Intensive hands-on microsurgery course provides a solid foundation for performing clinical microvascular surgery.

PURPOSE: Microvascular surgery requires highly specialized and individualized training; most surgical residency training programs are not equipped with microsurgery teaching expertise and/or facilities. The aim of this manuscript was to describe the methodology and clinical effectiveness of an international microsurgery course, currently taught year-round in eight countries. METHODS: In the 5-day microsurgery course trainees perform arterial and venous end-to-end, end-to-side, one-way-up, and continuous suture anastomoses and vein graft techniques in live animals, supported by video demonstrations and hands-on guidance by a full-time instructor. To assess and monitor each trainee's progress, the course's effectiveness is evaluated using "in-course" evaluations, and participant satisfaction and clinical relevance are assessed using a "post-course" survey. RESULTS: Between 2007 and 2017, more than 600 trainees participated in the microsurgery course. "In-course" evaluations of patency rates revealed 80.3% (arterial) and 39% (venous) performed in end-to-end, 82.7% in end-to-side, 72.6% in continuous suture, and 89.5% (arterial) and 62.5% (venous) one-way-up anastomoses, and 58.1% in vein graft technique. "Post-course" survey results indicated that participants considered the most important components of the microcourse to be "practicing on live animals", followed by "the presence of a full-time instructor". In addition, almost all respondents indicated that they were more confident performing clinical microsurgery cases after completing the course. CONCLUSIONS: Microvascular surgery requires highly specialized and individualized training to achieve the competences required to perform and master the delicate fine motor skills necessary to successfully handle and anastomose very small and delicate microvascular structures. The ever-expanding clinical applications of microvascular procedures has led to an increased demand for training opportunities. By teaching time-tested basic motor skills that form the foundation of microsurgical technique this international microsurgery-teaching course is helping to meet this demand.

Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size.

PURPOSE: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS). METHODS: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group). RESULTS: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115).  CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Novel cuff design to facilitate anastomosis of small vessels during cervical heterotopic heart transplantation in rats.

Cervical heterotopic heart transplantation in rodents is a useful tool for studying transplantation immunology. However, end-to-end anastomosis of small-diameter vessels by using standard microsurgical technique is technically difficult and can require prolonged graft ischemia. A novel cuff system was designed from polyethylene tubing to allow anastomosis of vessels with internal luminal diameters of 0.3 to 0.9 mm. Key features include a spring-like adjustable lumen to facilitate vessel eversion, a barb to hold vessel ends in place after eversion, and a handling system that allows easy manipulation and stabilization of cuffs by a single operator. After a training period, a single operator performed a series of 8 transplants in which the mean warm ischemic time of grafts was 8.5 ± 2.9 min. Here we provide a detailed description of how to construct and perform end-to-end vessel anastomosis by using our novel cuff system. The discussion of the technique is supplemented with tips learned during the process of developing a reliable experimental model.

Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: role of adenosine receptors.

BACKGROUND: Ischemia-reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. METHODS: The extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. RESULTS: PCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5'-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. CONCLUSIONS: Exogenous ATP administration to ischemic skeletal muscles appears to spare intracellular energy by acting primarily through adenosine receptors.

Factors influencing microsurgical skill acquisition during a dedicated training course.

BACKGROUND: Proficient microsurgical skills are considered essential in plastic and reconstructive surgery. Specialized courses offer trainees opportunity to improve their technical skills. Trainee aptitude may play an important role in the ability of a trainee to acquire proficient skills as individuals have differing fundamental abilities. We delivered an intensive 5-day microsurgical training course. We objectively assessed the impact of the course on microsurgical skill acquisition and whether aptitudes as assessed with psychometric tests were related to surgical performance. METHODS: Sixteen surgical trainees (male = 10 and female = 6) participated in the courses. Trainees' visual spatial, perceptual, and psychomotor aptitudes were assessed on day 1 of the course. The trainees' performance of an end-to-end arterial anastomosis was assessed on days 2 and 5. Surgical performance was assessed with objective structured assessment of technical skills(OSATS) and time to complete the task. RESULTS: The trainees showed a significant improvement in OSATS scores from days 2 to 5 (P < 0.001) and the time taken to complete the anastomosis (P < 0.001). Aptitude scores correlated strongly with objectively assessed microsurgical skill performance for male trainees but not for females. CONCLUSIONS: We demonstrated that participating in a microsurgical training course results in significant improvement in objectively assessed microvascular surgical skills. The degree of skills improvement was strongly correlated with psychomotor aptitude assessments scores for male trainees.

