Desferrioxamine treatment prevents chronic islet allograft damage

BALB/cByJ islet allografts are acutely rejected when transplanted into allogeneic mice (CBA/J). Culture of the tissue for 7 days in 95% O2 before grafting is a suboptimal treatment for the reduction of immunogenicity in this strain combination. Approximately half the animals reject these transplants in a chronic fashion. Chronic islet rejection differs from acute rejection of uncultured allogeneic islets. During chronic rejection, beta cells within the transplanted tissue degranulate but remain intact when the animal returns to the diabetic condition. Acute islet rejection is characterized by the destruction of beta cells that remain heavily granulated as long as they remain intact. We examined the effect of the iron chelating agent, desferrioxamine, on chronic islet allograft damage. Desferrioxamine inhibited chronic islet allograft damage but did not influence the process of rejection of uncultured islet tissue. This effect of desferrioxamine could not be attributed to a direct immunosuppressive effect of this agent.     

Cell repopulation in vascularized bone grafts.

Following vascularized bone autografts, osteocyte viability is largely maintained. Viable cells within a graft may be surviving graft-derived cells, their progeny, or host-derived cells from circulation or surrounding bone. This study was conducted to define the process of cell repopulation, within vascularized bone grafts. Using inbred Lewis rats, 30 female vascularized tibial bones were transplanted to syngeneic male recipients and 45 male grafts were transplanted to female recipients. Twenty-five female recipients were immunosuppressed with FK506 to prevent rejection caused by Y-chromosome related antigens. The grafts were assessed up to 24 weeks post-transplant by radiography, histology and semi-quantitative polymerase chain reaction (PCR) using both Y-chromosome and autosome gene-specific primers. The female to male isograft transplants were useful to measure low levels of repopulation with host-derived cells, while male to female transplants more accurately quantified higher levels of cellular replacement. No host-derived cells were detected in the transplanted bone before six weeks. Thereafter, the ratio of host-derived cells gradually increased. By 24 weeks only 0.1-1.0% of graft-derived cells remained in the transplanted tibias. This study demonstrates that Y-chromosome-specific PCR is a useful tool to detect the cell lineage and cell repopulation following rat sex-mismatch vascularized bone grafting. Our results showed that donor cells in vascularized bone grafts were gradually repopulated with recipient cells. Correlation with histologic findings suggests that the periosteal hypertrophy observed by six weeks post-transplant results from graft-derived cells, while later remodeling is associated with host-derived cells.     

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.     

Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation

Graft-versus-host disease (GvHD) and graft rejection have remained significant complications of allogeneic stem cell transplantation. Mesenchymal stem cells (MSCs) from the bone marrow have been shown to suppress T-cell activation in vitro and in vivo, and may be used to reduce GvHD in the recipient or to facilitate engraftment across MHC barriers. MSCs can be derived from a variety of tissues. Thus, we asked whether MSCs from different tissues might have differential effects on T-cell responses. We were particularly interested in MSCs derived from adipose tissue because of its abundance and accessibility. We investigated and compared the immunosuppressive potential of murine MSCs derived from muscle tissue, adipose tissue, omentum, and bone. Cells from the different tissues were enriched for MSCs and cultured for 2-3 weeks to deplete hematopoietic cells. Mixed lymphocyte reactions (MLRs) including MSCs were performed using concanavalin A or allogeneic T cells as inducers of T-cell activation. MSCs from all tissues differentiated into multiple lineages. Mitogen-induced T-cell activation, as well as allogeneic T-cell responses, was reduced in MLRs mediated by the addition of MSCs. Reduction of T-cell activation was most pronounced for muscle tissue in the mitogen-induced MLR and fat tissue during the allogeneic MLR. These data demonstrate that MSCs from multiple tissues efficiently reduce T-cell activation. The results suggest that MSCs from adipose tissue can serve as an alternative source for MSCs to bone or bone marrow for the modulation of GvHD after allogeneic stem cell transplantation or to enhance engraftment across MHC barriers.     

冷冻异体手指复合组织移植的临床评价

目的 评估自体拇甲皮瓣游离移植包冷冻异体手指骨－关节－肌腱－脾鞘复合组织再造拇、手术指的临床效果。方法 对再造拇、手指２７０例通过门诊复查、信访、Ｘ线睛、实验室检查、＾９９ｃＴｃＭＤＰ扫描及手术等方式进行随访,并对资料进行统计学处理与临床验证。随访时间为术后５个月～１６年,平均５年。结果 拇皮瓣携囊趾关节二块骨片包绕营养异体手指复合组织能造出外形良好的拇、手指,对掌功能优良率７１．９１％。感觉术后     

Recipient immature dendritic cells do not prolong allograft survival

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.     

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4(+) T cells

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A(u), were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20-100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from nai;ve animals. The proliferative response was primarily mediated by CD4(+) T cells recognizing the RT1.A(a) molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4(+) T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.     

