1 A Phase 2 Clinical Trial of Donor Specifi c Tolerance Induction in Recipients of HLA Disparate Living Donor Kidney Allografts by Donor Stem Cell Infusion. J. Leventhal,1 M. Abecassis,1 J. Miller,1 L. Gallon,1 R. Herzig,2 D. Tollerud,2 B. King,2 S. Ildstad.2 1Transplant Center, Northwestern Univ., Chicago, IL; 2Institute for Cellular Therapeutics, U of Louisville, Louisville, KY. Background: We have demonstrated that durable chimerism without GVHD can be established with minimal toxicity in highly-mismatched kidney Tx recipients through nonmyeloablative conditioning followed by infusion of a bioengineered stem cell graft. We herein provide intermediate-term F/U on our phase 2 trial. Methods: 14 HLA mismatched living donor renal Tx recipients entered into a tolerogenic protocol involving low intensity conditioning (fl udarabine, cyclophosphamide, 200cGy TBI days -4 to -1). Pts received a kidney Tx on day 0, then infusion of cryopreserved facilitating cell (FC)-enriched donor-derived CD34+ HSCT on Day +1. Subjects were discharged by POD 2 and managed as outpts. Maintenance IS consisted of Prograf and MMF w.o steroids. Weaning of IS occurred over a one year period. Results: Pt data is summarized below. All but one pt demonstrated peripheral blood macrochimerism post-Tx. Engraftment failure occurred in a highly sensitized (PRA > 40%) completely mismatched Tx recipient (NW9). Chimerism was lost in 4 pts at 2 (NW11), 3 (NW4), 6 (NW1) and 9 (NW12) months post-Tx. All pts demonstrated donor-specifi c hyporesponsiveness in the year post-Tx and were weaned from full dose IS. Complete IS withdrawal was successful in 6 of 10 pts with durable chimerism who are ≥ one year post-Tx; pts have been off IS for 2, 4, 7, 8, 9, & 19 months. There has been no GVHD or “engraftment syndrome”. Adverse events include herpes zoster reactivation in 3 pts; no CMV infection has occurred. Tx loss occurred in one pt (NW2) who developed aplastic anemia and sepsis following an atypical viral infection; successful rescue with banked autologous HSCT and subsequent re-Tx with a living donor kidney was performed. Conclusions: Low intensity conditioning + FC enriched HSCT can safely achieve durable chimerism in mismatched kidney Tx recipients, allowing for IS withdrawal. Prior sensitization represents an obstacle to successful induction of chimerism and tolerance induction. DISCLOSURE: King, B.: Employee, regenerex llc. Ildstad, S.: Employee, regenerex llc. Abstract# 2 B Cell Derived IL-10 Is Required for Co-Stimulatory Blockade Induced Tolerance. G. Lal,1 Y. Nakayama,2 B. E. Burrell,2 A. Sethi,1 Y. Ding,2 J. S. Bromberg.2 1National Centre for Cell Science, Pune, MH, India; 2University of Maryland, Baltimore, MD. Background: The anti-infl ammatory cytokine IL-10 plays a very important role in the maintenance of tolerance. Whether specifi c subsets of B cells and B cell derived IL-10 contribute to the generation of transplantation tolerance are not known. We hypothesized that specifi c subsets of B cells produce IL-10 that is required for tolerization. Methods: C57BL/6 mice received vascularized BALB/c cardiac allografts along with donor-specifi c splenocyte transfusion (DST, on day -7 prior to transplant) and anti-CD40L mAb (on days -7, -4, 0 and +4). One dose of anti-mouse CD20 mAb was administered on day +1 to deplete B cells. We created a novel mouse strain, CD19-cre+/+::IL-10fl /fl transgenic mice, in which IL-10 is specifi cally depleted in B cells, and they were used to investigate the function of B cell derived IL-10. Results: B cell depletion prevented co-stimulatory blockade induced allogeneic tolerance. Phenotypic analysis of different B cell subsets showed that tolerization with DST and anti-CD40L mAb did not significantly change the number of CD19+CD5+CD1d+ regulatory B cells (Breg) in spleen and lymph nodes. However, anti-CD20 mAb treatment significantly changed the number and percentage of the CD19+CD93-CD23+sIgMhisIgDhiCD21/CD35hi marginal zone precursor (MZP) subset in spleen and lymph nodes. DST plus anti-CD40L mAb treatment showed increased IL-10 mRNA expression in non-Breg cells, furthermore B cell depletion causes decreased IL-10 production in the residual MZP cells. Allogeneic transplantation under tolerogenic conditions in CD19-Cre+/+::IL-10fl /fl mice lead to acute rejection of the allograft, compared to in littermate controls, which resulted in tolerance. Immunohistochemical analysis of allografts showed signifi cantly increased perivascular and mononuclear infi ltration in the rejected grafts. CD19-Cre+/+::IL10fl /fl mice did not have any signifi cant change in the CD19+, CD4+, CD8+, Foxp3+ Treg, or Breg cell numbers or percentages in spleen and lymph nodes. However, MZP B cell number was reduced in the rejecting IL-10 defi cient mice compared to littermates in secondary lymphoid organs. Conclusion: IL-10 expression in B cell is required for the generation of transplantation tolerance. MZP subsets of B cell express IL-10 and MZP subset localization in the secondary lymphoid organs plays an important role in tolerance. These results demonstrate a novel subset of B cells is required for tolerization. 