Upper extremity transplantation in non-human primates: an orthotopic model for translational research

Vascularized composite allotransplantation (VCA) offers unparalleled restoration of function and form following devastating musculoskeletal and soft tissue injury. However, the potential adverse effects of life-long immunosuppression remain a significant cause for concern. Therefore, while the surgical techniques necessary for VCA have developed rapidly, the immunological aspects of these procedures and the potential functional significance of immunological processes on vascularized composite allografts remain areas in which further research is required. The functional complexity of these procedures, combined with the preclinical nature of many of the research questions, necessitates the use of large animal models to most effectively address some of the outstanding hypotheses. Cynomolgus macaques are among the premier large animal models for immunological research. This manuscript describes development of an orthotopic model of upper extremity transplantation in cynomolgus macaques. Following study of the anatomy to determine feasibility, in vivo proof of concept was achieved by autologous amputation and replantation in two animals, following which a preliminary series of four allotransplants was performed. The anatomy encountered and techniques required for successful transplantation are closely comparable to those in clinical upper extremity transplantation. This is a technically challenging model, but offers a rigorous pre-clinical platform for translational research in transplant immunology, and is suitable for detailed study of the impact of immunologic processes on functional outcomes following VCA

Concomitant face/upper extremity allotransplantation

Concomitant vascularized composite tissue allotransplantation (VCA) offers single stage restoration of complex anatomical units in a manner that would be difficult, if not impossible to accomplish using conventional reconstructive techniques. However, such extensive and complex transplant procedures have considerable inherent risk. Two cases of concomitant face and hand transplantation have been performed to date, both with significant early complications leading to significant morbidity, and in one case, mortality. These cases highlight some of the unknowns in both perioperative and immunological management that must be addressed if this complex subset of VCA procedures is to be more widely offered. Only once these acute challenges can be reliably managed will focus turn to rehabilitation and long-term outcomes. If these issues can be overcome, concomitant face and upper extremity transplantation could offer patients who have suffered severe, complex injuries unprecedented options for restoration of function and form

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

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8(+) T cells during HIV infection. The frequencies of effector CD8(+) T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4(+) T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8(+) T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8(+) T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology

Topical Delivery of Immunosuppression to Prolong Xenogeneic and Allogeneic Split-Thickness Skin Graft Survival

Cadaveric skin allograft is the current standard of treatment for temporary coverage of large burn wounds. Porcine xenografts are viable alternatives but undergo α-1,3-galactose (Gal)–mediated hyperacute rejection and are lost by POD 3 because of naturally occurring antibodies to Gal in primate recipients. Using baboons, we previously demonstrated that xenografts from GalT-KO swine (lacking Gal) provided wound coverage comparable with allografts with systemic immunosuppression. In this study, we investigate topical immunosuppression as an alternative to prolong xenograft survival. Full-thickness wounds in baboons were created and covered with xenogeneic and allogeneic split-thickness skin grafts (STSGs). Animals were treated with slow-release (TyroSphere-encapsulated) topical formulations (cyclosporine-A [CSA] or Tacrolimus) applied 1) directly to the STSGs only, or 2) additionally to the wound bed before STSG and 1). Topical CSA did not improve either xenograft or allograft survival (median: treated grafts = 12.5 days, control = 14 days; P = 0.27) with similar results when topical Tacrolimus was used. Pretreatment of wound beds resulted in a significant reduction of xenograft survival compared with controls (10 vs 14 days; P = 0.0002), with comparable results observed in allografts. This observation was associated with marked reduction of inflammation on histology with Tacrolimus and not CSA. Prolongation of allograft and xenograft survival after application to full-thickness wound beds was not achieved with the current formulation of topical immunosuppressants. Modulation of inflammation within the wound bed was effective with Tacrolimus pretreatment before STSG application and may serve as a treatment strategy in related fields

Transcriptomic, biochemical and individual markers in transplanted Daphnia magna to characterize impacts in the field

Daphnia magna individuals were transplanted across 12 sites from three Spanish river basins (Llobregat, Ebro, Jucar) showing different sources of pollution. Gene transcription, feeding and biochemical responses in the field were assessed and comparedwith those obtained in re-constitutedwater treatments spikedwith organic eluates obtained fromwater samples collected at the same locations and sampling periods. Up to 166 trace contaminants were detected in water and classified by their mode of action into 45 groups that included metals, pharmaceuticals, pesticides, illicit drugs, and other industrial compounds. Physicochemical water parameters differentiated the three river basins with Llobregat having the highest levels of conductivity, metals and pharmaceuticals, followed by Ebro, whereas the Jucar river had the greatest levels of illicit drugs. D. magna grazing rates and cholinesterase activity responded similarly than the diversity of riparian benthic communities. Transcription patterns of 13 different genes encoding for general stress, metabolismand energy processes,molting and xenobiotic transporters corroborate phenotypic responses differentiated siteswithin and across river basins. Principal Component Analysis and Partial Least Square Projections to Latent Structures regression analyses indicated that measured in situ responses ofmost genes and biomarkers and that of benthicmacroinvertebrate diversity indexeswere affected by distinct environmental factors. Conductivity, suspended solids and fungicideswere negatively relatedwith the diversity of macroinvertebrates cholinesterase, and feeding responses. Gene transcripts of heat shock protein and metallothionein were positively related with 11 classes of organic contaminants and 6 metals. Gene transcripts related with signaling paths of molting and reproduction, sugar, protein and xenobiotic metabolism responded similarly in field and lab exposures and were related with high residue concentrations of analgesics, diuretics, psychiatric drugs, β blockers, illicit drugs, trizoles, bisphenol A, caffeine and pesticides. These results indicate that application of omic technologies in the field is a promising subject in water management

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