Migration of mesenchymal stem cells to heart allografts during chronic rejection

BACKGROUND: Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and muscle cells. Their involvement in the tissue repair of allografts during the development of chronic rejection has been hypothesized, but not yet substantiated, by experimental evidence. METHODS: Rat MSC were isolated from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker gene. The -labeled cells were then transferred to F344 rats bearing Lewis (LEW) cardiac allografts to measure their localization and contribution to graft tissue repair. RESULTS: The MSC isolated from circulation exhibited multipotential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes, and fibroblasts. Intravenous engraftment of the -labeled cells into recipients of heart transplant resulted in migration of the beta-gal+ cells into the lesions of chronic rejection in the cardiac grafts and homing of the cells to the bone marrow. The majority of beta-gal+ cells present in the allografts exhibited fibroblast phenotypes, and a small number of the cells expressed desmin, indicative of myocyte differentiation. CONCLUSION: MSC vigorously migrated into the site of allograft rejection. This data suggests that they may be attracted to this site to actively participate in tissue repair during chronic rejection. In addition, given the robust migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.     

Heterotopic sternum transplant in rats: A new model of a vascularized bone marrow transplantation

We introduced the heterotopic vascularized sternum transplant as a more simple and pure alternative to allogeneic hind limb transplantation for the study of bone marrow transplantation. We report the clinical and histopathological manifestations after transplantation of syngeneic and allogeneic sternal grafts with and without immunosuppression with FK-506. Syngeneic grafts maintained normal histology, whereas allografts showed rejection, which was prevented by FK-506. FK-506-treated allografts developed chimerism that was present throughout the observation period. Transplantation of the sternum may be a valuable model to study vascularized bone marrow transplantation and its effects on repopulation of bone marrow of the host, chimerism, and tolerance.     

同种异体复合组织移植Banff移植病理学诊断标准及进展

同种异体复合组织移植（CTA）是一门新兴的用于治疗功能性组织或肢体缺损的移植学科,由于绝大多数CTA移植物为带血管的移植物,因此也称为带血管的复合组织移植（VCA）。CTA/VCA的移植物包含同种异体的皮肤、皮下组织、骨骼、肌肉、神经和血管等两种或多种组织结构成分。由于大多数CTA/VCA移植物中带有皮肤组织,而皮肤组织是抗原性最强的免疫成分,移植术后的急性排斥反应是导致CTA/VCA移植失败及其移植物失功的首要障碍, 因此CTA/VCA移植物中的皮肤排斥反应的组织病理学特征成为首要的关注点。本文就CTA/VCA的排斥反应等病理学特征、2007年Banff诊断标准及其研究进展予以综述,以期为CTA/VCA的排斥反应及其他并发症的诊断及治疗提供参考。     

Immunological transformations in the recipient of grafted allogeneic human bone

Twenty-one patients received allogeneic human bone grafts following deep freezing according to various orthopaedic indications. The HLA antigens of all donors and recipients had been determined preoperatively, and grafting was performed without any respect to the HLA match. The immunological follow-up of the recipients was managed by two different methods: MLC (mixed lymphocyte culture) and MAILA (monoclonal antibody-specific immobilisation of lymphocyte antigens). No immunosuppression was performed. The follow-up lasted up to 6 years. Allogeneic grafting of human cancellous bone induces specific immunological reactions in the recipient. The consequences of these observations are: (1) allogeneic bone grafting may induce second-set reactions following subsequent blood transfusion, tissue grafting or organ transplantation; (2) transplantation of fresh, perfused, vascularised allogeneic bone or joint may become a therapeutic approach in the near future. Then the employment of standard immunosuppressive protocols will be mandatory in order to fight acute rejection of the graft.The project Human Bone Transplantation is supported by the Karl -Wilder-Stiftung Munich, a grant of the German Life Insurances.     

UVB pretreatment of rat bone marrow allografts. Prevention of GVHD and induction of allochimerism and donor-specific unresponsiveness

Ultraviolet B irradiation has been used to pretreat blood and islets to prevent subsequent graft rejection. In this study the optimal dose of UVB irradiation of bone marrow was determined in syngeneic recipients and was subsequently applied to in-vitro treatment of bone marrow allografts. UVB pretreatment of donor bone marrow inoculum led to complete prevention of GVHD in allogeneic rat recipients without major marrow or other toxicity. Long-standing recipients of allogeneic UVB-BM became stable adult chimeras. The recipients of allogeneic BM were populated by donor-type peripheral blood lymphocytes and did not reject host or donor-type heart grafts. The BM allograft recipients were immunocompetent as measured by their ability to normally reject third-party cardiac allografts. We suggest that the prevention of GVHD and induction of stable chimerism in adult recipients of allogeneic UVB-BM may be mediated by suppressor mechanisms.     