2 B Cell Derived IL-10 Is Required for Co-Stimulatory Blockade Induced Tolerance. G. Lal,1 Y. Nakayama,2 B. E. Burrell,2 A. Sethi,1 Y. Ding,2 J. S. Bromberg.2 1National Centre for Cell Science, Pune, MH, India; 2University of Maryland, Baltimore, MD. Background: The anti-infl ammatory cytokine IL-10 plays a very important role in the maintenance of tolerance. Whether specifi c subsets of B cells and B cell derived IL-10 contribute to the generation of transplantation tolerance are not known. We hypothesized that specifi c subsets of B cells produce IL-10 that is required for tolerization. Methods: C57BL/6 mice received vascularized BALB/c cardiac allografts along with donor-specifi c splenocyte transfusion (DST, on day -7 prior to transplant) and anti-CD40L mAb (on days -7, -4, 0 and +4). One dose of anti-mouse CD20 mAb was administered on day +1 to deplete B cells. We created a novel mouse strain, CD19-cre+/+::IL-10fl /fl transgenic mice, in which IL-10 is specifi cally depleted in B cells, and they were used to investigate the function of B cell derived IL-10. Results: B cell depletion prevented co-stimulatory blockade induced allogeneic tolerance. Phenotypic analysis of different B cell subsets showed that tolerization with DST and anti-CD40L mAb did not significantly change the number of CD19+CD5+CD1d+ regulatory B cells (Breg) in spleen and lymph nodes. However, anti-CD20 mAb treatment significantly changed the number and percentage of the CD19+CD93-CD23+sIgMhisIgDhiCD21/CD35hi marginal zone precursor (MZP) subset in spleen and lymph nodes. DST plus anti-CD40L mAb treatment showed increased IL-10 mRNA expression in non-Breg cells, furthermore B cell depletion causes decreased IL-10 production in the residual MZP cells. Allogeneic transplantation under tolerogenic conditions in CD19-Cre+/+::IL-10fl /fl mice lead to acute rejection of the allograft, compared to in littermate controls, which resulted in tolerance. Immunohistochemical analysis of allografts showed signifi cantly increased perivascular and mononuclear infi ltration in the rejected grafts. CD19-Cre+/+::IL10fl /fl mice did not have any signifi cant change in the CD19+, CD4+, CD8+, Foxp3+ Treg, or Breg cell numbers or percentages in spleen and lymph nodes. However, MZP B cell number was reduced in the rejecting IL-10 defi cient mice compared to littermates in secondary lymphoid organs. Conclusion: IL-10 expression in B cell is required for the generation of transplantation tolerance. MZP subsets of B cell express IL-10 and MZP subset localization in the secondary lymphoid organs plays an important role in tolerance. These results demonstrate a novel subset of B cells is required for tolerization. Abstract# 3 Sotrastaurin + Reduced-Exposure Tacrolimus Prevents Acute Rejection and Preserves Renal Function after De Novo Kidney Transplantation – 6 Month Results. G. Russ,1 H. Tedesco-Silva,1 D. Kuypers,1 S. Cohney,1 R. Langer,1 E. S. Woodle,1 J. Gill,1 J. Ng,2 J. Klupp,2 L. Chodoff,2 K. Budde.1 1Sotrastaurin A2214 Study Group; 2Novartis Pharma AG. Purpose: Sotrastaurin (STN, AEB071) is a novel low molecular weight immunosuppressant which blocks early T-cell activation through protein kinase C inhibition. This study examined the effi cacy and safety of reduced-exposure tacrolimus (TAC) with STN in low-moderate risk renal transplant recipients. Methods: Recipients of a fi rst or second kidney with cold ischemia time (CIT) <30 hours were randomized to STN 100 or 200mg bid + standard TAC (sTAC; 5-12ng/ mL) or STN 300mg bid + reduced TAC (rTAC; 2-5ng/mL) vs. enteric-coated mycophenolic acid (MPA) + sTAC in a partially blinded study. All patients received basiliximab and steroids. Results: 298 patients were randomized (59% deceased donors). Key results at Month 6 are provided in the table. Most rejections were mild (Banff grade IA/IB 17, IIA 12, IIB 1, III 0); 3 were antibody-mediated (none on STN); 7 were antibody-treated. Frequency and pattern of infections were comparable between groups. A slight dose-proportional increase in heart rate (5-7 bpm) was observed for STN patients. No evidence of other ECG abnormalities or cardiac AEs and no between-group differences in blood pressure were observed. Leucopenia was signifi cantly less frequent in all STN treatment groups compared to the MPA group. Treatment with STN 300mg bid + TAC 2-5 ng/mL was associated with numerically higher eGFR compared to MPA + sTAC. In the STN 200 + sTAC group, longer median CIT and more DGF may have contributed to reduced eGFR. Conclusion: STN 200mg bid + sTAC and STN 300mg bid + rTAC provided effective rejection prophylaxis through Month 6. STN-TAC regimens were well tolerated; discontinuation due to AEs was dose-proportional. The rTAC regimen was associated with the highest GFR. 3 Sotrastaurin + Redu

