Limited efficacy of rapamycin monotherapy in vascularized composite allotransplantation.

BACKGROUND: Vascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells. However, we found high dose RPM monotherapy prolonged VCA survival by only a few days, leading us to explore the mechanisms responsible. METHODS: A mouse orthotopic forelimb transplantation model (BALB/c- > C57BL/6) was established using WT mice, as well as C57BL/6 recipients with conditional deletion of T-bet within their Treg cells. Events in untreated VCA recipients or those receiving RPM or FK506 therapy were analyzed by flow-cytometry, histopathology and real-time qPCR. RESULTS: Therapy with RPM (2 mg/kg/d, p < .005) or FK506 (2 mg/kg/d, p < .005) each prolonged VCA survival. In contrast to FK506, RPM increased the ratio of splenic Treg to Teff cells (p < .05) by suppressing Teff and expanding Treg cells. While the proportion of activated splenic CD4 + Foxp3- T cells expressing IFN-γ were similar in control and RPM-treated groups, RPM decreased the proportions ICOS+ and CD8+ IFN-γ + splenic T cells. However, RPM also downregulated CXCR3+ expression by Tregs, and forelimb allografts had reduced infiltration by CXCR3+ Treg cells. In addition, allograft recipients whose Tregs lacked T-bet underwent accelerated rejection compared to WT mice despite RPM therapy. CONCLUSIONS: We demonstrate that while RPM increased the ratio of Treg to Teff cells and suppressed CD8+ T cell allo-activation, it failed to prevent CD4 Teff cell activation and impaired CXCR3-dependent Treg graft homing, thereby limiting the efficacy of RPM in VCA recipients.

Extended ex vivo normothermic perfusion for preservation of vascularized composite allografts.

Ischemia and reperfusion injury remains a significant limiting factor for the successful revascularization of amputated extremities. Ex vivo normothermic perfusion is a novel approach to prolong the viability of the amputated limbs by maintaining physiologic cellular metabolism. This study aimed to evaluate the outcomes of extended ex vivo normothermic limb perfusion (EVNLP) in preserving the viability of amputated limbs for over 24 hours. A total of 10 porcine forelimbs underwent EVNLP. Limbs were perfused using an oxygenated colloid solution at 38°C containing washed RBCs. Five forelimbs (Group A) were perfused for 12 hours and the following 5 (Group B) until the vascular resistance increased. Contralateral forelimbs in each group were preserved at 4°C as a cold storage control group. Limb viability was compared between the 2 groups through assessment of muscle contractility, compartment pressure, tissue oxygen saturation, indocyanine green (ICG) angiography and thermography. EVNLP was performed for 12 hours in group A and up to 44 hours (24-44 hours) in group B. The final weight increase (-1.28 ± 8.59% vs. 7.28 ± 15.05%, P = .548) and compartment pressure (16.50 ± 8.60 vs. 24.00 ± 9.10) (P = .151) were not significantly different between the two groups. Final myoglobin and CK mean values in group A and B were: 875.0 ± 325.8 ng/mL (A) versus 1133.8 ± 537.7 ng/mL (B) (P = .056) and 53 344.0 ± 16 603.0 U/L versus 64 333.3 ± 32 481.8 U/L (P = .286). Tissue oxygen saturation was stable until the end in both groups. Infra-red thermography and ICG-angiography detected variations of peripheral limb perfusion. Our results suggest that extended normothermic preservation of amputated limbs is feasible and that the outcomes of prolonged EVNLP (>24 hours) are not significantly different from short EVNLP (12 hours).

Self-organized formation of developing appendages from murine pluripotent stem cells.

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.

Graft-implanted, enzyme responsive, tacrolimus-eluting hydrogel enables long-term survival of orthotopic porcine limb vascularized composite allografts: A proof of concept study.

