Split tolerance to a composite tissue allograft in a swine model

BACKGROUND: The antigenicity of skin is a major obstacle to expanding human composite tissue transplantation. For example, multiple rejection episodes of the skin have been noted in clinical hand transplant patients. We have previously demonstrated tolerance to vascularized musculoskeletal allografts in major histocompatibility complex (MHC)-matched miniature swine treated with 12 days of cyclosporine. This regimen did not reproducibly lead to tolerance to subsequent frozen donor skin grafts. However, such skin grafts did not have a primary vascular supply. The aim of this study was to determine if tolerance to limb allografts with a vascularized skin component could be achieved with MHC matching and a 12-day course of immunosuppression. METHODS: Hind limb grafts harvested with a 100 cm(2) cutaneous paddle were transplanted heterotopically into six MHC-matched, minor antigen-mismatched miniature swine. All animals received a 12-day course of cyclosporine. One control animal was not immunosuppressed. Grafts were evaluated with biweekly biopsies and tissue viability determined by histologic analysis. To test for sensitization, frozen donor skin grafts were applied to all animals that survived to postoperative day 100. RESULTS: All treated animals (n=6) were tolerant to their musculoskeletal allografts at the time of necropsy (>100 days) regardless of the status of the epidermis. One animal demonstrated tolerance to the skin for more than 180 days. The other five animals demonstrated prolonged survival of the epidermal portion of the graft. The control animal rejected the graft epidermis at 10 days postoperatively. Frozen donor skin grafts demonstrated accelerated rejection (<10 days) in three of the animals and led to simultaneous rejection of both the epidermis of the allograft and the skin graft in the long-term tolerant animal. The rejection of the skin grafts did not break tolerance to the musculoskeletal portion in any of the animals. CONCLUSIONS: All animals exhibited indefinite survival of the musculoskeletal portion of their allografts but only prolonged survival of the epidermis. The loss of the graft skin appears to be the result of an isolated immune reaction to the skin, and, in particular, the epidermis. This observation is further substantiated by the accelerated rejection of secondarily placed frozen donor skin grafts.     

Induction of tolerance to composite tissue allograft in a rat model

The goal of this study was to establish a rat model that can be used to determine the variables in influencing induction of tolerance to composite tissue allografts. An anti T-cell depleting agent (R73) and 15-deoxyspergualin were given in different doses and schedule to four groups of Lewis rats receiving a limb transplant from Brown-Norway donors. Graft survival prolongation was maximal combining a single dose of R73 and a 20-day administration of 15-deoxyspergualin. Long-term survivors accepted a skin graft from Brown-Norway donors at 80 days, but rejected grafts from an unrelated donor. Skin grafting did not influence survival of the transplanted limb. Mixed allogeneic chimerism was not detectable in peripheral blood by flow cytometry, but immunohistochemistry identified donor-derived cells in the thymus of tolerant recipients at 100 days. These results suggest a state of donor-specific, dynamic tolerance, with potential for future application in human composite tissue allotransplantation.     

Impact of donor bone marrow on survival of composite tissue allografts

Composite tissue allotransplants (CTA) involves transplantation of various tissues including vessels, nerves, skin, and immune cells and bears significant antigenic load. Different immunosuppressive protocols are used for experimental and clinical CTA. Immunosuppressive agents maintain survival of the different components of composite tissue allografts. However, the potential side effects of chronic immunosuppression currently limit the widespread application of CTA transplants. Bone marrow therapy in many tolerance induction protocols therefore provides a guide to reaching the target of permanent immunotolerance. Multiple studies suggest that bone marrow is immunomodulatory and may facilitate allograft acceptance.In this review, bone marrow-based therapy protocols of experimental and clinical models are presented in composite tissue transplantation.     