Spectacular success in preventing renal allograft rejection in rats was obtained over 40 yr ago using only the reactants of the response: donor-type antigen and homologous antiserum directed against donor-type antigen. Tolerance was antigen specific and sustained by persistent antigen of the graft. The model has never been tested rigorously in a large species, though the rationale for why the procedures should work applies across species including humans. Confirming the results in a large species would have profound impact on research for treating multiple immune mediated diseases, in addition to providing a way for treating some transplant recipients. This is a propitious time to confirm the applicability to larger species. If successful, only the lack of imagination limits the potential impact.

This study examined alemtuzumab (anti-CD 52, Campath-1H) and basiliximab (anti-CD 25, Simulect) as induction immunosuppression in kidney transplantation. We used a single-center, nonrandomized, retrospective, sequential study design to evaluate outcomes in kidney transplant recipients given either alemtuzumab (n = 123) or basiliximab (n = 155) induction in combination with a prednisone-free maintenance protocol using tacrolimus and mycophenolate mofetil. Kaplan-Meier analyses of long-term patient and graft survivals and rejection rates were determined according to induction agent, donor source and recipient ethnicity. Secondary endpoints included the quality of renal allograft function and the etiology of infectious complications. Overall long-term patient and graft survival rates did not significantly differ between patients treated with alemtuzumab and basiliximab. A lower rate of early (<3 months) rejection was observed in the alemtuzumab (4.1%) versus the basiliximab (11.6%) group, but the rates for both groups were equivalent at 1 year. Patient and kidney survival and rejection rates were nearly identical between Caucasians and African Americans that received alemtuzumab. Quality of renal function and incidence of infectious complications were similar in the two groups. Alemtuzumab induction therapy was similar in efficacy to basiliximab in a prednisone-free maintenance immunosuppressive protocol for an ethnically diverse population of kidney transplant recipients.