BACKGROUND: Currently, patients receiving vascularized composite allotransplantation (VCA) grafts must take long-term systemic immunosuppressive therapy to prevent immunologic rejection. The morbidity and mortality associated with these medications is the single greatest barrier to more patients being able to receive these life-enhancing transplants. In contrast to solid organs, VCA, exemplified by hand or face transplants, allow visual diagnosis of clinical acute rejection (AR), directed biopsy and targeted graft therapies. Local immunosuppression in VCA could reduce systemic drug exposure and limit adverse effects. This proof of concept study evaluated, in a large animal forelimb VCA model, the efficacy and tolerability of a novel graft-implanted enzyme-responsive, tacrolimus (TAC)-eluting hydrogel platform, in achieving long-term graft survival. METHODS: Orthotopic forelimb VCA were performed in single haplotype mismatched mini-swine. Controls (n = 2) received no treatment. Two groups received TAC hydrogel: high dose (n = 4, 91 mg TAC) and low dose (n = 4, 49 mg TAC). The goal was to find a dose that was tolerable and resulted in long-term graft survival. Limbs were evaluated for clinical and histopathological signs of AR. TAC levels were measured in serial blood and skin tissue samples. Tolerability of the dose was evaluated by monitoring animal feeding behavior and weight. RESULTS: Control limbs underwent Banff Grade IV AR by post-operative day six. Low dose TAC hydrogel treatment resulted in long-term graft survival time to onset of Grade IV AR ranging from 56 days to 93 days. High dose TAC hydrogel also resulted in long-term graft survival (24 to 42 days), but was not well tolerated. CONCLUSION: Graft-implanted TAC-loaded hydrogel delays the onset of Grade IV AR of mismatched porcine forelimb VCA grafts, resulting in long term graft survival and demonstrates dose-dependent tolerability.

Utility of IL-2 Complexes in Promoting the Survival of Murine Orthotopic Forelimb Vascularized Composite Allografts.

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.

Experimental Forelimb Allotransplantation in Canine Model.

As reconstructive transplantation is gaining popularity as a viable alternative for upper limb amputees, it is becoming increasingly important for plastic surgeons to renew surgical skills and knowledge of this area. Forelimb allotransplantation research has been performed previously in rodent and swine models. However, preclinical canine forelimb allotransplantation studies are lacking in the literature. The purpose of this paper is to provide an overview of the surgical skills necessary to successfully perform forelimb transplantation in canines as a means to prepare for clinical application. A total of 18 transplantation operations on canines were performed. The recipient limb was shortened at the one-third proximal forearm level. The operation was performed in the following order: bones (two reconstructive plates), muscles and tendons (separately sutured), nerves (median, ulnar, and radial nerve), arteries (two), and veins (two). The total mean time of transplantation was 5 hours ± 30 minutes. All of the animals that received transplantation were treated with FK-506 (tacrolimus, 2 mg/kg) for 7 days after surgery. Most allografts survived with perfect viability without vascular problems during the early postoperative period. The canine forelimb allotransplantation model is well qualified to be a suitable training model for standard transplantation and future research work.

A Porcine Orthotopic Forelimb Vascularized Composite Allotransplantation Model: Technical Considerations and Translational Implications.

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.

Orthotopic forelimb allotransplantation in the rat model.

In this report, we present a rat orthotopic forelimb allotransplantation model. Eight forelimbs were transplanted from Brown Norway rats to Lewis rats. Axillary vessels of transplant were used as the vascular pedicles, which were anastomosed to the external jugular vein and common carotid artery of the recipient rat. The ulnar, radial, and median nerves were also repaired. Among rats, a tapered dose of cyclosporine was administered in five rats. In other three rats, no immunosuppressive therapy was given. The viability and signs of rejection of transplanted forelimbs, sensation recovery, bone healing, and histology were assessed up to the 90th postoperative day. All of rats but one survived surgery. All of transplanted forelimbs survived. In the rats treated with cyclosporine the transplanted forelimbs achieved long-term survival with motion and sensation recovery. On 90th day after surgery, bone healing was achieved. There was no sign of rejection in histology. In the rats without cyclosporine treatment, the transplanted forelimbs experienced tissue necrosis started from day 12 postoperatively. This experimental study showed the feasibility of orthotopic forelimb allotransplantation in the rat model. © 2015 Wiley Periodicals, Inc. Microsurgery 36:672-675, 2016.

The Effect of Ex Situ Perfusion in a Swine Limb Vascularized Composite Tissue Allograft on Survival up to 24 Hours.