Sublethal fractionated total-body irradiation and donor bone marrow infusion for induction of allograft tolerance

Tolerance to skin allografts across the strong histocompatibility barrier H-2b to H-2d was achieved with sublethal fractionated total-body irradiation, FTBI, delivered to H-2d mice in 3 doses of 250 rads within 24 hr, followed by transfusion of 3 X 10(7) H-2b donor bone marrow (BM) cells. H-2b skin allografts were applied within 48 hr after the initial radiation. 70% of the mice became long-term (greater than 180-day) survivors with fur-bearing grafts. Marked interexperiment variability in survival rates suggested that infection was the major cause of death in this model and lower weight gain and survival rates for allogenic BM vs. media-treated controls suggested that graft-versus-host disease (GVHD) was also a factor. The observation, however, that long-term survivors (70% of all mice) gained weight and appeared healthy suggested that the GVHD might be self-limiting. Chimeric analysis revealed that approximately 25% of spleen cells were of donor origin, both at short-term (6 weeks) and long-term (greater than 1 year) intervals after tolerance induction. In spite of hematopoietic chimerism, a low incidence of spontaneous tumors, less than 1%, occurred in the long-term survivors.     

吻合血管同种异体骨移植后存活状况研究

目的报道吻合血管同种异体骨移植术后不同时段存活状况。方法建立吻合血管同种异体股骨干移植动物模型，在术后不同时段进行活体解剖，观察血管的通畅度．并切取移植骨进行组织学、电镜及SDH染色检测。结果术后4周对照组血管基本完全闭塞，而实验组术后血管保持通畅。组织学检查显示对照组术后移植骨骨陷窝内骨细胞缺失，哈弗斯管内血管亦消失；而实验组术后骨陷窝内始终有骨细胞充填。电镜表现为对照组术后移植骨出现骨细胞核浓缩．核碎裂，直至骨陷窝内骨细胞丢失；实验组术后骨细胞超微结构正常、对照组术后2周．骨组织SDH染色已无蓝染的骨细胞，而实验组术后可见同心圆排列蓝染的骨细胞。结论在免疫调控下，吻合血管的同种异体骨移植术后供体始终保持活力状态。     

Use of cryopreserved donor bone marrow in cadaver kidney allograft recipients

Donor-specific unresponsiveness to organ allografts remains an elusive goal in clinical transplantation, as most successful experimental protocols for the production of antigen-specific immunosuppression require lengthy recipient pretreatment. The use of an induction course of antilymphocyte serum (ALS) beginning at the time of transplantation, followed by the transfusion to the recipient of donor-specific bone marrow, has been shown in animals to induce prolonged allograft survival and is applicable for use in cadaver donor clinical transplantation. Our preliminary data in humans suggest that the transfusion of cryopreserved cadaver donor bone marrow following a short course of ALS is safe and does not induce graft-versus-host disease or allograft rejection. Twenty patients have been included in the protocol and 19 have been discharged from the hospital with functioning kidney transplants. One graft failed at 3 months. Eight patients have been withdrawn entirely from prednisone immunosuppression 3-6 months following transplantation. The contralateral kidneys from the marrow donors were transplanted into an additional 20 patients who received sequential immunosuppressive therapy without marrow transfusion. Three of these grafts have failed within 3 months due to acute rejection. Donor marrow transfusion may give rise to improved allograft and patient survival in clinical transplantation while at the same time allow for reduced requirements for nonspecific immunosuppressive agents with their undesirable side effects.     

Thymus-mediated immune tolerance to renal allograft is donor but not tissue specific.

Studies were conducted in Lewis (RT1l) rats to determine whether the process of unresponsiveness to kidney graft induced by the intrathymic glomerular transplantation were donor-strain specific as suggested by previous studies (Remuzzi et al., Lancet 1991;337:750-752). When glomeruli from Sprague-Dawley rats were injected in the thymus of Lewis rats, the subsequent kidney graft from a "third party" Brown-Norway (RT1n) rejected within 9 to 14 days. Moreover, an alternative site for glomerular antigen inoculation, such as i.p. administration, failed to induce a state of unresponsiveness to renal allograft. Whether tolerance was tissue specific was investigated by intrathymic injection of a preparation of donor blood cells that only included white cells. Such a maneuver, followed 10 days later by a kidney transplant, allowed indefinite renal graft survival in all animals, whereas all rats injected intrathymically with blood cell medium alone rejected the kidney graft in 8 to 11 days. Shortening the time interval between intrathymic injection of blood cells and kidney transplantation still allowed the graft to survive indefinitely. Finally, Lewis (RT1l) rats with chronic renal failure injected intrathymically with blood cells from Brown-Norway (RT1n) rats tolerated indefinitely a subsequent kidney graft from the same donor. These findings indicate that (1) the induction of immune tolerance to renal allograft induced by intrathymic injection of antigens is donor but not tissue specific; (2) the time interval between intrathymic injection of donor cells and the subsequent kidney transplantation can be reduced to 24 h; and (3) uremia does not preclude the possibility of renal allograft tolerance after the thymus procedure.     

