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To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure.Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol").Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring.All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates.Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

BACKGROUND Immunologic monitoring of pediatric transplant (Tx) recipients, who are at increased risk of Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disease, is an important goal in clinical transplantation. Here, we investigated the impact of EBV load on T-cell immunity from pediatric Tx recipients, using clinically applicable tests for improved assessment of T-cell immune competence. METHODS Thirty-five asymptomatic pediatric thoracic Tx patients were categorized into three groups according to their EBV load levels as follows: undetectable viral load (UVL), chronic low viral load (LVL) and chronic high viral load (HVL). Global and EBV-specific T-cell immunity were assessed by ATP release using Cylex Immuknow and T Cell Memory assays. RESULTS UVL patients exhibited normal ATP release to Concanavalin A (ConA) and phytohemagglutinin (PHA; 190+/-86 ng/mL, 328+/-163 ng/mL) and detectable EBV-specific (37+/-34 ng/mL) ATP responses. LVL patients displayed significantly stronger responses to ConA (373+/-174 ng/mL), PHA (498+/-196 ng/mL) and EBV (152+/-179 ng/mL), when compared with UVL or to HVL patients (ConA 185+/-114 ng/mL, PHA 318+/-173 ng/mL, and EBV 33+/-42 ng/mL). Moreover, HVL patients displayed significant inverse correlation between CD4+ T-cell ATP levels and EBV loads. CONCLUSIONS Evaluation of global and EBV-specific T-cell immunity provides a rapid assessment of patients' immune competence. It is still unclear whether selective oversuppressed ATP release by CD4+ T cells reflects HVL patients at risk of posttransplant lymphoproliferative disease. Further longitudinal studies will determine the importance of Immuknow test in identifying asymptomatic HVL patients vulnerable to EBV complications.

Background Multiple drug immunosuppression has allowed the near elimination of rejection, but without commensurate improvements in longterm graft survival and at the cost of quality of life. We have suggested that transplantation outcomes can be improved by modifying the timing and dosage of immunosuppression to facilitate natural mechanisms of alloengraftment and acquired tolerance. Study design Two therapeutic principles were applied for kidney transplantation: pretransplant recipient conditioning with antilymphoid antibody preparations (Thymoglobulin [Sangstat] or Campath [ILEX Pharmaceuticals]), and minimal posttransplant immunosuppression with tacrolimus monotherapy including "spaced weaning" of maintenance doses when possible. The results in Thymoglobulin- (n = 101) and Campath-pretreated renal transplantation recipients (n = 90) were compared with those in 152 conventionally immunosuppressed recipients in the immediately preceding era. Results Spaced weaning was attempted in more than 90% of the kidney transplant recipients after pretreatment with both lymphoid-depleting agents, and is currently in effect in two-thirds of the survivors. Although there was a much higher rate of acute rejection in the Thymoglobulin-pretreated recipients than in either the Campath-pretreated or historic control recipients, patient and graft survival in both lymphoid depletion groups is at least equivalent to that of historic control patients. In the Thymoglobulin-conditioned patients for whom followups are now 24 to 40 months, chronic allograft nephropathy (CAN) progressed at the same rate as in historic control patients. Selected patients on weaning developed donor-specific nonreactivity. Conclusions After lymphoid depletion, kidney transplantation can be readily accomplished under minimal immunosuppression with less dependence on late maintenance immunosuppression and a better quality of life. Campath was the more effective agent for pretreatment. Guidelines for spaced weaning need additional refinement.

Background Although readily accomplished from immunocompetent Epstein-Barr virus- (EBV) seropositive individuals, the effective ex vivo generation of EBV-specific cytotoxic T lymphocytes (CTL) from the peripheral blood mononuclear cells (PBMC) of EBV-seronegative subjects has proven to be a challenge. The focus of our study was to ascertain optimized culture conditions required for the ex vivo generation of EBV-reactive autologous CTL from the PBMC of EBV-seronegative volunteers. Method Freshly isolated PBMC obtained from immunocompetent EBV-seronegative and -seropositive individuals were used to generate EBV-specific autologous CTL lines using both conventional and a novel, modified ex vivo culture technique. Results In contrast to responses observed in EBV-seropositives after two to three rounds of ex vivo stimulation, gamma-irradiated autologous lymphoblastoid cell lines (LCL) were incapable of eliciting an effective anti-EBV cytotoxic response when freshly-isolated PBMC from EBV-seronegative individuals were used as responders. Under these culture conditions, CD4+ T cells with preferential expression of the Th2-type cytokine IL-4 were predominantly expanded in the PBMC obtained from EBV-seronegative individuals. However, the addition of recombinant human (rh) IL-12 during the primary phase of ex vivo stimulation resulted in augmentation of EBV-specific cytolysis of autologous LCL by CD8+ T cells. Furthermore, there was down-regulation in the secretion of IL-4 and up-regulation in that of the Th1-type cytokine IFN-gamma by responder CD4+ and CD8+ T cells. Conclusions Taken together these data suggest that the addition of rhIL-12 during the primary phase of ex vivo stimulation of freshly isolated PBMC from EBV-seronegative individuals results in skewing of the immune response predominantly towards a CD4+ Th1-type (IFN-gamma) with the generation of an efficacious CTL-mediated anti-EBV reactivity. This novel ex vivo approach for generating effective autologous EBV-specific CTL could be adopted to treat refractory post-transplant lymphoproliferative disorders, which may be encountered in EBV-seropositive-->EBV-seronegative organ transplant recipients. Additionally, these ex vivo generated anti-EBV T cells could also be infused perioperatively to enhance prophylactically immunity against EBV infection in high-risk EBV-seronegative organ allograft recipients.