Vijay S. Gorantla

Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.

Objective: To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. Background: 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”). Methods: 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. Results: 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. Conclusions: 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.

Outcomes of the First 2 American Hand Transplants at 8 and 6 Years Posttransplant

PURPOSE The feasibility of hand allotransplantation has been demonstrated. The purpose of the article is to report the (1) functional return, (2) psychosocial outcomes, (3) clinical and histological assessment for rejection, (4) complications, and (5) graft survival in the 2 American hand transplant recipients. METHODS We present 2 patients 106 and 81 months, respectively, after unilateral transplantation of an allogeneic hand and forearm. We analyzed clinical course, number of rejection episodes, adverse events, function of the allograft, and quality of life. Clinical laboratory results, biopsy histology, and patient clinical examinations were used to compare the clinical course. Standard hand function tests were used to evaluate function. Psychological interviews were used to assess acceptance and quality of life. RESULTS Our patients have allograft survival with improvements in intrinsic muscle activity, total active motion and return of functional grip, pinch strength, and sensibility. Rejection episodes were restricted primarily to the first 6 months after transplantation, and all responded to treatment. The major posttransplantation complications were a cytomegalovirus infection in patient 1 and osteonecrosis of the hip requiring both hips to be replaced, 1 at year 4 and the other at year 6, as well as transient immunosuppression-related diabetes in patient 2. Recently we have weaned both patients off maintenance steroids. Current Carroll scores are fair for patient 1 (72/99) and fair for patient 2 (55/99), although patient 2 has not had good recovery of intrinsic function. Both patients are back at work and report an excellent quality of life at nearly 9 and 7 years, respectively, after transplantation. CONCLUSIONS Our intermediate long-term results of hand transplants have demonstrated functional return similar to that of replants. Graft survival and quality of life after hand transplantation has far exceeded initial expectations. We conclude that allogeneic hand transplant is feasible and holds promise as a treatment modality for catastrophic upper extremity loss. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic IV.

Atypical Acute Rejection After Hand Transplantation

Skin rejection after hand transplantation is characterized by a maculopapular erythematous rash that may be diffuse, patchy or focal, and distributed over forearms and dorsum of the hands. This ‘classical’ pattern of rejection usually spares the skin of the palm and does not affect the nails. Herein, we report the experience on four cases presenting with an ‘atypical’ pattern of rejection that is novel in involving the palmar skin and the nails. All patients were young and exposed to repetitive and persistent mechanical stress of the palm. Characteristic features of rejection included a desquamative rash associated with dry skin, red papules, scaling and lichenification localized to the palm. Skin lesions were associated with nail dystrophy, degeneration, deformation or loss. Histology of the skin and nail bed revealed a lymphocytic infiltrate with predominance of T cells (CD3+, CD4+ and CD8+), with small numbers of B cells (CD20+ and CD79a+) and a low number of Forkhead transcription factor 3 (FOXP3)‐positive cells in one patient. The lesions persisted over weeks to months, responded poorly to steroid treatment and were managed with antithymocyte globulin (ATG; Thymoglobulin, Genzyme, Cambridge, MA), alemtuzumab and/or intensified maintenance immunosuppression.

Mixed allogeneic chimerism and tolerance to composite tissue allografts

The development of effective immunosuppressive drugs has made solid organ allotransplantation the preferred approach for treatment of end‐organ failure. The benefits of these immunosuppressants outweigh their risks in preventing rejection of lifesaving solid‐organ allografts. On the contrary, composite tissue allotransplants are non‐lifesaving and whether the risks of immunosuppressants justify their benefits is a subject of debate. Hence, composite tissue allografts (CTA) have not enjoyed widespread clinical application for reconstruction of large tissue defects. Therefore, a method of preventing rejection that would eliminate the need for toxic immunosuppressants is of particular importance in CTA. Bone marrow transplantation (BMT) to establish mixed chimerism induces tolerance to a variety of allografts in animal models. This article reviews mixed chimerism‐based tolerance protocols. Their limitations and their relevance to CTA are discussed, highlighting some unique characteristics (high antigenicity and the presence of active bone marrow) that make CTAs different from solid organ allografts.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

Background. Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. Methods. [ACI→WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. Results. [ACI→WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4±2.8 days after limb transplantation. [ACI→WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. Conclusions. Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI→WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Composite tissue allotransplantation in chimeric hosts part II. A clinically relevant protocol to induce tolerance in a rat model