New composite tissue allograft model of vascularized bone marrow transplant: the iliac osteomyocutaneous flap

Vascularized bone marrow transplant (VBMT) induces donor-specific chimerism in experimental models across the major histocompatibility barrier. An experimental model for immunotolerance studies should sustain a high antigenicity with low morbidity. Accordingly, we introduced an iliac bone osteomusculocutaneous (IBOMC) transplant model in rat. It consists of a large skin component and an abundant bone marrow cells (BMC) population. We tested this model with isograft transplantations between Lewis rats (RT1^l) and with allograft transplantation between Lewis-Brown Norway (LBN RT1^l + n) donors and Lewis (RT1^l) recipients under low dose of cyclosporine A monotherapy. Immunologic responses were tested for donor cell engraftment and chimerism induction. All isografts survived indefinitely and allografts were viable at 200 days post-transplant under low dose of cyclosporine A. Microangiography of the graft revealed preservation of skin, muscle, and bone components. Histologic examination confirmed viability of all allograft components without signs of rejection. Long-term engraftment of donor-origin (RT1ⁿ) BMC was confirmed by donor-specific chimerism (1.2%) in peripheral blood and bone marrow (1.65%) compartments and by engraftment into lymphoid organs of recipients. The IBOMC transplant proved to be a reliable composite tissue allotransplantation (CTA) model. Moreover, because of its robust bone marrow component and large skin component, it is applicable to studies on immunologic responses in CTA.     

Skin allograft survival following intrathymic injection of donor bone marrow.

BACKGROUND: Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity. MATERIALS AND METHODS: Lewis rats were injected intrathymically with 1 x 10(8) Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS. RESULTS: Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes. CONCLUSION: This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models.     

A delay in bone marrow transplantation after partial conditioning improves engraftment

BACKGROUND: In the present study we examined the effect of the timing of marrow infusion on engraftment in nonmyeloablatively conditioned mice. METHODS: B10 mice were conditioned with decreasing doses of total body irradiation (TBI) and reconstituted with bone marrow cells (BMCs) from major histocompatibility complex-disparate donor B10.BR mice at 0 or 6 hr, or on days 1, 2, 3, 4, 5, 8, and 12 with respect to TBI. RESULTS: After undergoing conditioning with 700 cGy TBI and transplantation with 15 x 10(6) BMCs, 100% of recipients engrafted if the marrow was infused between 0 and 4 days after TBI. For lower doses of TBI, a delay in infusion of the marrow after TBI conditioning was associated with a significant increase in engraftment. Significantly less engraftment was achieved in animals conditioned with 600 cGy TBI if the marrow was infused at 0 or 6 hr compared with a 1- to 4-day delay. When the TBI was decreased to 500 cGy, engraftment occurred only when the transplant was performed between days 2 and 8. The highest proportion of recipients engrafted when the marrow was infused on day 4. This enhanced engraftment after a delay in marrow infusion is associated with a significant reduction in host mixed lymphocyte reaction reactivity and is correlated inversely with serum levels of interleukin-6 in the recipient. CONCLUSIONS: These data demonstrate for the first time that a delay between conditioning and marrow infusion significantly improves allogeneic engraftment in nonmyeloablatively conditioned recipients and reduces the total conditioning required.     

