Radbeh Torabi

Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

We previously reported life-supporting α1,3-galactosyltransferase knockout (GalTKO) thymokidney xenograft survival of >2 months in baboons. However, despite otherwise normal renal function, recipients developed proteinuria with morphologic changes (podocyte effacement), a condition that presents a major obstacle to long-term studies in this model. A recent clinical study showed that rituximab therapy after allogeneic transplant prevented proteinuria possibly associated with loss of sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b). Here, we demonstrate that rituximab prevents the disruption of pig podocytes in an SMPDL-3b-dependent manner in vitro and the early development of proteinuria after xenogeneic kidney transplantation in baboons. Immunofluorescence showed SMPDL-3b expression in pig glomerular epithelium; immunoprecipitation demonstrated rituximab binding to SMPDL-3b in glomeruli. Culture of isolated pig podocytes with naive baboon sera, which has preformed antipig natural antibodies, reduced SMPDL-3b expression, disrupted podocyte morphology, and decreased podocyte proliferation, whereas pretreatment with rituximab prevented these effects. Six baboons received rituximab before transplantation to deplete B cells and again in the peri-transplant period; 18 baboons treated only before transplantation served as historical controls. The onset of post-transplant proteinuria was significantly delayed in a B cell-independent manner in the animals that received peri-transplant rituximab treatment. Although further optimization of this protocol is required, these data provide intriguing clues to the mechanisms of post-transplant proteinuria in xenogeneic kidney transplantation and a potential strategy for its prevention.

The gracilis myocutaneous free flap in swine: An advantageous preclinical model for vascularized composite allograft transplantation research

Vascularized composite allotransplantation (VCA) has become a clinical reality, prompting research aimed at improving the risk‐benefit ratio of such transplants. Here, we report our experience with a gracilis myocutaneous free flap in Massachusetts General Hospital miniature swine as a preclinical VCA model. Fourteen animals underwent free transfer of a gracilis myocutaneous flap comprised of the gracilis muscle and overlying skin, each tissue supplied by independent branches of the femoral vessels. End‐to‐end anastomoses were performed to the common carotid artery and internal jugular vein, or to the femoral vessels of the recipients. Thirteen of fourteen flaps were successful. A single flap was lost due to compromise of venous outflow. This model allows transplantation of a substantial volume of skin, subcutaneous tissue, and muscle. The anatomy is reliable and easily identified and harvest incurs minimal donor morbidity. We find this gracilis myocutaneous flap an excellent pre‐clinical model for the study of vascularized composite allotransplantation.

Genetically modified porcine split-thickness skin grafts as an alternative to allograft for provision of temporary wound coverage: preliminary characterization

Temporary coverage of severely burned patients with cadaver allograft skin represents an important component of burn care, but is limited by availability and cost. Porcine skin shares many physical properties with human skin, but is susceptible to hyperacute rejection due to preformed antibodies to α-1,3-galactose (Gal), a carbohydrate on all porcine cells. Our preliminary studies have suggested that skin grafts from α-1,3-galactosyltransferase knock out (GalT-KO) miniature swine might provide temporary wound coverage comparable to allografts, since GalT-KO swine lack this carbohydrate. To further evaluate this possibility, eight non-human primates received primary autologous, allogeneic, GalT-KO, and GalT+xenogeneic skin grafts. Additionally, secondary grafts were placed to assess whether sensitization would affect the rejection time course of identical-type grafts. We demonstrate that both GalT-KO xenografts and allografts provide temporary coverage of partial- and full-thickness wounds for up to 11 days. In contrast, GalT+xenografts displayed hyperacute rejection, with no signs of vascularization and rapid avulsion from wounds. Furthermore, secondary GalT-KO transplants failed to vascularize, demonstrating that primary graft rejection sensitizes the recipient. We conclude that GalT-KO xenografts may provide temporary coverage of wounds for a duration equivalent to allografts, and thus, could serve as a readily available alternative treatment of severe burns.

