Tissue Allograft Transplantation Research Articles

Cytomegalovirus-Related Complications in Human Hand Transplantation

Up to date, 24 hands/thumbs have been transplanted in 18 patients. We herein report on cytomegalovirus (CMV) infection, disease, and the adopted treatment. Immunosuppression consisted of tacrolimus-based triple-drug therapy with antithymocyte globuline or CD25-receptor antagonist induction. Donor/recipient CMV match was negative/negative (n=8), negative/positive (n=3), positive/positive (n=3), positive/negative (n=3) and unknown in one case. Six patients (three +/-, two +/+, and one -/+) received gancyclovir i.v. followed by oral gancyclovir or valgancyclovir for prophylaxis. Patient and graft survival at a mean follow-up of 42.9 months were 100% and 91%, respectively. Of all patients tested for CMV, 45.5% developed CMV infection or disease. Two patients that were given a CMV-positive graft showed very high viral loads (550 and 1200/200000 leukocytes) after transplantation. Gancyclovir treatment failed to permanently control CMV in 80% of the patients experiencing CMV infection. Those patients requiring more toxic second-line therapies (foscarnet/cidofovir) suffered from side effects such as nephrotoxicity, nausea, vomiting, and diarrhea. CMV infection/disease complicated the postoperative course after composite tissue allograft (CTA) transplantation in five of nine recipients challenged with the virus. The close time correlation suggests an association between virus replication and rejection in some cases. CMV represents the major infectious threat in CTA transplantation. Therefore, CMV-mismatch should be avoided and prophylaxis with valgancyclovir and anti-CMV hyperimmunoglobulin should be mandatory.

Development and Maintenance of Donor-Specific Chimerism in Semi-Allogenic and Fully Major Histocompatibility Complex Mismatched Facial Allograft Transplants

The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.

Composite Tissue Allograft Transplantation of Cephalocervical Skin Flap and Two Ears

Composite Tissue Allograft Transplantation of Cephalocervical Skin Flap and Two Ears

Tolerance Induction in Composite Facial Allograft Transplantation in the Rat Model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.

Bone and tissue allograft use by orthopaedic surgeons

The purpose of our study was to determine the involvement of orthopaedic surgeons in the process of acquiring allografts they transplant. A questionnaire regarding allograft acquisition and use was directed to 340 hospitals. In approximately 85% of the institutions, nonorthopaedic personnel selected and acquired the allografts. In most, those responsible for providing surgeons with allografts had little or no knowledge of the practices of tissue banking and allograft transplantation biology. In about 15% of the hospitals, the surgeon was involved in the selection of the source of allografts. It is imperative that orthopaedic surgeons who transplant bone and tissue allografts become actively involved in determining the source and processing of tissue transplants they place in their patients.

New composite tissue allograft transplantation model in mouse with intravital microscopic evaluation of microcirculation.

A new mouse composite tissue allograft (CTA) transplantation model was developed to study the microcirculatory changes during acute allograft rejection and ischemia/reperfusion (I/R) injury. The donor cremaster muscle allografts were prepared as a tube flap, harvested on the common iliac vessels, transplanted to the neck region of the recipient, and anastomosed to the recipient's ipsilateral carotid artery and external jugular vein using standard end-to-end microsurgical technique. In Group 1 (n=6), the hemodynamics of cremasteric muscle microcirculation was measured in C57BL/6N mice without transplantation for baseline data. In Group 2 (n=6), isograft transplantations were performed between C57BL/6N mice. In Group 3 (n=5), allograft transplantations were performed across a high histocompatibility barrier between C3H and C57BL/6N mice. Following transplantation, cremaster muscle tube flaps were prepared for standard microcirculatory measurements of functional capillary perfusion, diameters, and red blood cell (RBC) velocities of 1 (st), 2 (nd), and 3 (rd) order arterioles and venules, and numbers of rolling, adhering, and transmigrating leukocytes and lymphocytes. Hemodynamic parameters of microcirculation did not differ significantly between the three groups. However, the number of rolling, adhering, and transmigrating polymorphonuclear leukocytes and lymphocytes was significantly increased in the allograft group ( p<0.001) as early as 2 hr following transplantation. Cremaster muscle transplantation in mice is a reliable and reproducible model with a 95 percent immediate success rate. The model offers the unique possibility of studying leukocyte-endothelial interaction during acute allograft rejection and I/R injury in mouse.