PURPOSE: To test the potential for the ex situ limb perfusion system to prolong limb allograft survival up to 24 hours. METHODS: We used 20 swine for the study. In group 1 (control), 4 limbs were perfused with heparin solution and preserved at 4°C for 6 hours. In group 2, 4 limbs were perfused with autologous blood at 27°C to 32°C for 24 hours. In both groups, limbs were transplanted orthotopically to recipients and monitored for 12 hours. In addition to perfusion parameters, we recorded perfusate gases and electrolytes (pH, pCO2, pO2, O2 saturation, Na, K, Cl, Ca, HCO3, glucose, and lactate) and obtained functional electrostimulation hourly throughout the experiment. Histology samples were obtained for TUNEL staining and single-muscle fiber contractility testing. RESULTS: In both groups, hemodynamic variables of circulation remained stable throughout the experiment. Neuromuscular electrical stimulation remained intact until the end of reperfusion in group 2 vs no response in group 1. In group 2, a gradual increase in lactate levels during pump perfusion returned to normal after transplantation. Compared with the contralateral limb in group 2, single-muscle fiber contractility testing showed no significant difference at the end of the experiment. CONCLUSIONS: We demonstrated extended limb survival up to 24 hours using normothermic pulsatile perfusion and autologous blood. CLINICAL RELEVANCE: Successful prolongation of limb survival using ex situ perfusion methods provides with more time for revascularization of an extremity.

Evaluation of a porcine whole-limb heterotopic autotransplantation model.

BACKGROUND: Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field. MATERIALS AND METHODS: In seven large white pigs, one forelimb was amputated under anesthesia and autotransplanted heterotopically with an arc of rotation of 180° and partially placed in a subcutaneous pocket. Clinical parameters were monitored and muscle biopsies were analyzed using ultrastructural morphological assessment of mitochondria quality after an observation period of 7 days. RESULTS: All animals could fully mobilize postoperatively without restrictions. At sacrifice, the anastomosed pedicle vessels of the limb were patent in six animals. In one pig, venous thrombosis could be observed. Muscle response was triggered following direct electrostimulation in six replanted limbs. The replanted extremities gained 12.97% weight within 7 days postreplantation compared with the amputation baseline values (P = 0.464 while maintaining normal compartment pressures at sacrifice (8.25 ± 5.31 cmH(2)O, P = 0.60). The ultrastructural evaluation of mitochondria morphology revealed intact mitochondria without signs of ischemia/reperfusion damage. CONCLUSION: This porcine model proved feasible, reliable, and reproducible for whole-limb autotransplantation. It presents significant potential in future preclinical research of whole-limb CTA transplantation.

Embryological evidence identifies wing digits in birds as digits 1, 2, and 3.

The identities of the digits of the avian forelimb are disputed. Whereas paleontological findings support the position that the digits correspond to digits one, two, and three, embryological evidence points to digit two, three, and four identities. By using transplantation and cell-labeling experiments, we found that the posteriormost digit in the wing does not correspond to digit four in the hindlimb; its progenitor segregates early from the zone of polarizing activity, placing it in the domain of digit three specification. We suggest that an avian-specific shift uncouples the digit anlagen from the molecular mechanisms that pattern them, resulting in the imposition of digit one, two, and three identities on the second, third, and fourth anlagens.

Bone quality in swine composite tissue allografts: effects of combination immunotherapy.

BACKGROUND: Tacrolimus (FK506)/mycophenolate mofetil (MMF)/prednisone combination immunosuppression therapy has been found to effectively prevent composite tissue allograft (CTA) rejection with minimal toxicity in a preclinical porcine model. These findings have been reproduced in 24 human hands transplanted in 18 patients. In CTAs containing bone, adequate bone quality and healing are essential for long-term functional success. The purpose of this study was to determine the effect FK506/MMF/prednisone immunotherapy has on bone quality and healing. METHODS: Forelimb CTA-flaps were transplanted in nine pigs. Recipient animals received FK506/MMF/prednisone therapy for 3 months. Bone quality was studied pre- and posttransplant by measuring acoustic velocity and density and by calculating elastic coefficients. Additional bone quality analyses were performed on unoperated limbs, and in bone grafts from two pigs that had autograft procedures performed. Bone healing was assessed using radiographic analysis. RESULTS: Three animals were lost to immunosuppression-related complications before the endpoint of the study. The bone component of all six CTA-flaps showed normal healing. Although results of the bone density measurements were not significantly different when comparing pre- to posttransplant values, acoustic velocity and elastic coefficient measurements showed a significant decrease posttransplant indicating a decrease in bone quality. CONCLUSIONS: FK506/MMF/prednisone combination therapy prevented rejection, did not adversely affect bone quality, and showed normal bone healing. The transplant procedure itself decreased bone quality more than the immunosuppression regimen did over the observation period in this study. Based on these findings, we conclude to prevent CTA failure it is important to monitor bone quality posttransplant.

