Rolf N. Barth

THE INNATE IMMUNE RESPONSE AND ACTIVATION OF COAGULATION IN α1,3-GALACTOSYLTRANSFERASE GENE-KNOCKOUT XENOGRAFT RECIPIENTS

Background. The role of the innate immune system in the development of thrombotic microangiopathy (TM) after α1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain. Methods. Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out. Results. Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft. Conclusions. Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.

The Maryland aggregate pathology index: a deceased donor kidney biopsy scoring system for predicting graft failure.

Despite the common use of diagnostic pretransplant deceased donor kidney biopsy, there is no consensus on the prognostic significance of the pathologic findings. In order to assist clinicians with interpretation we analyzed 371 pretransplant biopsies and correlated the findings with graft failure. Glomerular pathology was assessed with percent glomerulosclerosis (GS), glomerular size and periglomerular fibrosis (PGF); vascular pathology with arterial wall‐to‐lumen ratio (WLR) and arteriolar hyalinosis and interstitial pathology with measurement of cumulative fibrosis and presence of scar. Using two‐thirds of the study population as a model‐development cohort, we found that biopsy features independently associated with an increased risk of graft failure were GS ≥15%, interlobular arterial WLR ≥0.5 and the presence of PGF, arteriolar hyalinosis or scar. The Maryland Aggregate Pathology Index (MAPI), was developed from these parameters and validated on the remaining one‐third of the population. Five‐year actuarial graft survival was 90% for kidneys with MAPI scores between 0 and 7, 63% for scores from 8 to 11 and 53% for scores from 12 to 15 (p < 0.001). We conclude MAPI may help transplant physicians estimate graft survival from the preimplantation biopsy findings, in clinical situations similar to this study population (cold ischemia over 24 h, GS < 25%).

Activation of Cytomegalovirus in Pig-to-Primate Organ Xenotransplantation

ABSTRACT Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.

Total face, double jaw, and tongue transplantation: An evolutionary concept

Background: The central face high-energy avulsive injury has been frequently encountered and predictably managed at the R Adams Cowley Shock Trauma Center. However, despite significant surgical advances and multiple surgical procedures, the ultimate outcome continues to reveal an inanimate, insensate, and suboptimal aesthetic result. Methods: To effectively address this challenging deformity, a comprehensive multidisciplinary approach was devised. The strategy involved the foundation of a basic science laboratory, the cultivation of a supportive institutional clinical environment, the innovative application of technologies, cadaveric simulations, a real-time clinical rehearsal, and an informed and willing recipient who had the characteristic deformity. Results: After institutional review board and organ procurement organization approval, a total face, double jaw, and tongue transplantation was performed on a 37-year-old man with a central face high-energy avulsive ballistic injury. Conclusions: This facial transplant represents the most comprehensive transplant performed to date. Through a systematic approach and clinical adherence to fundamental principles of aesthetic surgery, craniofacial surgery, and microsurgery and the innovative application of technologies, restoration of human appearance and function for individuals with a devastating composite disfigurement is now a reality. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Thymic transplantation in miniature swine. I. Development and function of the "thymokidney".

BACKGROUND Previous studies in our laboratory have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. The focus of the present study was to explore the feasibility of autologous thymic transplantation to produce a new transplantable organ (thymokidney) and to examine the function of subsequent vascularized thymokidney transplants in T cell development. MATERIALS AND METHODS Eight juvenile swine received autologous thymic grafts under the renal capsule. Thymic tissue was obtained through a partial (n=6) or complete (n=2) thymectomy, and growth of the autologous thymic graft was compared between partially and completely thymectomized animals. Two of the partially thymectomized animals received irradiated (1000 cGy) as well as non-irradiated autologous thymic grafts. Graft survival, growth and evidence of thymocyte development was determined by (a) macroscopic examination of the implanted tissue, (b) histological examination, and (c) flow cytometry. Naive CD4 SP T cells were identified by CD45RA-expression. RESULTS Growth of transplanted thymic tissue was demonstrated in all thymic graft recipients. No difference was seen between partially and completely thymectomized animals. By POD 60, the thymic grafts exhibited normal macroscopic and microscopic structure, and normal thymocyte composition. Irradiated thymic tissue displayed a similar pattern of development, but growth was markedly delayed. To evaluate thymic function of the graft, a composite thymokidney was transplanted into a recipient which had previously been thymectomized, had few circulating CD4-single positive cells and had lost MLR reactivity. The number of CD4+/CD45RA+ cells in this animal increased steadily from POD 30 to POD 150, indicating that the thymus of the composite thymokidney allograft was functional; in addition, MLR assays demonstrated that the recipient recovered immunocompetence. CONCLUSIONS The establishment of a thymokidney by thymic autografting to the renal subcapsular space results in normal thymic growth and function, and may provide a valuable tool for studying the role of the thymus in tolerance induction. As far as we are aware, we provide the first evidence of functional vascularized thymic graft reconstituting T cells and leading to a return of a immunocompetence in a large animal model.

Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.

Background. Inbred miniature swine provide a large animal model inwhich the effects of selective major histocompatibility complex (MHC) matchingcan be reproducibly studied. We have previously demonstrated that although a12-day course of cyclosporine uniformly induces tolerance to classI-mismatched renal allografts, it does not induce tolerance across full MHCbarriers. In this study, we assessed whether and at what dose tacrolimus mightpermit allografts to induce tolerance across different MHCbarriers. Methods. Recipients of MHC disparate renal allografts were treatedwith a 12-day course of tacrolimus by continuous intravenous infusion. Groupswere divided as follows: (1) class I-mismatched kidneys with 0.3 mg/kg/daytacrolimus (n=3); (2) fully MHC-mismatched kidneys with 0.3 mg/kg/daytacrolimus (n=2); and (3) fully MHC-mismatched kidneys with0.12–0.16 mg/kg/day tacrolimus(n=4). Results. In groups 1 and 2, recipients with tacrolimus levels of45–80 ng/ml accepted renal allografts long-term with stable renalfunction. Donor-specific hyporesponsiveness was demonstrated by cell-mediatedlymphocytotoxicity and mixed lymphocyte response, and subsequent donor-matchedgrafts were also accepted, without further immunosuppression (n=4),confirming systemic tolerance. In group 3, recipients that achieved tacrolimuslevels of 35 ng/ml (n=2) accepted their grafts without chronic changes,whereas recipients with levels of 20–26 ng/ml (n=2) developedchronic allograft glomerulopathy, suggesting 35 ng/ml as the threshold bloodlevel for tolerance induction. In vitro assays demonstrated that peripheralblood lymphocytes from tolerant animals produced inhibitory cytokines,suggesting the involvement of regulatorymechanisms. Conclusions. To our knowledge, this study represents the firstdemonstration of the induction of transplant tolerance across a two-haplotypefull MHC barrier with a short course of immunosuppression in a large animalmodel. These studies may also have clinical applicability, because the timecourse required to induce tolerance was sufficiently short that the high druglevels required might be expected to be tolerated clinically with onlytransienttoxicity.

Thrombotic Microangiopathic Glomerulopathy in Human Decay Accelerating Factor–Transgenic Swine-to-Baboon Kidney Xenografts

Models of pig-to-baboon xenografting were examined to identify the mechanisms and pathologic characteristics of acute humoral xenograft rejection (AHXR). Thymus and kidney (composite thymokidney) from human decay accelerating factor-transgenic swine were transplanted into baboons (n = 16) that were treated with an immunosuppressive regimen that included extracorporeal immunoadsorption of anti-alphaGal antibody and inhibition of complement activation. Morphologic and immunohistochemical studies were performed on protocol biopsies and graftectomy samples. All renal xenografts avoided hyperacute rejection. However, graft rejection coincided with the increase of anti-alphaGal antibody in the recipients circulation. The 16 xenografts studied were divided into two groups dependent on the rapid return (group 1) or gradual return (group 2) of anti-alphaGal antibody after immunoadsorption. In group 1 (n = 6), grafts were rejected to day 27 with development of typical AHXR, characterized by marked interstitial hemorrhage and thrombotic microangiopathy in the renal vasculature. In group 2 (n = 10), grafts also developed thrombotic microangiopathy affecting mainly the glomeruli by day 30 but also showed minimal evidence of interstitial injury and hemorrhage. In the injured glomeruli, IgM and C4d deposition, subsequent endothelial cell death and activation with upregulation of von Willebrand factor and tissue factor, and a decrease of CD39 expression developed with the formation of fibrin-platelet multiple microthrombi. In this model, the kidney xenografts, from human decay accelerating factor-transgenic swine, in baboons undergo AHXR. In slowly evolving AHXR, graft loss is associated with the development of thrombotic microangiopathic glomerulopathy. Also, anti-alphaGal IgM deposition and subsequent complement activation play an important role in the mechanism of glomerular endothelial injury and activation and the formation of multiple microthrombi.

Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness.

Background. The potential of xenotransplantation for clinical application will require overcoming barriers of humoral and cellular rejection, through strategies using immune suppression or tolerance induction. This laboratory has previously reported the induction of tolerance in the discordant xenogeneic model of pig-to-rodent thymic transplantation. We also have described a miniature swine model of fully mismatched allogeneic composite vascularized thymokidney transplantation that induced transplantation tolerance. We tested a combination of these approaches in a clinically relevant pig-to-primate model of xenotransplantation. Methods. Composite thymokidney grafts were prepared 40 to 80 days before transplantation by the autologous implantation of thymic tissue under the renal capsule of human decay-accelerating factor transgenic swine. Baboons received xenotransplants of both human decay-accelerating factor composite thymokidneys and omental implants of thymic tissue. Recipients were treated with an immunosuppressive-conditioning regimen including thymectomy or thymic irradiation, extracorporeal immunoadsorption of anti-&agr;Gal antibodies and T-cell depletion. Recipients were followed for indicators of xenograft rejection, T-cell depletion and reconstitution, anti-&agr;Gal antibody levels, and mixed lymphocyte responses. Immunologic responses were studied in those animals that survived for more than 3 weeks. Results. Thymokidney xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall’s corpuscles under the renal capsule and in the omental implants, and with evidence of few host lymphocytes. Three animals demonstrated donor-specific unresponsiveness, while maintaining normal alloresponses, in mixed–lymphocyte-response assays performed after immunosuppression had been stopped. Rejected grafts demonstrated humoral damage without evidence of cellular infiltrates. After graftectomy, one animal maintained donor-specific cellular unresponsiveness and stable anti-&agr;Gal antibody levels for more than 2 months. Conclusions. We concluded that composite thymokidney and thymic-tissue xenotransplantation from swine to baboons can induce donor-specific cellular unresponsiveness and stable anti-&agr;Gal antibody levels, suggesting avoidance of sensitization after xenotransplantation. The presence of viable donor-swine thymic epithelium could have a role in the development of donor-specific T-cell tolerance. Further strategies to address humoral rejection could prolong graft survival and result in long-term tolerance to xenografts.

Vascularized Bone Marrow-Based Immunosuppression Inhibits Rejection of Vascularized Composite Allografts in Nonhuman Primates

Vascularized composite allograft (VCA) transplantation (also referred to as composite tissue allotransplantation) has demonstrated clinical success in cases of hand, arm and face transplantation despite prior belief that skin provides an insurmountable barrier to allograft rejection. These overall good outcomes are facilitated by substantial immunosuppressive requirements in otherwise healthy patients, yet still demonstrate frequent rejection episodes. We developed a nonhuman primate model of facial segment allotransplantation to elucidate the unique pathophysiology and immunosuppressive requirements of VCA with addition of concomitant vascularized bone marrow (VBM). Heterotopically transplanted facial segment VCA with VBM treated only with tacrolimus and mycophenolate mofetil (MMF) demonstrated prolonged rejection‐free survival, compared to VCA without VBM that demonstrated early rejection episodes and graft loss. While VCA with VBM demonstrated sporadic macrochimerism, acute and chronic rejection and graft loss occurred after discontinuation of immunosuppression. These data support an immunomodulatory role of VBM in VCA that reduces immunosuppressive requirements while providing improved outcomes.

Activation of Porcine Cytomegalovirus, but Not Porcine Lymphotropic Herpesvirus, in Pig-to-Baboon Xenotransplantation

Tissue-invasive disease due to porcine cytomegalovirus (PCMV) has been demonstrated after pig-to-baboon solid-organ xenotransplantation. Porcine lymphotropic herpesvirus (PLHV)-1 is associated with B cell proliferation and posttransplant lymphoproliferative disorder after allogeneic bone marrow transplantation in swine but has not been observed in pig-to-primate xenotransplantation. Activation of PCMV and PLHV-1 was investigated in 22 pig-to-baboon xenotransplants by use of quantitative polymerase chain reaction. PCMV was found in all xenografts; increased viral replication occurred in 68% of xenografts during immunosuppression. PLHV-1 was found in 12 xenografts (55%); no increases in viral replication occurred during immunosuppression. Control immunosuppressed swine coinfected with PCMV and PLHV-1 had activation of PCMV but not PLHV-1. PCMV, but not PLHV-1, is activated in solid-organ xenotransplantation.