Background. We and others have shown that mixed allogeneic chimerism induces donor-specific tolerance to composite tissue allografts across major histocompatibility complex barriers without the need for immunosuppression. However, a delay period between bone marrow transplantation and limb allotransplantation is required, making such protocols impractical for clinical application. This study eliminates this delay period in a rat hind limb allotransplantation model by performing mixed allogeneic chimerism induction and transplantation “simultaneously.” Methods. Group 1 included controls in which naïve Wistar Furth (WF) hosts received ACI hind limbs. Group 2 included (ACI→WF) chimeras that received limbs from third-party donors (Fisher), and group 3 included chimeras that received irradiated (1,050 cGy) ACI limbs. In group 4, WF hosts conditioned with 950 cGy received irradiated (1,050 cGy) ACI limbs followed by infusion of 100×106 ACI T-cell–depleted bone marrow cells and immunotherapy (tacrolimus and mycophenolate mofetil) for 28 days. Group 5 animals received the same treatment as group 4 animals without immunotherapy. Results. The rats in groups 1 and 2 rejected their limbs within 10 days. Only one rat in group 4 survived to the end of the study. Groups 3 and 5 demonstrated long-term limb survival without rejection or graft-versus-host disease. High levels of donor chimerism (>80%) were achieved and maintained throughout the study. Mixed lymphocyte reaction assays in both groups revealed donor-specific hyporesponsiveness with vigorous third-party reactivity. Conclusions. This study demonstrated that infusion of donor bone marrow cells into conditioned hosts immediately after limb transplantation results in stable mixed chimerism, robust tolerance, and reliable limb allograft survival.

Immunosuppression and rejection in human hand transplantation.

Avoidance or at least minimization of maintenance immunosuppression represents the key step for promoting wider applicability of reconstructive transplantation. Understanding the mechanisms of composite tissue allograft rejection is essential in working toward that goal. We herein review the current knowledge on acute rejection in reconstructive transplantation and discuss findings in the light of novel immunosuppressive and immunomodulatory strategies.

Composite tissue allotransplantation: hand transplantation and beyond.

Recent advances in transplant immunology are shifting the focus from immunosuppression to immunoregulation, making composite tissue allotransplantation with novel and less potent immunosuppressive regimens a possibility. Hand transplantation has been the most frequently performed human composite tissue allotransplantation, with more than 50 upper extremity-based transplants done worldwide. Further research is needed regarding immunomodulating protocols, and careful oversight and individualized screening procedures will be required as patients seeking improved quality of life through human composite tissue allotransplantation come to accept a certain level of risk in these experimental procedures. Still, composite tissue allotransplantation offers to advance transplant medicine and reconstructive surgery.

Targeting of glycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation.

Background. Glycosaminoglycans (GAGs) are heteropolysaccharides present as integral components of the extracellular matrix (ECM), cell and basement membranes. GAGs play an important role in immune and inflammatory responses because of their ability to interact with cytokines and chemokines, promoting the localization of these molecules onto the ECM or cell membranes at specific anatomical sites. The main goal of these studies was to test the hypothesis that interference with the binding of cytokines/chemokines to GAGs will interfere with a graft rejection response. Methods. MC-2, a cationic peptide derived from the sequence of the heparin-binding domain of mouse interferon gamma, was used as an inhibitor of the binding of cytokines/chemokines to GAGs. The effects of this peptide were studied in an allogeneic transplantation model involving vascularized rat skin flaps. Results. The MC-2 peptide was found to inhibit binding of interferon-&ggr;, as well as that of the chemokines, interleukin-8, interferon gamma inducible protein-10, and regulated on activation normal T cell expressed and secreted (RANTES), to GAGs in vitro. Direct administration of MC-2 in an allogeneic skin flap transplantation model resulted in a significantly delayed time of rejection, from 5.4 ± 0.5 days (control; n=6) to 12.6 ± 1.6 days (treated animals; n=10). Histopathologic analysis of the skin biopsies was consistent with the delayed rejection process in those animals receiving the peptide, showing only mild signs of rejection up to day 11 (in contrast, all control animals had rejected their flaps by day 6). Conclusions. These results are consistent with the idea that GAG-cytokine interactions constitute valid therapeutic targets and suggest the potential applicability of such an approach in the prevention of graft rejection.

Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells Prolong Graft Survival in Vascularized Composite Allotransplantation

Background Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)–derived MSCs in a rat VCA model. Methods Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 106 or 5 × 106 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. Results AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. Conclusions AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.