Immunomodulatory Strategies Directed Toward Tolerance of Vascularized Composite Allografts.

Background Achieving tolerance of vascularized composite allografts (VCAs) would improve the risk-to-benefit ratio in patients who undergo this life-enhancing, though not lifesaving, transplant. Kidney cotransplantation along with a short course of high-dose immunosuppression enables tolerance of heart allografts across a full major histocompatibility complex (MHC) mismatch. In this study, we investigated whether tolerance of VCAs across full MHC disparities could be achieved in animals already tolerant of heart and kidney allografts. Methods Miniature swine that were tolerant of heart and/or kidney allografts long term underwent transplantation of myocutaneous VCA across the same MHC barrier. Before VCA transplant, group 1 (n = 3) underwent class I–mismatched kidney transplantation; group 2 (n = 3) underwent 2 sequential class I–mismatched kidney transplantations; group 3 (n = 2) underwent haploidentical MHC-mismatched heart/kidney transplantation; and group 4 (n = 2) underwent full MHC-mismatched heart/kidney transplantation. Results All 3 animals in group 1 and 2 of 3 animals in group 2 showed skin rejection within 85 days; 1 animal in group 2 showed prolonged skin survival longer than 200 days. Animals in groups 3 and 4 showed skin rejection within 30 days and regained in vitro evidence of donor responsiveness. Conclusions This is the first preclinical study in which hearts, kidneys, and VCAs have been transplanted into the same recipient. Despite VCA rejection, tolerance of heart and kidney allografts was maintained. These results suggest that regulatory tolerance of skin is possible but not generally achieved by the same level of immunomodulation that is capable of inducing tolerance of heart and kidney allografts. Achieving tolerance of skin may require additional immunomodulatory therapies.

Abrogation of Renal Allograft Tolerance in MGH Miniature Swine: The Role of Intra-Graft and Peripheral Factors in Long-Term Tolerance

We have previously demonstrated that long‐term tolerance (LTT) of an MHC class‐I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class‐I mismatched primary kidneys were subjected to a treatment consisting of donor‐specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor‐MHC‐matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4+Foxp3+ Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class‐I mismatched allotransplants.

VASCULARIZED COMPOSITE ALLOGRAFT TRANSPLANT SURVIVAL IN MINIATURE SWINE: IS MHC TOLERANCE SUFFICIENT FOR ACCEPTANCE OF EPIDERMIS?

Background We have previously reported that Massachusetts General Hospital miniature swine, which had accepted class I–mismatched kidneys long-term after 12 days of high-dose cyclosporine A, uniformly accepted donor-major histocompatibility complex (MHC)–matched kidneys without immunosuppression but rejected donor MHC-matched split-thickness skin grafts by day 25, without changes in renal graft function or antidonor in vitro responses. We have now tested whether this “split tolerance” would also be observed for the primarily vascularized skin of vascularized composite allografts (VCAs). Methods Group 1 animals (n=3) received donor MHC-matched VCAs less than 70 days after primary kidney transplant (KTx). Group 2 animals (n=3) received a second donor-matched kidney transplant followed by a donor-matched VCA more than 200 days after primary KTx. Results Animals in Group 1 lost the epidermis on days 28, 30, and 40, with all other components of the VCAs remaining viable. Histology showed cellular infiltration localized to dermal-epidermal junction. One of three recipients of VCAs in Group 2, accepted all components of the VCA, including epidermis (>200 days). The other two recipients lost only the epidermis on days 45 and 85, with survival of the remainder of the VCA long-term. Conclusions All tissues of a VCA are accepted long-term on animals tolerant of class I–mismatched kidneys, with the exception of epidermis, the survival of which is markedly prolonged compared with split-thickness skin grafts but not indefinite. Exposure of tolerant animals to second donor-matched kidneys before VCA increases the longevity of the VCA epidermis, suggesting an increase in the immunomodulatory mechanisms associated with tolerance of the kidney.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Our recent studies in an inbred swine model demonstrated that both peripheral and intra‐graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor‐matched kidney. Here, we have asked whether both peripheral and intra‐graft regulatory elements are required for adoptive transfer of tolerance when only a long‐term tolerant (LTT) kidney is transplanted. Nine highly‐inbred swine underwent a tolerance‐inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor‐matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance‐inducing and/or tolerance‐maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor‐matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Effects of Transient Donor Chimerism on Rejection of MHC-Mismatched Vascularized Composite Allografts in Swine