A novel liposome-based therapy to reduce complement-mediated injury in revascularized tissues.

BACKGROUND: Ischemia/reperfusion (IR) injury is an unavoidable consequence of tissue transplantation or replantation that often leads to inflammation and cell death. Excessive complement activation following IR induces endothelial cell injury, altering vascular and endothelial barrier function causing tissue dysfunction. To mitigate the IR response, various systemic anti-complement therapies have been tried. Recently, we developed a localized therapy that uses biotinylated fusogenic lipid vesicles (BioFLVs) to first incorporate biotin tethers onto cell membranes, which are then used to bind therapeutic fusion proteins containing streptavidin (SA) resulting in the decoration of cell membranes. The therapy is applied in two steps using solutions delivered intra-arterially. MATERIALS AND METHODS: Alteration of formulation, concentration and duration of incubation of BioFLVs were conducted to demonstrate the ability of the system to modulate biotin tether incorporation in cultured cells. Using a rat hind limb model, the ability of BioFLVs to decorate endothelium of femoral vessels with FITC-labeled SA for 48 h of reperfusion was demonstrated. The feasibility of a BioFLV-based anti-complement therapy was tested in cultured cells using SA fused with vaccinia virus complement control protein (SA-VCP), a C3 convertase inhibitor. Human ovarian carcinoma (SKOV-3) cells were incubated with BioFLVs first and then with SA-VCP. To activate complement the cells were treated with a SKOV-3-specific antibody (trastuzumab) and incubated in human serum. RESULTS: Decoration of cells with SA-VCP effectively reduced complement deposition. CONCLUSIONS: We conclude that BioFLV-mediated decoration of cell membranes with anti-complement proteins reduces complement activation and deposition in vitro and has the potential for application against inappropropriate complement activation in vivo.

Ethical considerations in face transplantation.

Human face transplantation is now a clinical reality. The surgical techniques necessary to perform these procedures have been used routinely in reconstructive microsurgery for many years. From an immunological standpoint since face and hand contain mostly the same tissues it is reasonable to assume that the same immunosuppressive regimen found to be effective in human hand transplants should also work in face transplantation. It is the ethical issues associated with the risks and benefits of performing facial transplantation that have posed the greatest challenges leading up to performing this new procedure. In this editorial, we will review some of the main events that have led to the recently performed human face transplants, specifically focusing on the key ethical issues at the center of this debate. We will discuss how the research and clinical experience in human hand transplantation laid the foundation for performing face transplantation and describe the research and the ethical guidelines upon which a team at the University of Louisville based their position "to move ahead" in spite of much criticism. Finally we will outline some of the key arguments against face transplantation, and conclude with a discussion on what comes next now that the first human face transplants have been performed.

Psychosocial implications of disfigurement and the future of human face transplantation.

Although the first face transplants have been attempted, the social and psychological debates concerning the ethics and desirability of the procedure continue. Critics contend that these issues have not yet been sufficiently addressed. With this in mind, the present article seeks to elaborate on key psychological and social factors that will be central for addressing the ethical and psychosocial challenges necessary to move face transplantation into mainstream medicine. The goals of this article are to (1) discuss the psychosocial sequelae of facial disfiguration and how face transplantation may relieve those problems, and (2) delineate inclusion and exclusion criteria for the selection of research subjects for face transplantation. The article uses concepts from symbolic interaction theory in sociology to articulate a theoretically coherent scheme for comprehending the psychosocial difficulties of facial disfiguration and the advantages offered by facial transplantation. The authors conclude that the psychosocial implications of disfigurement warrant surgical intervention and that research in the area of face transplantation should continue.