The effect of G-CSF-stimulated donor marrow on engraftment and incidence of graft-versus-host disease in allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic bone marrow transplantation (allo-BMT). In an attempt to improve the results of HLA-identical sibling BMT, we investigated the effect of accelerating hemopoietic reconstitution and reducing acute GVHD (aGVHD) in allo-BMT receiving G-CSF-stimulated donor marrow and the preliminary biological mechanism. The donors of 30 patients (study group) with leukemia were given G-CSF 3-4 microg/kg/d for 7 doses prior to marrow harvest. The results of subsequent engraftment in the recipients were compared with those of 18 patients without G-CSF (control group). Five donors themselves were studied to assess the effects of G-CSF on the hematopoietic progenitor cells and lymphocyte subsets in the bone marrow (BM). We observed that the stimulated BM yielded higher numbers of nucleated cells as well as CFU-GM and CD34+ cells (p<0.01), and that hemopoietic reconstitution was accelerated. The median number of days of granulocyte count exceeding 0.5x10(9)/L and platelet count exceeding 20x10(9)/L was 16 (range 10-23 d) and 18.5 (range 13-31 d), respectively (control group: median 22 d, range 13-29 d and median 23 d, range 17-34 d; p=0.001). The incidence of grade II-IV severe aGVHD was very low, with only 1 case (3.3%) with acute grade II aGVHD limited to the skin in the study group. Five of 18 patients in the control group manifested grade II-IV severe aGVHD (27.8%, p=0.02). The number of T-lymphocyte subsets in the harvested BM using G-CSF stimulation was changed. In the G-CSF-stimulated marrow group, CD4+ decreased and CD8+ increased significantly (p=0.02). The changes of progenitor cells and T-lymphocyte subsets in donors' BM from pre- and post-G-CSF stimulation showed that the percentage of CD4+ reduced (p=0.04) and that of CD8+ increased (p=0.06), while that of CD34+ also increased (p=0.002). The incidence of chronic GVHD and relapse had no significant difference between both groups. These results indicate that allo-BMT in BM G-CSF priming can accelerate engraftment and minimize the incidence of severe aGVHD. There is a trend in favor of improved transplantation-related mortality.     

Immunologic approaches to composite tissue allograft

This article discusses the immunologic principles and the most promising immunologic approaches for composite tissue allograft tolerance. We have previously reviewed some of the pharmacologic approaches for composite tissue allo-transplantation. In this review, we will summarize the range of options that may address the challenge of transplantation in reconstructive surgery.     

Pharmacologic approaches to composite tissue allograft

This article discusses the pharmacologic approaches and the most promising new compounds for composite tissue allograft tolerance. Although some approaches rely on a combination of immunosuppressive agents that act synergistically against rejection, other strategies use immunologic manipulation, including major histocompatibility complex matching, induction of chimerism, and use of monoclonal antibodies to abrogate the immune response. There is still a need, however, to reproduce these findings in species phylogenetically closer to humans. This may be the target of future research efforts, which may overcome the challenge of limb and face transplant rejection.     

Establishing a composite tissue allograft registry

Currently there is no international registry for composite tissue allografts (CTA), but there have been discussions about creating such a registry. This article discusses the advantages/benefits, and disadvantages of establishing an international composite tissue registry. It also looks at some of the potential pitfalls that may hinder the long-term survival of the effort and makes recommendations as to how to avoid them based on the experience of other registries. It is our belief that now is the time for the formation of a CTA registry. There is a strong consensus among the transplanting centers for its formation. If properly constructed, a scientific registry on CTA will be a true attribute to the scientific and medical communities.     

组织工程化异体骨复合BMP和自体骨髓治疗股骨头缺血性坏死

目的探讨非细胞型组织工程化异体骨复合骨形态发生蛋白(BMP)和自体骨髓植入治疗青壮年股骨头缺血性坏死的疗效。方法首先进行股骨头髓芯减压,彻底刮除死骨。将异体松质骨混合bBMP和自体骨髓植入死骨刮除区,部分脱钙冻干异体腓骨植入髓芯减压孔道,直接支撑股骨头病变区软骨下骨。采用该方法治疗64例(78髋)股骨头缺血性坏死患者。结果所有患者均获随访,随访时间3个月～6年,平均44个月。随访3年以上28例(36髋),其中18例(22髋)FicatⅠ、Ⅱ期病例,患髋无明显疼痛及功能受限,CT显示形成密度较高的新骨,病变范围及程度无发展。4例(6髋)FicatⅠ、Ⅱ期病例,患髋症状无加重,但病变进展了一期。6例(8髋)FicatⅢ期病例,2例无明显疼痛及活动受限,4例因症状无明显改善而行人工关节置换术。结论异体腓骨可提供直接的机械支撑力,阻止股骨头病变发展和塌陷;BMP可诱导骨髓和异体骨以增强其成骨能力。异体骨复合BMP和自体骨髓可作为治疗青壮年FicatⅠ、Ⅱ期股骨头缺血性坏死的一种选择。