Faculty Bibliography

The discrepancy between organ need and organ availability represents one of the major limitations in the field of transplantation. One possible solution to this problem is xenotransplantation. Research in this field has identified several obstacles that have so far prevented the successful development of clinical xenotransplantation protocols. The main immunologic barriers include strong T-cell and B-cell responses to solid organ and cellular xenografts. In addition, components of the innate immune system can mediate xenograft rejection. Here, we review these immunologic and physiologic barriers and describe some of the strategies that we and others have developed to overcome them. We also describe the development of two strategies to induce tolerance across the xenogeneic barrier, namely thymus transplantation and mixed chimerism, from their inception in rodent models through their current progress in preclinical large animal models. We believe that the addition of further beneficial transgenes to Gal knockout swine, combined with new therapies such as Treg administration, will allow for successful clinical application of xenotransplantation.

Delayed gastric emptying is a significant postoperative complication of living donor hepatectomy for liver transplantation and may require endoscopic or surgical intervention in severe cases. Although the mechanism of posthepatectomy delayed gastric emptying remains unknown, vagal nerve injury during intraoperative dissection and adhesion formation postoperatively between the stomach and cut liver surface are possible explanations. Here, we present the first reported case of delayed gastric emptying following fully laparoscopic hepatectomy for living donor liver transplantation. Additionally, we also present a case in which symptoms developed after open right hepatectomy, but for which dissection for left hepatectomy was first performed. Through our experience and these two specific cases, we favor a neurovascular etiology for delayed gastric emptying after hepatectomy.

In recent years different minimal access strategies have been designed in order to perform living donor liver surgery for adult recipients with less morbidity. Techniques involve shortening the length of the incision with or without previous laparoscopic mobilization of the liver. Herein we present two cases of totally laparoscopic living donor left hepatectomy, with and without removal of the middle hepatic vein, respectively. We describe in detail the anatomical and technical aspects of the procedure focusing on relevant points to enhance safety.

BACKGROUND:Long-term tolerance of class I disparate renal allografts in miniature swine can be induced by a short course of cyclosporine and persists for 3 to 4 months after grafts are removed. Donor class I peptide immunization 6 weeks after graftectomy of tolerated kidneys leads to sensitization, but donor skin grafts do not. Here, we tested the hypothesis that skin grafts prevent rejection after simultaneous peptide administration and skin grafting. METHODS:Miniature swine underwent bilateral nephrectomy and class I-mismatched renal transplantation with a 12-day course of cyclosporine A to induce long-term tolerance. Tolerated allografts were then replaced with recipient-matched kidneys, and animals were challenged with simultaneous donor-type skin grafts and peptide. Six weeks later, second donor-matched kidneys were transplanted without immunosuppression, and immune responses were characterized. RESULTS:Animals treated only with peptide (n=2) rejected subsequent renal transplants in 3 to 5 days with strong in vitro antidonor responses. Of five recipients of skin-plus-peptide regimen, two accepted kidneys long term, one demonstrated a modestly prolonged survival (11 days), and two rejected rapidly (5-7 days). The two long-term acceptors maintained donor-specific hyporesponsiveness in vitro. CONCLUSIONS:Sensitization by class I peptide in previously tolerant swine could be prevented by simultaneous class I skin grafts. These data suggest that skin grafts may actually augment rather than abrogate downregulation in some cases. A mechanistic hypothesis for this surprising result is that recognition of class I antigens through the direct rather than the indirect pathway of antigen presentation promotes tolerance by expanding regulatory T cells.

We have previously reported that transplantation (Tx) of prevascularized donor islets as composite islet-kidneys (IK) reverses diabetic hyperglycemia in miniature swine. In order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. IKs were prepared in baboons by isolating islets from 50% to 70% partial pancreatectomies and injecting them under the autologous renal capsule, allowing vascularization before allogeneic Tx. Baboons with diabetes induced by stereptozotocin or total pancreatectomy, received composite IKs (n = 3) or free islets under the renal capsule or intraportally (n = 3), across fully allogeneic barriers with an immunosuppressive regimen consisting of ATG followed by MMF and tacrolimus. FBS of two of IK recipients decreased immediately after Tx and no insulin therapy was required throughout the experimental period (225 and 301 days). In contrast, all recipients of allogeneic free islets showed unstable FBS levels and required insulin within 2 months. We conclude that in addition to maintaining creatinine in the normal range, fully allogeneic IKs from single primate donors can achieve glucose regulation without insulin therapy, while free islets do not. These results support the feasibility of composite allogeneic IK Tx as a potential cure for end-stage diabetic nephropathy.

BACKGROUND:Transplantation of vascularized donor thymic tissue along with a kidney transplant has markedly improved graft survival across the discordant pig-to-baboon xenogeneic barrier. To quantify the production of baboon T cells by the porcine thymic tissue, we recently developed an assay to measure the excised DNA products of baboon T-cell receptor (TCR) gene rearrangement (signal-joining TCR excision circles, sjTREC). METHODS:Initial polymerase chain reaction (PCR) analysis documented that TCR δREC-ψJα rearrangement occurs in baboons. Primers, specific to baboon sjTREC sequence were designed and used to quantify sjTREC molecules in peripheral blood mononuclear cells and thymic tissue using a quantitative PCR assay. RESULTS:sjTREC levels were higher in phenotypically naïve (CD3CD45RA) T cells (650 copies/100,000 cells) than in phenotypically memory (CD3CD45RA) T cells, with sjTREC below the limit of detection (40 copies/100,000 cells). Surgical removal of the native thymus in two baboons led to a significant decrease of sjTREC in peripheral blood (from 1104 and 920 copies to 184 and 190 copies/100,000 cells, respectively), confirming the role of the thymus in maintaining the peripheral T-cell pool. In two thymectomized baboons that received porcine thymokidney xenografts, sjTREC levels remained low in the peripheral blood (<40 copies/100,000 cells), but increased to 52 and 192 copies/100,000 cells in thymic biopsies, implying that baboon thymopoiesis had begun to occur in the porcine thymic xenografts. CONCLUSIONS:Baboon sjTREC can be quantified by quantitative PCR using primers specific to baboon sequence. Initial results suggest that baboon thymopoiesis occurs in vascularized porcine thymus xenografts.

UNLABELLED:BACKGROUND AND AIMS OF STUDY: We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH). MATERIALS AND METHODS/METHODS:30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70-80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays. RESULTS:Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment. CONCLUSION/CONCLUSIONS:Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients.

The thymus serves as the central organ of immunologic self-nonself discrimination. Thymocytes undergo both positive and negative selection, resulting in T cells with a broad range of reactivity to foreign antigens but with a lack of reactivity to self-antigens. The thymus is also the source of a subset of regulatory T cells that inhibit autoreactivity of T-cell clones that may escape negative selection. As a result of these functions, the thymus has been shown to be essential for the induction of tolerance in many rodent and large animal models. Proper donor antigen presentation in the thymus after bone marrow, dendritic cell, or solid organ transplantation has been shown to induce tolerance to allografts. The molecular mechanisms of positive and negative selection and regulatory T-cell development must be understood if a tolerance-inducing therapeutic intervention is to be designed effectively. In this brief and selective review, we present some of the known information on T-cell development and on the role of the thymus in experimental models of transplant tolerance. We also cite some clinical attempts to induce tolerance to allografts using pharmacologic or biologic interventions.