Electrically stimulated rectus abdominis muscle flap to achieve enterostomal continence: development of a functional canine model.

BACKGROUND: Dynamic myoplasty has many clinical applications and has proven to be a versatile surgical procedure with great promise. This procedure has been used to achieve fecal/urinary continence, as in the dynamic graciloplasty, and to augment cardiac ventricular function, as is commonly seen with dynamic latissimus cardiomyoplasty. In the present study, the authors describe a functional innovative island flap sphincter created from the rectus abdominis muscle in a large-animal model to provide stomal continence for future clinical studies. METHODS: The caudal region of the rectus abdominis muscle in eight mongrel canines (23 to 25 kg) was investigated through anatomical dissections during which the location of the neurovascular pedicles and the intersegmental muscle dimensions between the muscle inscriptions were noted. The rectus abdominis muscle was used to create functional dynamic stomal sphincters that were trained with subcutaneously implanted pulse stimulators. RESULTS: The neurovascular pedicles were consistently found in similar locations along the posterior medial aspect of the caudal portion of the canine's rectus abdominis muscle. The vertical height of the deep inferior epigastric pedicle and caudal intercostal nerve muscular mean entry points were 6.75 +/- 1.89 cm and 7.44 +/- 0.86 cm, respectively. The mean caudal intersegmental muscle length of the rectus abdominis muscle used to create the sphincter was 9.69 +/- 1.81 cm. CONCLUSIONS: The canine rectus abdominis muscle has reliable anatomical locations where the neurovascular pedicle may be found. This canine muscle may be used to create a continent island flap stomal sphincter. This large-animal sphincter model is versatile, durable, and easy to manipulate, with minimal morbidity to the animal.

Composite tissue allotransplantation of the hand and face: a new frontier in transplant and reconstructive surgery.

Each year an estimated 7-million people in the USA need composite tissue reconstruction because of surgical excision of tumors, accidents and congenital malformations. Limb amputees alone comprise over 1.2 million of these. This figure is more than double the number of solid organs needed for transplantation. Composite tissue allotransplantation in the form of hand and facial tissue transplantation are now a clinical reality. The discovery, in the late 1990s, that the same immunotherapy used routinely in kidney transplantation was also effective in preventing skin rejection made this possible. While these new treatments seem like major advancements most of the surgical, immunological and ethical methods used are not new at all and have been around and routinely used in clinical practice for some time. In this review of composite tissue allotransplantation, we: (i) outline the limitations of conventional reconstructive methods for treating severe facial disfigurement, (ii) review the history of composite tissue allotransplantation, (iii) discuss the chronological scientific advances that have made it possible, (iv) focus on the two unique clinical scenarios of hand and face transplantation, and (v) reflect on the critical issues that must be addressed as we move this new frontier toward becoming a treatment in mainstream medicine.

Risk acceptance in laryngeal transplantation.

PURPOSE: Advancements in the fields of head and neck surgery and immunology have paved the way for new quality of life-improving procedures such as larynx transplantation. To quantitatively assess the risks versus benefits in larynx transplantation, we used a questionnaire-based survey (Louisville Instrument For Transplantation [LIFT]) to measure the degree of risk individuals are willing to accept to receive different types of transplantation procedures. METHODS: The LIFT contains 237 standardized questions incorporating standard gamble and time tradeoff outcome measures as well as questions assessing body image perception, depression, self-esteem, optimism, socially desirable responding, and demographics. Respondents were questioned on the extent to which they would trade off specific numbers of life-years, or sustain other costs, in exchange for receiving seven different types of transplant procedures. For this study, we questioned 243 individuals in three study populations with differing life experiences: healthy individuals, organ transplant recipients, and laryngectomees. RESULTS: All populations questioned perceived risks differently based on their varied life experiences and would accept differing degrees of risk for the different transplant procedures. Organ transplant recipients were the most risk-tolerant group, whereas laryngectomees were the least risk-tolerant. CONCLUSIONS: By questioning individuals with life experiences directly relevant to the risks and benefits associated with larynx transplantation, this study provides an empiric basis for assessing risk versus benefit in this new quality of life-improving procedure.