217. Thymic dependence of tolerance in vascularized composite tissue allografts under Cyclosporine A and ab-T Cell Receptor monoclonal antibody protocol

Background In our previous studies, we have demonstrated that vascularized composite tissue allograft transplants treated with ab-TCR) monoclonal antibody (mAb) and Cyclosporine A (CsA) for 7 days developed robust tolerance across MHC barrier. In this paper we have investigated the role of host thymus in the induction ot tolerance in rat hind-limb allograft model. Material and Methods Five weeks old, euthymic (group A) and thymectomized (group B) Lewis recipients (LEW,RT1l) received vascularized hind-limb allografts from Lewis-Brown-Norway (LBNŕF1, RT1l+n) donors. Group lA (n=6) and 1B (n=6) and Group IIA (n=6) and IIB (n=6) served as the isograft and the allograft control groups, respectively. Groups IIIA (n=5) IIIB (n=5) and IVA (n=5) and IVB (n=5) received monotherapies of either ab-TCR mAb or CsA tor 7 days, respectively. In Groups VA(n=6) and VB (n=6) a treatment protocol of combined ab-TCR/CsA was applied for 7 days. Mixed lymphocyte reaction (MLR) and skin grafting were performed for the evaluation of the donor specific tolerance in vitro and in vivo, respectively. Flow cytometry analysis was applied for the determination of the efficacy ot immunosuppressive treatment and the presence of mixed donor specific, hematopoietic chimerism. Results Combined treatment with ab-TCR/CsA protocol resulted in indefinite survival of the euthymic rats from group VB (>100 days) and extended the survival of the animals from group VA up to 51 days, post-transplant. MLR showed donor specific tolerance in the group VA at day 100. Flow cytometry revealed stable, multillinege chimerism in the peripheral blood of euthymic rats (CD4+/RT1n–17.3% and CD8+/RT11n–13.9%) and transient chimerism in thymectiomized limb allograft recipients. Conclusions Thymus is necessary for the induction ot donor specific tolerance under ab-TCR/CsA protocol in vascularized composite tissue allograft model. Tolerogenic activity of the thymus resulted in the creation ot stable mixed hematopoietic macrochimeric state within recipient.

Induction of donor-specific tolerance in rat hind-limb allografts under antilymphocyte serum and cyclosporine a protocol

Composite tissue allograft (CTA) transplantation became a clinical reality despite major side effects associated with the administration of chronic immunosuppression. Development of new treatment modalities eliminating life-long immunosuppression is essential for the future of CTA transplantation. In this study, combined use of cyclosporine A (CsA) and antilymphocyte serum (ALS) was tested for the potential to induce tolerance in the rat hind-limb allograft recipients across a major histocompatibility (MHC) barrier (Lewis-Brown-Norway [LBN, RT1(l+n)] to Lewis [LEW, RT1(l)] rats). Thirty transplantations were performed in 5 experimental groups. Animals received CsA and ALS 12 hours before surgery for 21 days thereafter. Although the allograft controls rejected their limbs at day 7 combined treatment of CsA and ALS resulted in indefinite survival (over 420 d) in all allograft recipients. Long-term survivors showed 35% to 42% of donor-specific chimerism in the peripheral blood. Clinical tolerance was confirmed by acceptance of the donor-specific skin grafts and immunocompetence was confirmed by rejection of the third-party grafts. Mixed lymphocyte reaction revealed suppressed response against donor-type antigens and increased response to third-party antigens. Donor-specific tolerance across MHC barrier was induced in CTA allografts under 21 days protocol of ALS/CsA.

Concerns about human hand transplantation in the 21st century.

The decision to perform a human hand transplant was justified perhaps on less than an ideal scientific basis-only approximately 60 rat limb transplants and 2 primate limb transplants have survived for longer than 200 days and only 8 of 19 pig limb osteomyocutaneous transplants showed no signs of rejection at 90 days. It seems unlikely that the survival of a human hand transplant will be any better than the survival of a kidney transplant, which has a half-life of approximately 7.5 to 9.5 years. Fourteen hand transplants, however, have now been performed in 11 humans with the skin component of 1 remaining viable up to 3 years after surgery. Intermittent episodes of acute rejection seem to have been relatively simple to reverse by temporarily increasing the dose of immunosuppressive agents and steroids. Chronic rejection has occurred in 1 patient, necessitating re-amputation of the transplanted hand. Active range of motion of the digits has been surprisingly better than would have been expected based on previous results of replantation, but return of sensibility has been less than optimal. The immunosuppression has been well tolerated without any major medical problems or life-threatening episodes, but some patients have developed chronic viral and fungal infections and several have developed posttransplant diabetes. Extrapolating from the previous experience of solid-organ transplants, chronic immunosuppression may predispose a hand transplant patient to an 80% chance of developing an infection, a 20% potential risk of developing posttransplant diabetes, and a 4% to 18% potential risk of developing a malignancy. Even though there is universal agreement that composite tissue allograft transplantation will become the ultimate reconstructive option, no one can predict the eventual role of hand transplantation in the future, but perhaps an international database of these hand transplant patients should be established so that independent reviewers can more objectively evaluate their functional outcome, the incidence of chronic rejection, and the risks of long-term immunosuppression.