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts.

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.

Migration of LHRH neurons into the spinal cord: evidence for axon-dependent migration from the transplanted chick olfactory placode.

In the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons originate in the olfactory placode and migrate along the olfactory nerve to the forebrain. In previous studies, we demonstrated that LHRH neurons followed the trigeminal nerve when the olfactory nerve was physically interrupted. To examine whether LHRH neurons possess the capacity to migrate along the different type of axons, the olfactory placode was transplanted into the base of the forelimb. Three to five days after the transplantation, LHRH neurons were detectable in the spinal nerve, the dorsal root ganglion, the sympathetic ganglion and the spinal cord. Double or triple labelling studies for LHRH, somatostatin and/or axonin-1 showed that LHRH neurons entered the spinal nerve in contact with the olfactory axons, which are specifically immunoreactive to somatostatin. Migrating LHRH neurons continued to associate closely with the olfactory axons in the spinal nerve. However, some LHRH neurons often migrated along with the axonin-1 positive spinal sensory axons, maintaining a distance from the olfactory axons. Furthermore, a few LHRH neurons were observed in the ventral root and the ventral funiculus independent of olfactory axons. As LHRH neurons were observed in the motor component of the spinal nerve, it is probable that LHRH neurons also invaded the spinal cord using the motor axons as a guiding substrate for their migration. These results suggest that the migration mode of LHRH neurons is axon dependent in the peripheral region, however, chemical identity with regard to axonal substrate choice for migration was not specified in the present study.

Swine composite tissue allotransplant model for preclinical hand transplant studies.

Our laboratory previously developed and used an orthotopic radial forelimb osteomyocutaneous flap in the pig as a preclinical composite tissue allograft (CTA) model. To ensure that it mimicked the clinical situation as closely as possible we developed this model taking many immunologic and reconstructive considerations into account. While our original pig CTA model was ideal for studying the methods of preventing skin, muscle, bone, vessel and nerve rejection, and systemic toxicity, it did not include specialized tissues/structures of a joint and digit. Therefore, we were unable to evaluate rejection of these specialized tissues and their functional properties. Recognizing the importance of assessing joint rejection and function in hand transplantation research we developed a new swine forelimb CTA model that included the animal's medial digit. The present study describes the anatomy and the transplantation technique used in this new preclinical CTA model. We transplanted a radial osteomyocutaneous flap that included the medial digit between two size- (17-21 kg) and age- (6-8-week) matched farm pigs. We removed the digit from the recipient pig's forelimb in continuity with a section of the radial bone and replaced it with the same structure transplanted from a donor pig. After transplantation, a full-length cast was placed on the recipient pig's operated limb and changes in flap color, temperature and the presence of edema were monitored continuously for 6 h, and then regularly at predetermined intervals over 4 days. No weight bearing restrictions were placed on the animal's operated limb. After 4 days, the animal was euthanized. Direct visual monitoring of the allograft during 4 days revealed it was viable with no signs of graft failure due to technical complications associated with the transplant procedure. Upon waking from anesthesia, the animal stood and wandered freely about its cage with no apparent difficulty. Based on the animal's high level of activity at this time, we concluded that the procedure caused it minimal morbidity. At 4 days after the operation, early signs of rejection (skin erythema and edema) were observed. By incorporating a digit into our original CTA pig forelimb model we have made it a better model for performing preclinical hand transplant studies. The added advantage of being able to assess methods of preventing rejection in the specialized joint/digital tissues (articular cartilage, digital flexor and extensor systems, the nail complex) and assess long-term function of these structures is important. The fact that the procedure does not cause major morbidity to the animal makes it possible to conduct long-term graft survival and functional studies.