Neuroregeneration in composite tissue allografts: effect of low-dose FK506 and mycophenolate mofetil immunotherapy.

BACKGROUND: The immunosuppressant FK506 has been reported to increase the rate of peripheral nerve regeneration in nerve crush injury and nerve allograft models. The purpose of this study was to determine whether low doses of FK506 and mycophenolate mofetil had a neuroregenerative effect in revascularized peripheral nerve allografts in a rat hind limb transplantation model. METHODS: Wistar Furth rat recipients received limbs from syngeneic Wistar Furth donors (group 1, n = 4) or from allogeneic August X Copenhagen Irish rat donors (group 2, n = 6). Wistar Furth recipients received limbs from August X Copenhagen Irish donors and were treated with FK506/mycophenolate mofetil for 5 months (group 3, n = 7). At the end of the follow-up period, histomorphometric analysis of sciatic and tibial nerves from transplanted and intact hind limbs was conducted. Sciatic and tibial nerves were examined at the level of coaptation and near the neuromuscular junction, respectively. RESULTS: Transplanted limbs in groups 1 and 3 completed the study without rejection, while the limbs in group 2 were rejected within a few days. Sciatic and tibial nerve analysis in groups 1 and 3 limbs showed myelinated axons of various diameters but in significantly fewer numbers than in nontransplanted contralateral nerves. The number and size of myelinated axons of transplanted nerves at corresponding levels were not significantly different between syngeneic and allogeneic (FK506/mycophenolate mofetil-treated) transplants. CONCLUSIONS: The authors conclude that long-term neuroregeneration of revascularized peripheral nerves using low-dose FK506/mycophenolate mofetil was similar to that of syngeneic transplants. The occurrence of acute rejection episodes with low-dose FK506/mycophenolate mofetil did not appear to benefit nor impair neuroregeneration.

Investigation of risk acceptance in facial transplantation.

BACKGROUND: The surgical techniques necessary to transplant a human face are well established, and the early success of human hand transplants suggests that the immunological hurdles of transplanting human facial tissues have largely been overcome. Therefore, it is the ethical barriers that pose the greatest challenge to performing facial transplantation. At the center of the ethical debate is the question, "Do the risks posed by the life-long immunosuppression that a recipient would have to take justify the benefits of receiving a face transplant?" In this study, the authors answer this question by assessing the degree of risk individuals would be willing to accept to receive a face transplant. METHODS: To quantitatively assess risks versus benefits in facial transplantation, the authors developed the Louisville Instrument for Transplantation, or LIFT, which contains 237 standardized questions. Respondents in three study populations (healthy individuals, n = 150; organ transplant recipients, n = 42; and individuals with facial disfigurement, n = 34) were questioned about the extent to which they would trade off specific numbers of life-years, or sustain other costs, in exchange for receiving seven different transplant procedures. RESULTS: The authors found that the three populations would accept differing degrees of risk for the seven transplant procedures. Organ transplant recipients were the most risk-tolerant group, while facially disfigured individuals were the least risk tolerant. All groups questioned would accept the highest degree of risk to receive a face transplant compared with the six other procedures. CONCLUSIONS: This study presents an empirical basis for assessing risk versus benefit in facial transplantation. In doing so, it provides a more solid foundation upon which to introduce this exciting new reconstructive modality into the clinical arena.

Risk acceptance in composite-tissue allotransplantation reconstructive procedures.

