -No -Yes 17 4 1 4 5 -- 1 -- 1 -- 20 2 14 1 0 1 -- 0 -- 0 -- 14 1 12 1 0 1 -- 0 -- 0 -- 12 1 0.578 0.792 0.964

25. Table (8) Relation between results, Banff classification of the graft biopsies; results of protocol biopsies and IL-10 production IL-10 production P value Low (n=22) Intermediate (n=15) High (n=13) *Result of 1st biopsy -Insufficient -Normal -ACR1 -Acute FK toxicity *Banff classification of 1st biopsy -BLR2 -ACR1grade 1 *Result of 2nd biopsy -ACR1 -Acute FK toxicity *Banff classification of 2nd biopsy -BLR -ACR1 grade 1 *Result of protocol biopsy -Normal -Chronic tubulo-interstitial fibrosis (mild degree) -Chronic tubulo-interstitial fibrosis (moderate degree) 1 2 4 1 4 -- 2 0 2 (n=20) 5 14 1 0 1 0 1 0 -- 1 0 1 0 (n=13) 5 7 1 0 2 0 -- 1 0 1 0 (n=10) 1 9 0 0.385 0.595 0.135 0.465

26. Table (9) Relation between graft function at serial follow up intervals and IL-10 production IL-10 production P value Low (n=22) Intermediate (n=15) High (n=13) Creatinine at 14 days M+SD (range), mg/dl Creatinine at 1 month M+SD (range), mg/dl * Creatinine at 3 months M+SD (range), mg/dl * Creatinine at 6 months M+SD (range), mg/dl * Creatinine at 12 months M+SD (range), mg/dl * Creatinine at 24 months M+SD (range), mg/dl * Creatinine at 36 months M+SD (range), mg/dl * Creatinine at 48 months M+SD (range), mg/dl * Creatinine at 60 months M+SD (range), mg/dl * Creatinine at 72 months M+SD (range), mg/dl * Creatinine at last follow up M+SD (range), mg/dl 1.11±0.20 1.12±0.21 1.21±0.27 1.23±0.34 1.19±0.34 1.28±0.37 1.25±0.34 1.28±0.32 1.29±0.32 1.34±0.37 1.36±0.36 1.04±0.22 1.10±0.28 1.11±0.27 1.11±0.26 1.14±0.19 1.14±0.28 1.23±0.25 1.23±0.24 1.24±0.24 1.25±0.25 1.25±0.27 1.18±0.27 1.22±0.17 1.12±0.19 1.13±0.21 1.13±0.23 1.16±0.18 1.23±0.22 1.31±0.27 1.31±0.29 1.35±0.37 1.32±0.43 0.357 0.341 0.387 0.380 0.751 0.349 0.967 0.785 0.816 0.708 0.671

27. Table (10) Relation between graft & patient survival and IL-10 production IL-10 production P value Low (n=22) Intermediate (n=15) High (n=13) *Period of functioning graft M+SD (range), months *Condition at last follow up -alive with functioning graft -alive with graft failure -died with functioning graft -died with graft failure 73.68±6.63 22 -- 71.53±5.36 15 -- 71.54±6.78 13 -- 0.496

28. III Analysis of the data in relation to combined IL-2 & IL-10 production

29. Group 1: High IL-2 producers & High IL-10 producers. Group 2: High IL-2 producers & Intermediate IL-10 producers. Group 3: High IL-2 producers & Low IL-10 producers. Group 4: Intermediate IL-2 producers & High IL-10 producers. Group 5: Intermediate IL-2 producers & Intermediate IL-10 producers. Group 6: Intermediate IL-2 producers & Low IL-10 producers. Group 7: Low IL-2 producers & High IL-10 producers. Group 8: Low IL-2 producers & Intermediate IL-10 producers. Group 9: Low IL-2 producers & Low IL-10 producers.