Background: Despite encouraging outcomes in vascularized composite allograft (VCA) transplantation, the risks of chronic immunosuppression limit widespread applicability. It has been suggested that infusion of donor bone marrow along with the VCA may reduce the level of immunosuppression required to prevent clinical VCA rejection. However, no clear evidence has yet been presented to confirm the role of donor bone marrow in the prevention of rejection. In this study we investigated the immunologic effects of concurrent bone marrow transplantation in a large animal VCA model. Methods: MGH miniature swine (n=4) received a non-myeloablative conditioning regimen consisting of low-dose total body irradiation, T-cell depletion, a short course of Cyclosporine A, with or without varying doses of donor bone marrow cells in combination with a complete MHC-mismatched VCA. Animals were monitored daily for signs of rejection or graft versus host disease. Chimerism levels were assessed using flow cytometry and in vitro assays were performed to assess for donor-specific responses. Results: Transient chimerism was prolonged with increased bone marrow cell doses and total body irradiation. While animals that received BMC infusions did not have significantly prolonged VCA acceptance following cessation of immunosuppression compared to animals that received conditioning without BMCs, they demonstrated better early clinical outcomes and demonstrated donor-specific unresponsiveness during the presence of detectable chimerism. Conclusions: Detectable mixed chimerism following bone marrow transplantation and VCA mitigates donor-specific responses and acute rejection episodes, but does not appear to be sufficient for tolerance induction.

The rejuvenating effects of leuprolide acetate on the aged baboon's thymus

BACKGROUND We have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. METHODS AND RESULTS Thymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25mg; LI) and were followed for 3 months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4(+)/CD45RA(hi+) population in the peripheral blood occurred for 50 days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4(+)/CD8(+) cells. TREC assay corroborated enhancement in thymic function. CONCLUSION These data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

Blockade of IgM-Mediated Inflammation Alters Wound Progression in a Swine Model of Partial-Thickness Burn

In a mouse model, a second-degree burn elicits a severe inflammatory response that is mediated by circulating autoantibody specific for a neoantigen (nonmuscle myosin). Nonmuscle myosin is expressed by injured tissue, leading to amplified ulceration and scarring. We hypothesize that a synthetic peptide (N2) can mimic the neoantigen and competitively inhibit the autoantibody, decreasing inflammation, and reducing the extent of burn injury in a preclinical swine model of burn. Second-degree burns were created on young swine using brass cylinders, warmed to varying temperatures before skin contact. Animals were treated in double-blind fashion with normal saline, control peptide, or blocking peptide. Biopsies were taken at 2 hours, 1, 4, 7, and 14 days after burn injury. Burn wound healing parameters were assessed. Immunohistochemical staining for Ki-67, immunoglobulin (Ig)M, and interleukin (IL)-8 were also performed. N2 blocking peptide administration decreased dermal injury at 4 days with increased reepithelization, indicating more rapid healing. N2 normalized skin histology by 14 days and showed improved epidermal healing. Granulation tissue thickness was decreased, and there was an accompanying decrease in neutrophil infiltration. The basal layer of epidermis in N2-treated animals displayed more cells positive for Ki-67, suggesting a prompter regenerative capacity. Immunohistochemical staining demonstrated decreased deposition of immunoglobulin M and interleukin-8 after thermal injury in animals treated with N2 peptide, in comparison to controls. The findings of this study identify N2 blocking a specific inflammatory pathway, as a novel therapeutic approach, preventing the evolution of cutaneous burn injuries in a preclinical animal model.