Composite-tissue allotransplantation (CTA) is a new therapeutic modality to reconstruct major tissue defects of the face, larynx, and extremities. Unlike most life-saving organ-transplantation procedures, CTA is considered to improve quality of life. Therefore, the question arises, do the risks posed by the immunosuppression drugs that patients must take to prevent rejection justify the benefits of these procedures? The purpose of this study was to assess the relative risk that individuals are willing to accept in order to receive the benefits of CTA procedures. We used a psychometrically reliable and valid instrument to question two primary populations of individuals: those who live with the risks of immunosuppression, and healthy individuals. The level of risk acceptance for the seven transplant procedures tested (foot, single hand, double hand, larynx, kidney, hemiface, and full face) showed significant differences in research participants' risk acceptance for the different transplant procedures, but no significant differences between groups. Based on these findings, we conclude that certain CTA procedures convey benefits to recipients that are perceived by subjects, including individuals who live with the risks of immunosuppression, to warrant the risks of these procedures.

Ischemic preconditioning of skeletal muscle: duration of late-phase protection.

BACKGROUND: The time course of the late phase of ischemic preconditioning (IPC) was determined in latissimus dorsi muscle (LDM) flaps using viability and function as the endpoints. MATERIALS AND METHODS: LDM flaps from Sprague-Dawley rats were allocated into 6 groups. LDMs were preconditioned with 2 30-minute periods of ischemia separated by 10 minutes of reperfusion and subjected to a 4-hour ischemic insult after 24, 48, 72, and 96 hours from IPC. LDMs were evaluated for percent necrosis and muscle contractile function and compared with controls. RESULTS: The late phase of IPC provides significant protection against necrosis up to 72 hours. Conversely, when the end point used was muscle contractile function, the protection only lasted 48 hours. CONCLUSION: The time course of late-phase protection in skeletal muscle is 2-3 days. Late phase IPC appears to protect muscle flaps during the most critical time period following elevation.

Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy.

Although vascularized bone and joint allotransplantation is a promising new treatment option for reconstructing large bone defects, the need for immunosuppressive agents to prevent rejection in these procedures poses a major problem. This problem stems from the fact that several of these agents can cause harmful side effects, such as alterations in bone quality and healing. Therefore, the purpose of this study was to determine what effect the commonly used immunosuppressant regimen cyclosporine A-based combination therapy has on bone quality and healing. In 10 pigs, vascularized bone allografts with skin and muscle components (osteomyocutaneous free flaps) were transplanted from size-matched donor animals. Recipient animals received oral cyclosporine A/mycophenolate mofetil/prednisone therapy for 90 days. Bone quality was studied before and after transplantation by measuring the bone's acoustic velocity and density and calculating the bone's elastic coefficient. Bone healing was assessed using radiographic analysis. Four animals were lost as a result of graft rejection or immunosuppression-related complications before the 90-day endpoint of the study. Although bone specimens taken from the six animals that completed the 90-day protocol had histological signs of rejection, they all seemed to have normal bone healing. Posttransplant bone density values were significantly decreased (p < 0.05) (1544.7 +/- 47.5 kg/m3) as compared with pretransplant values (1722.7 +/- 44.1 kg/m3). Results of the acoustic velocity and elastic coefficients measurements showed a significant decrease (p < 0.05) in posttransplant values (from 3503.0 +/- 165.1 meters/sec to 2963.0 +/- 54.6 meters/sec and from 21.6 +/- 2.2 GPa to 13.6 +/- 0.5 GPa, respectively), indicating diminished bone quality. The findings indicate that cyclosporine A/mycophenolate mofetil/prednisone combination therapy is ineffective in preventing bone rejection, that it decreases bone quality, and that it is associated with systemic toxicity, suggesting that this immunosuppressive regimen at the doses used in this study is not ideal for vascularized bone allotransplantation procedures.

