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BACKGROUND: Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplantation (Tx). However, host immune responses to the various VCA components, especially those involving skin, are complex and make selection of appropriate therapy challenging. Although the interplay between Foxp3+ T regulatory (Treg) cells and CD4 and CD8 effector T cells is of central importance in determining the acceptance or rejection of solid organ allografts, there is little information available concerning the contribution of Treg cells to VCA survival. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. METHODS: We established a fully major histocompatibility complex-disparate (BALB/c- > C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre- and post-Tx IL-2/anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival. RESULTS: Both strategies expanded the Treg cell population in vivo and prolonged VCA survival (P < 0.001), but IL-2C administration pre-Tx led to significantly longer survival compared with IL-2C administration post-Tx (P < 0.01). In addition, compared with post-Tx therapy, pre-Tx therapy resulted in an increased ratio of Treg cells to CD8+ T cells (P < 0.001), reduced proliferation of CD4 and CD8 effector T cells, and reduced production of IFN-γ. Optimal effects were seen when combined with rapamycin therapy, whereas the combination of IL-2C therapy plus calcineurin inhibitor was counterproductive. CONCLUSIONS: Our studies involving different IL-2C-mediated Treg cell expansion strategies demonstrate that pre-Tx IL-2C therapy may be a useful component for developing strategies to promote VCA survival.

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BACKGROUND: The restoration of complex tissue deficits with vascularized composite allotransplantation is a paradigm shift in reconstructive surgery. Clinical adoption of vascularized composite allotransplantation is limited by the need for systemic immunosuppression, with associated morbidity and mortality. Small-animal models lack the biological fidelity and preclinical relevance to enable translation of immunologic insights to humans. Large-animal models have been described; however, limitations persist, including the inability of heterotopic models to evaluate functional nerve regeneration, and the sensitivity of primates to toxicity of immunosuppressive drugs. The authors' novel orthotopic porcine limb transplant model has broad applicability and translational relevance to both immunologic and functional outcomes after vascularized composite allotransplantation. METHODS: Recipients underwent amputation at a level corresponding to the mid forearm. Replantation or transplantation of grafts was performed by plate fixation of the radio-ulna, microsurgical repair of brachial artery and median nerve, and extensor and flexor tendon repairs. Viability of replants was monitored clinically and radiologically. Transplants were monitored for clinicopathologic signs of rejection. Animals mobilized freely postoperatively. RESULTS: Replantations remained viable until the endpoint of 14 days. Transplants developed Banff grade 4 acute rejection by postoperative day 7. Doppler sonography and angiography confirmed vascular patency. Serial biopsy specimens of skin and histopathology of replants at endpoint confirmed tissue viability and bone healing. CONCLUSIONS: An orthotopic load-bearing porcine forelimb vascularized composite allotransplantation model was successfully established. Technical, procedural, and logistic considerations were optimized to allow model use for immunologic, bone healing, functional nerve regeneration, and other translational studies.

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Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.

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Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from controlateral grafts of limb buds and regeneration blastemas in the axolotl has been analyzed. Grafts were made so as to appose anterior and posterior limb positions. Overall, the contribution from graft and stump tissue was found to be approximately equal although the position of the boundary between the two was variable from limb to limb. This result is consistent with models which suggest that intercalary regeneration is the driving force for patterning of the vertebrate limb. In addition, the pattern of cellular contribution to supernumerary limbs was consistently found to be asymmetrical in the dorsal-ventral axis. Hence, posterior limb tissue predominantly contributed cells to the posterior and dorsal part of the supernumerary limb whereas anterior limb tissue predominantly contributed cells to the anterior and ventral part of the supernumerary limb. The reason for this asymmetrical pattern remains unknown, but we suggest that it might result from a directional bias in intercalary regeneration, similar to that observed during intercalation in the proximal-distal axis of the urodele limb. Using the triploid cell marker in conjunction with a black/white pigmentation marker, the relationship between the cellular contribution boundary and the pigmentation boundary in supernumerary limbs has also been analyzed. It has been found that the positions of the two boundaries do not coincide, a result which suggests that the eventual location of pigment cells is not a good indicator of the location of nonpigment cells derived from graft and stump.

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Symmetrical forelimbs were created in the axoltl by performing surgery on embryos at stages 32-34. The technique of J.M.W. Slack (J. Embryol. Exp. Morphol., 39:151-168, 1977) was utilized. Several experiments were then performed to test the ability of these symmetrical forelimbs to participate in pattern formation. When symmetrical limbs were amputated without previous surgery, 58% failed to regenerate. When symmetrical limbs were wounded in the plane of symmetry and permitted to heal for 30 days prior to amputation, 75% failed to regenerate. When the anterior half of the symmetrical limb was exchanged with the posterior half of the contralateral forelimb followed by amputation 30 days later, both limbs failed to regenerate. When the anterior half of the symmetrical limb was exchanged with the anterior half of an asymmetrical limb followed by amputation 30 days later, the previously symmetrical limbs regenerated asymmetrical hands, and previously asymmetrical limbs failed to regenerate. These results indicate that wounding increases the occurrence of regenerative failure in embryonically produced symmetrical forelimbs. The anterior half of the embryonically produced symmetrical forelimb behaves unpredictably and in a manner not easily described with any of the current models of pattern regulation. The posterior half of the embryonically produced symmetrical forelimb behaves predictably during pattern formation.

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Various experiments were performed on the limb buds of axolotls to compare the behaviour of amphibian limbs with that previously reported for chick limbs. Following removal of the tip or whole limb bud, extensive powers of regulation were observed since complete limbs always formed. Similarly after distal to proximal grafts intercalary regulation occurred to produce perfect limbs and after proximal to distal grafts serial repetitions resulted. Transplantation and rotation of limb buds to reverse either the dorso-ventral, antero-posterior or both axes resulted in the induction of supernumerary limbs in a large proportion of cases. Such regulatory behaviour of axolotl limb buds is in contrast to the mosaic nature of chick limbs and as a result, theories such as the progress-zone theory which have been formulated on the basis of data from chick limbs are not relevant to general principles of vertebrate limb development. Possible reasons for the diverse behaviour between the two systems are discussed.

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