Hand transplantation: pertinent data and future outlook.

Transplantation of composite tissue allografts, such as a hand, offers immense potential in reconstructive surgery. Review of current replantation literature suggests the prospect for significant functional return following hand transplant, provided appropriate patient is selected and allograft rejection is prevented. Experimental studies of limb transplantation in rodents have demonstrated the efficacy of combination therapy using multiple immunosuppressants. However, long-term survival of limb allografts could not be achieved in large animal models without significant drug toxicity. Given the potential for organ failure, opportunistic infection, and malignancy resulting from long-term immunosuppression, the risk-benefit ratio for hand transplant must be carefully weighed. Our laboratory has been able to achieve allograft survival with minimum immunosuppression by MHC matching or donor antigen exposure prior to immune maturity in the swine. Future transplantation of composite tissue allografts, therefore, may depend upon such modalities to induce host tolerance without long-term immunosuppression.

Susceptibility to cyclophosphamide and thalidomide of fetal rat limb buds grafted in athymic (nude) mice.

Limb buds of day-14 rat fetuses were cut into pieces and transplanted into athymic (nude) mice. On the 7th and 9th days after grafting, the host nude mice were given cyclophosphamide intraperitoneally (10-120 mg/kg) or thalidomide orally (30-240 mg/kg). On the 20th day, the grafted tissue was examined macroscopically and histiologically. The grafts maintained in vehicle-treated nude mice showed considerable growth and tissue differentiation similar to in vivo. Growth and histogenesis of the grafts were significantly inhibited by treatment with cyclophosphamide (greater than or equal to 20 mg/kg). There was no indication that treatment with thalidomide (less than or equal to 240 mg/kg) adversely affects the development of grafted limbs. Thus, the susceptibility of transplanted rat limb buds to these two human teratogens was identical to the susceptibility of living rat fetuses. The heterotransplantation method of embryonic tissues may be of potential use for the study of teratogenic mechanisms and for the screening of human teratogens.

Growth-promoting properties of the internal milieu of pregnant and lactating rats.

The growth-promoting properties of the internal milieu of pregnant and lactating rats were investigated using transplanted whole rat embryos or fetal paws. When placed under the kidney capsule of intact nonpregnant hosts and incubated for 12 days, such transplants grow rapidly, and tissues differentiate normally. Thus they provide an accurate means of assessing the growth-promoting properties of the internal environment of host animals in different physiological states. Transplant growth during days 0-11 of pregnancy was similar to that observed in age-matched virgin control hosts during an equivalent 12-day period. However, growth of transplants was decreased by 40% in hosts during days 10-22 of pregnancy and by approximately 30% in hosts during days 1-13 or days 11-23 of lactation. Increase in tail length, which was used as an index of maternal skeletal growth, was reduced by 50% during the second half of pregnancy and the early and late periods of lactation compared with age-matched virgin females. No such inhibition was recorded during the first half of pregnancy. Compared with virgins, serum insulin-like growth factor I (IGF-I) levels were reduced by approximately 20% on days 6 and 10 of pregnancy, and by 63-66% during the second half of gestation. Serum IGF-I levels rose during lactation to reach prepregnancy levels by day 12, but a second decline occurred by day 18 postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of forelimb allografts in young rabbits immunosuppressed with cyclosporine.

Seventeen forelimbs were transplanted orthotopically from young Dutch rabbits to young New Zealand rabbits treated with cyclosporine. The transplanted limbs demonstrated significant bone growth. The growth in the transplanted limbs was about 75 to 80% of that observed in the unoperated limb. The long bones of the 3 longest surviving rabbits (133 days, 150 days, 150 days) studied radiographically demonstrated increases in length over their original lengths (humerus 22%, ulna 26%, and radius 31%). Hair and nail growth were noted at about day 10. Response to pain stimuli (withdrawal of forelimb) and functional use (ambulation with 50% weight bearing) was seen at two to three months. Permanent survival was not achieved because of a species-specific toxic wasting syndrome from cyclosporine.