Lymphadenectomy prior to rat hind limb allotransplantation prevents graft-versus-host disease in chimeric hosts.

In previous rat studies, the use of mixed allogeneic chimerism (MAC) to induce host tolerance to hind limb allografts has resulted in severe graft-versus-host disease (GVHD). The purpose of this study was to determine if immunocompetent cells in bone marrow (BM) and/or lymph nodes (LNs) of transplanted limbs were responsible for inducing GVHD in mixed chimeric hosts. [ACI-->Wistar Furth] chimeric rats received ACI hind limbs that were non-irradiated, irradiated (1050 cGy) or lymphadenectomized. Rejection, GVHD and donor chimerism was assessed. Chimeric hosts rejected none of their limbs. However, hosts of non-irradiated hind limbs succumbed to GVHD 22.4+/-0.8 days after transplantation. In contrast, chimeras that received irradiated or lymphadenectomized ACI hind limbs showed no clinical or histological signs of GVHD at 5 months. We conclude that mixed chimeric hosts are susceptible to GVHD due to the immunocompetent cell load provided by the LNs, not the BM, of hind limb allografts.

On the ethics of facial transplantation research.

Transplantation continues to push the frontiers of medicine into domains that summon forth troublesome ethical questions. Looming on the frontier today is human facial transplantation. We develop criteria that, we maintain, must be satisfied in order to ethically undertake this as-yet-untried transplant procedure. We draw on the criteria advanced by Dr. Francis Moore in the late 1980s for introducing innovative procedures in transplant surgery. In addition to these we also insist that human face transplantation must meet all the ethical requirements usually applied to health care research. We summarize the achievements of transplant surgery to date, focusing in particular on the safety and efficacy of immunosuppressive medications. We also emphasize the importance of risk/benefit assessments that take into account the physical, aesthetic, psychological, and social dimensions of facial disfiguration, reconstruction, and transplantation. Finally, we maintain that the time has come to move facial transplantation research into the clinical phase.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

BACKGROUND: Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. METHODS: [ACI-->WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. RESULTS: [ACI-->WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4+/-2.8 days after limb transplantation. [ACI-->WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. CONCLUSIONS: Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI-->WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Composite tissue allotransplantation in chimeric hosts part II. A clinically relevant protocol to induce tolerance in a rat model.

BACKGROUND: We and others have shown that mixed allogeneic chimerism induces donor-specific tolerance to composite tissue allografts across major histocompatibility complex barriers without the need for immunosuppression. However, a delay period between bone marrow transplantation and limb allotransplantation is required, making such protocols impractical for clinical application. This study eliminates this delay period in a rat hind limb allotransplantation model by performing mixed allogeneic chimerism induction and transplantation "simultaneously." METHODS: Group 1 included controls in which naïve Wistar Furth (WF) hosts received ACI hind limbs. Group 2 included (ACI-->WF) chimeras that received limbs from third-party donors (Fisher), and group 3 included chimeras that received irradiated (1,050 cGy) ACI limbs. In group 4, WF hosts conditioned with 950 cGy received irradiated (1,050 cGy) ACI limbs followed by infusion of 100 x 10(6) ACI T-cell-depleted bone marrow cells and immunotherapy (tacrolimus and mycophenolate mofetil) for 28 days. Group 5 animals received the same treatment as group 4 animals without immunotherapy. RESULTS: The rats in groups 1 and 2 rejected their limbs within 10 days. Only one rat in group 4 survived to the end of the study. Groups 3 and 5 demonstrated long-term limb survival without rejection or graft-versus-host disease. High levels of donor chimerism (>80%) were achieved and maintained throughout the study. Mixed lymphocyte reaction assays in both groups revealed donor-specific hyporesponsiveness with vigorous third-party reactivity. CONCLUSIONS: This study demonstrated that infusion of donor bone marrow cells into conditioned hosts immediately after limb transplantation results in stable mixed chimerism, robust tolerance, and reliable limb allograft survival.