30. Table (11) Relation between number, degree & management in acute rejection episodes; incidence of chronic allograft nephropathy and combined IL production IL Combination P value 1 (n=3) 2 (n=2) 3 (n=5) 4 (n=2) 5 (n=4) 6 (n=6) 7 (n=8) 8 (n=9) 9 (n=11) *Number of AR episodes Zero - one - two *Degree of 1st AR -ACR grade 1 -BLR *Management of 1st AR -steroids -other therapy *Degree of 2nd AR -ACR grade 1 -BLR *Management of 2nd AR -steroids -other therapy *CAN -No -Yes 3 0 -- 0 -- 0 -- 3 0 2 0 -- 0 -- 0 -- 2 0 3 1 0 2 -- 1 -- 1 -- 5 0 2 0 -- 0 -- 0 -- 2 0 4 0 -- 0 -- 0 -- 4 0 5 1 0 1 -- 0 -- 0 -- 6 0 7 1 0 1 -- 0 -- 0 -- 7 1 8 1 0 1 -- 0 -- 0 -- 8 1 9 2 0 1 2 -- 0 -- 0 -- 9 2 0.762 0.572 0.875

31. Table (12) Relation between graft function at serial follow up intervals and combined IL production IL Combination P value 1 (n=3) 2 (n=2) 3 (n=5) 4 (n=2) 5 (n=4) 6 (n=6) 7 (n=8) 8 (n=9) 9 (n=11) Creatinine at 14 days M+SD (range), mg/dl Creatinine at 1 month M+SD (range), mg/dl * Creatinine at 3 months M+SD (range), mg/dl * Creatinine at 6 months M+SD (range), mg/dl * Creatinine at 12 months M+SD (range), mg/dl * Creatinine at 24 months M+SD (range), mg/dl * Creatinine at 36 months M+SD (range), mg/dl * Creatinine at 48 months M+SD (range), mg/dl * Creatinine at 60 months M+SD (range), mg/dl * Creatinine at 72 months M+SD (range), mg/dl * Creatinine at last follow up M+SD (range), mg/dl 1.3±0.4 1.3±0.2 1.1±0.2 1.2±0.2 1.2±0.3 1.3±0.2 1.4±0.2 1.3±0.2 1.2±0.2 1.2±0.1 0.7±0.3 0.8±0.3 0.8±0.4 0.8±0.2 1.0±0.2 1.0±0.3 1.1±0.1 1.2±0.4 1.1±0.3 1.2±0.6 1.2±0.1 1.1±0.2 1.4±0.2 1.3±0.2 1.4±0.2 1.3±0.1 1.4±0.1 1.5±0.2 1.2±0.4 1.1±0.3 1.2±0.4 1.0±0.3 1.0±0.2 1.1±0.2 1.1±0.1 1.2±0.4 1.3±0.4 1.2±0.3 1.0±0.1 0.9±0.2 0.9±0.1 1.0±0.1 1.3±0.1 0.9±0.9 1.1±0.2 1.0±0.1 1.5±0.1 1.1±0.1 1.1±0.2 1.2±0.3 1.2±0.4 1.2±0.1 1.2±0.2 1.2±0.3 1.3±0.4 1.1±0.1 1.2±0.1 1.1±0.1 1.1±0.2 1.1±0.1 1.3±0.2 1.3±0.3 1.3±0.4 1.3±0.5 1.1±0.1 1.2±0.2 1.2±0.1 1.2±0.3 1.3±0.2 1.3±0.1 1.3±0.2 1.5±0.2 1.1±0.2 1.1±0.3 1.2±0.4 1.1±0.3 1.2±0.4 1.2±0.3 1.2±0.4 1.3±0.3 0.175 0.354 0.204 0.468 0.769 0.643 0.530 0.825 0.677 0.769 0.912

32. Table (13) Relation between graft & patient survival and combined IL production IL Combination P value 1 (n=3) 2 (n=2) 3 (n=5) 4 (n=2) 5 (n=4) 6 (n=6) 7 (n=8) 8 (n=9) 9 (n=11) *Period of functioning graft M+SD (range), months *Condition at last follow up -alive with functioning graft -alive with graft failure -died with functioning graft -died with graft failure 68.7±8.9 3 -- 74.0±5.6 2 -- 72.6±6.9 5 -- 69.5±3.5 2 -- 67.0±3.5 4 -- 74.5±5.3 6 -- 73.1±6.8 8 -- 73.0±5.2 9 -- 73.7±7.6 11 -- 0.686

33. CONCLUSIONS

34. 1- IL-2 & IL-10 gene polymorphism had no impact on the number or the degree of acute rejection episodes among the study groups. 2- IL-2 & IL-10 gene polymorphism had no impact on the incidence of chronic allograft rejection or the pathological findings in protocol biopsies among the study groups.

35. 3 - il-2 & IL-10 gene polymorphism had no impact on either patient or graft survival. 4- In our study, we found no impact of IL-2 & IL-10 different combinations on incidence and degree of acute rejection episodes, incidence of chronic allograft nephropathy, pathological changes in protocol biopsies, graft function and graft and patient survivals.

36. THANK YOU