O141* Aims: To determine the effect of Campath 1-H induction therapy compared to basiliximab on outcomes of kidney transplant recipients followed for 2 years. Methods: This is a single-center, retrospective study. The Campath 1-H [n=123 (cads 31, LD 92)] and basiliximab [n=155 (cads 58, LD 97)] cohorts were sequentially treated groups. The demographics of the groups were similar. The mean±SD follow-up time for the Campath group was 21.8±4.7 mo. (tx dates: 10/01-9/02) and the basiliximab group was 45.8±10.0 mo. (tx dates: 9/98-9/01). The induction dose of Campath was 30 mg as a single dose intra-operatively, and Basiliximab 20 mg days 0 and 2. All kidney transplant recipients received a prednisone-free maintenance immunosuppression protocol of tacrolimus and MMF. Patient and graft survival and rejection rates were determined by Kaplan-Meier life table analysis. Rejections were biopsy-proven. Results: The table below shows the pertinent results. There were no statistically significant differences between groups in 2-year patient and graft survival rates. The 1-year rejection rates were similar, however, the mean time to rejection in the first year was significantly earlier in the basiliximab group (33.4 d.) vs Campath (116 d). Throughout the 2-year follow-up interval the Campath recipients consistently required less MMF immunosuppression. Important viral and fungal infectious disease occurred in 10% of Campath recipients and 15% of basiliximab recipients. One PTLD occurred in each group.FigureStatistical analysis: ap=0.10; bp=0.15; cp=0.52; dp<0.001. Conclusions: This series is the largest long-term experience with Campath in kidney transplantation. The 2-year patient and graft survival results using Campath in a prednisone-free maintenance protocol compared favourably with basiliximab. The advantages of Campath were: it prevented very early rejection, a lower dose of maintenance MMF was required, a slight decrease in important infectious complications was observed, and it was less costly than basiliximab. Campath is a safe and effective induction agent for kidney transplant recipients.

Kaufman, Dixon B. MD, PhD; Leventhal, Joseph R. MD, PhD; Gallon, Lorenzo G. MD; Parker, Michele A. MS; Stuart, Frank P. MD Author Information

Islet transplantation is becoming established as a treatment option for type I diabetes in select patients. Individuals with type I diabetes who have previously received a successful kidney allograft may be good candidates for islet transplantation. They have already assumed the risks of chronic immunosuppression, so the added procedural risk of a subsequent islet transplant would be minimal. Furthermore, because of the preimmunosuppressed state it is possible that islet-after-kidney transplantation may result in a more efficient early islet engraftment. Consequently, insulin independence might be achieved with significantly fewer islets than the approximately 8-10,000 islet equivalents/kg/b.w. currently required. A mass that usually demands two or more cadaveric donors. A case of successful islet-after-kidney transplantation is described using the steroid-free Edmonton immunosuppression protocol. Characteristics of the final islet product are: a) islet equivalents: 265,888 (4100 islet equivalents/kg/b.w.); b) islet purity: 75-80%; c) viability: >95% (trypan blue exclusion); and d) mean islet potency (static low-high glucose challenge): 4.16 +/- 1.91-fold increase. Post-transplant the patient's hypoglycemic episodes abated. Exogenous insulin requirements were eliminated at week 12 post-transplant as basal and Ensure (Abbott Laboratories, Abbott Park, IL, USA) oral glucose stimulated C-peptide levels peaked and stabilized. Twenty-four-hour continuous glucose monitoring confirmed moment-to-moment glycemic control, and periodic nonfasting finger stick glucose determinations over the next month confirmed glycemia was controlled. Hemoglobin A1c levels declined from a pretransplant level of 6.9% to 5.3%. Renal allograft function remained changed.

Background. We examined the feasibility of rapid corticosteroid elimination in simultaneous pancreas kidney transplantation.Methods. Forty consecutive simultaneous pancreas-kidney (SPK) transplant recipients were enrolled in a prospective study in which antithymocyte globulin induction and 6 days of corticosteroids were administered along with tacrolimus and MMF (n=20) or tacrolimus and sirolimus (n=20). Mean+/-SD follow-up for recipients receiving tacrolimus/MMF and tacrolimus/sirolimus were 12.7+/-3.9 and 13.4+/-2.9 months, respectively. Patient and graft survival, and rejection rates were compared to an historical control group (n=86; mean follow-up 41.5+/-15.4 months) of SPK recipients that received induction and tacrolimus, MMF, and corticosteroids.Results. Demographic characteristics of recipient and donor variables were similar among all groups. The 1-year actuarial patient, kidney, and pancreas survival rates in the 40 SPK transplant recipients with rapid corticosteroid elimination were 100, 100, and 100%, respectively. In the historical control group the 1-year actual patient, kidney, and pancreas survival rates were 96.5, 93.0, and 91.9%, respectively. The 1-year rejection-free survival rate recipients in the rapid steroid elimination group collectively was 97.5 vs 80.2% in the historical control group (P=0.034). At 6 and 12 months posttransplant the serum creatinine values remained stable in all groups.Conclusions. We conclude that chronic corticosteroid exposure is not required in SPK transplant recipients receiving antithymocyte globulin induction and maintenance immuno-suppression consisting of either tacrolimus and mycophenolate mofetil or tacrolimus and sirolimus.