Advances in composite tissue allograft transplantation as related to the hand and upper extremity

The clinical transplantation of composite tissue allografts (CTA) such as human hand or larynx is stimulating discussions among surgeons at national and international forums on the indications, ethical aspects, toxic effects of immunosuppression, and functional results of the first reported cases of unilateral and bilateral hand transplantation. This Clinical Perspective article presents the latest advances in clinical and experimental research related to the field of CTAs. The article presents the historic aspects of CTA, a broad view of the current state of composite tissue transplantation, the mechanism of allograft rejection, current experimental and clinical protocols, and, finally, the future prospects of the standard use of CTAs. It is clear that there is a substantial demand for routine use of CTAs but the treatment protocols need to be optimized and the functional outcomes need to be improved.

Tolerance to limb tissue allografts between swine matched for major histocompatibility complex antigens.

Transplantation of limb tissue allografts would greatly expand the realm of reconstructive surgery. However, the toxicity of chronic immunosuppression has adversely tilted the risk-benefit balance for clinical transplant. In this study, a procedure was sought to achieve host tolerance to limb tissue allografts through matching of the major histocompatibility complex (MHC) antigens between donor and host swine using only a 12-day course of cyclosporine. Massachusetts General Hospital (MGH) miniature swine were used as a large animal model with defined MHC, and musculoskeletal grafts from the donor hind limb were transplanted heterotopically to the recipient femoral vessels. Allografts from MHC-mismatched donors treated with cyclosporine (n = 4) were rejected in less than 6 weeks by gross inspection and histologic sections. Allografts from MHC-matched, minor antigen mismatched donors not treated with cyclosporine (n = 4) were rejected between 9 and 12 weeks. Allografts from similarly matched donors treated with 12 days of cyclosporine (n = 7) showed no evidence of rejection until sacrifice between 25 and 47 weeks. Thus allograft tolerance was achieved between MHC-matched swine using a limited course of cyclosporine. Demonstration of limb tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward clinical transplantation.

Use of swine model in transplantation of vascularized skeletal tissue allografts

Permanent tolerance to vascularized skeletal tissue allografts can be induced in miniature swine with minor antigen differences using a 12-day course of CsA. Demonstration of skeletal tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward transplantation of skeletal tissue allografts in humans.

Withdrawal of immunosuppression: implications for composite tissue allograft transplantation

Complete or partial withdrawal of immunosuppression is a desirable goal for physicians managing solid organ transplant recipients and has particular appeal for the management of composite tissue allograft recipients. Experience to date with steroid withdrawal or cyclosporine withdrawal in organ transplant recipients suggests that the risks of acute rejection are minimized with slow tapering of the drugs and when drug withdrawal is attempted many months or years after transplantation. Unfortunately, the full benefits of withdrawing any component of a multidrug immunosuppression regimen can probably be achieved only when the drug is withdrawn relatively early after transplantation. Thus, there is a need for improved immunologic monitoring to facilitate withdrawal of immunosuppression in any setting. Because steroid withdrawal might be particularly advantageous to the recipient of a composite tissue allograft, further experience is needed to determine the safety of steroid withdrawal with newer immunosuppressants such as tacrolimus, mycophenolate mofetil, and sirolimus.

Transplantation of Vascularized Composite Mandibular Allografts in Young Cynomolgus Monkeys

In the search for donor tissue for massive craniofacial defects, the transplantation of somatic tissue allografts was explored. Four young, out-bred cynomolgus monkeys were the recipients of orthotopic hemimandibular allotransplants from nonrelated cynomolgus monkeys. The transplant consisted of one-half of the mandible with attached muscle, skin, and mucosa. Cyclosporine 15 mg/kg/day was given subcutaneously each day. The 4 monkeys were observed for 13, 27, 63, and 65 days, respectively. All transplants showed primary wound healing and hair growth. The 2 longest survivors chewed, ate a normal diet, and gained weight. Two allografts showed severe rejection signs at 2 to 3 weeks, and the monkeys were euthanized. One monkey had a second episode of rejection that could not be reversed, and it was killed. The fourth monkey died of undetermined causes.