BACKGROUND:The relevance of cytomegalovirus (CMV) in simultaneous pancreas kidney (SPK) transplant recipients in the modern era of immunosuppression and antiviral therapeutics is largely unquantified. We sought to determine the risk factors of CMV disease and its impact on SPK transplant outcomes in recipients all receiving a consistent regime of maintenance immunosuppression and CMV prophylaxis.METHODS:This is a retrospective, single center study of 100 consecutive SPK transplant recipients. All received maintenance immunosuppression with mycophenolate mofetil, tacrolimus, and prednisone. CMV prophylaxis consisted of a short course of parenteral gancyclovir followed by oral gancyclovir. Recipients at high-risk (D+/R-) for CMV also received CMV hyperimmune globulin. Multivariate analysis of risk factors for CMV disease and risk factors for adverse outcomes in SPK transplantation were determined. The effect of duration of prophylaxis on timing and severity of CMV disease in high-risk (D+/R-) SPK transplant recipients was also evaluated.RESULTS:The actual 1-year rate of CMV disease was 17.0% (12.0% noninvasive, 5.0% tissue invasive); and according to donor and recipient CMV serological status was: D-/R+: 0%; D-/R-: 2.8%; D+/R+: 25.6%; and D+/R-: 40.6%. Multivariate analysis showed transplantation of organs from a donor with positive CMV serology to be predictive of CMV disease with a relative risk of 63.37 (P=0.0052). In the high-risk (D+/R-) subgroup, the duration of prophylactic therapy delayed onset of CMV disease, but had minimal effect on severity. Invasive CMV disease was an independent predictor of mortality but did not decrease kidney or pancreas allograft survival.CONCLUSIONS:Outcomes of SPK transplantation have improved in the current era of modern immunosuppression, yet CMV remains an important pathogen. The serological status of the organ donor and the duration of CMV prophylaxis are predictive of who and when CMV disease may occur. Nevertheless, new strategies that reduce risk and severity of CMV disease are still needed.

As a rare postoperative complication, renal artery aneurysm has been reported in 0.95% of kidney transplants. A renal artery aneurysm was repaired prior to transplantation of the kidney.

Tishler, D. S.1; Fryer, J. P.1; Anderson, B. E.1; Ivancic, D. Z.1; Leventhal, J. R.1; Kaufman, D. B.1; Abecassis, M. I.1; Stuart, F. P.1; Zhang, Z. J.1 Author Information

BACKGROUNDLaparoscopic live donor nephrectomy (LDN) is a less invasive alternative to open nephrectomy (ODN) for living kidney donation. Concerns have been raised regarding the safety of LDN, the short and long term function of kidneys removed by LDN, and a potential higher incidence of urologic complications in LDN transplant recipients.METHODSBetween October 1997 and May 1999, 80 LDNs were performed at our center. All patients were followed longitudinally with office visits and telephone interviews. These LDNs were compared with 50 ODN performed from January 1996 to October 1997.RESULTSLDN procedures took significantly longer than ODN (4.6 vs. 3.1 hr). However, LDN was associated with significant reduction in i.v. narcotic use, a rapid return to diet, and shorter hospital stay. Of the 80 LDN procedures, a total of 75 (94%) were completed laparoscopically. Five patients were converted to laparotomy: three for hemorrhage and two for complex vascular anatomy. ODN conversion was associated with large donor body habitus and/or obesity. Seven LDN patients had minor complications and 4 had major complications. All major complications consisted of vascular injuries (2 lumbar vein injuries, 1 renal artery, and 1 aortic injury). All patients made complete recoveries. All LDN kidneys functioned immediately posttransplant. We have observed 100% patient and 97% 1-year actuarial graft survival in LDN transplant recipients. There have been no short-or long-term urologic complications in this series.CONCLUSIONWith increasing experience and standardization of technique, LDN is a safe and effective procedure. Patients undergoing LDN demonstrate clinically significant, more rapid postoperative recoveries and shorter hospital stays than ODN patients. Excellent initial graft function and long-term graft survival have been observed with LDN kidneys. Urologic complications can be avoided. LDN has become the preferred surgical approach for living kidney donation at our center.