Host-derived neoangiogenesis with short-term immunosuppression allows incorporation and remodeling of vascularized diaphyseal allogeneic rabbit femur transplants

The purpose of this study was to demonstrate that living bone allotransplants can incorporate, remodel, and maintain mechanical properties without long-term immunosuppression in a fashion comparable to living autotransplants. For this, viability is maintained by repair of nutrient vessels and neovascularization from implanted host-derived vasculature. Microsurgically revascularized femoral diaphysis allotransplants were transferred from young male New-Zealand-White (NZW) into 4 groups of male Dutch-Belted (DB) rabbits. Short-term immunosuppression by tacrolimus (IS, groups 4 and 5) and host-derived neovascularization (NV) from implanted fascial flaps was used to maintain viability (groups 3 and 5) as independent variables. Group 2 received neither IS nor NV. Vascularized pedicled autotransplants were orthotopically transplanted in group 1. After 16 weeks, transplants were evaluated using radiologic, histologic, biomechanical, and histomorphometric parameters. Vascularized bone allotransplants treated with both short-term IS and host-derived NV (group 5) healed in a fashion similar to pedicled autotransplants (group 1). Their radiographic scores were higher than other groups. Groups with patent fascial flaps (3 and 5) showed significantly greater neoangiogenesis than ligated controls (2 and 4). Tacrolimus administration did not affect neoangiogenesis. Elastic modulus and ultimate stress were significantly greater in autogenous bone than in allotransplanted femora. Biomechanical properties were not significantly different among allotransplants. Bone turnover was decreased with IS, but increased with NV by the implanted fascial flaps. Living allogeneic femoral allotransplants treated with short-term IS and host-derived neoangiogenesis can lead to stable transplant incorporation in this rabbit model. The combination of both factors optimizes bone healing. Transplant mineralization is improved with neoangiogenesis but diminished with IS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 763–770, 2009     

Bone grafts in T-cell deficient rats

Revascularization, new bone formation, and resorption of fresh syngeneic and allogeneic cancellous bone that were transplanted to an intramuscular pouch have been studied in athymic and normal rats. Revascularization was evaluated with radioactive microspheres; formation of new bone was assessed with 85Sr incorporation; and resorption was measured by the graft weight reduction. Animals were killed 2, 6, or 12 weeks after transplantation. The circulation and bone formation in allogeneic grafts were greatly impaired in normal rats as compared with the athymic group and the syngeneic grafts. The allografts in normal rats had a smaller weight reduction than the allografts in athymic rats, suggesting impaired resorption. We conclude that the T-lymphocyte system is at least partly responsible for the difference between syngeneic and allogeneic bone grafts, and that the thymus-dependent primary rejection mechanism probably is important for the vitality of allogeneic bone grafts.     

NK Cells Rapidly Reject Allogeneic Bone Marrow in the Spleen Through a Perforin- and Ly49D-Dependent, but NKG2D-Independent Mechanism

We have used a sensitive and specific in vivo killing assay to monitor the kinetics, anatomic location and mechanisms controlling NK-mediated rejection of Balb/c bone marrow by C57BL/6 natural killer (NK) cells. We find that NK killing of fully allogeneic bone marrow is a rapid, highly efficient process, leading to substantial rejection of transplanted marrow within 6 h of transplant and elimination of 85% of the transplanted cells within 2 days. NK-mediated rejection occurred predominantly in the spleen, with sparing of rejection in the bone marrow and lymph nodes. Rejection was dependent on Perforin gene function, but was independent of interferon-gamma. Finally, rejection of Balb/c bone marrow by B6 NK cells required signaling through the Ly49D receptor, but occurred despite blockade of NKG2D, which distinguishes these results from previous studies using semiallogeneic transplant pairs. These results identify NK cells as highly active mediators of bone marrow rejection, and suggest that inhibiting NK function early during transplantation may increase the efficiency of engraftment and allow successful engraftment of limiting doses of donor bone marrow.     

Bone grafts in T-cell deficient rats

Revascularization, new bone formation, and resorption of fresh syngeneic and allogeneic cancellous bone that were transplanted to an intramuscular pouch have been studied in athymic and normal rats. Revascularization was evaluated with radioactive microspheres; formation of new bone was assessed with ⁸⁵Sr incorporation; and resorption was measured by the graft weight reduction. Animals were killed 2, 6, or 12 weeks after transplantation. the circulation and bone formation in allogeneic grafts were greatly impaired in normal rats as compared with the athymic group and the syngeneic grafts. the allografts in normal rats had a smaller weight reduction than the allografts in athymic rats, suggesting impaired resorption. We conclude that the T-lymphocyte system is at least partly responsible for the difference between syngeneic and allogeneic bone grafts, and that the thymus-dependent primary rejection mechanism probably is important for the vitality of allogeneic bone grafts.     

异体胎骨移植治疗小儿四肢肿瘤性骨缺损

胎儿骨移植是近几年国内采用的一种新的植骨方法，我院采用胎儿骨移植治疗小儿四肢肿瘤性骨缺损４２例，取得了较好的效果，治疗结果表明胎儿骨免疫排斥反应弱，骨诱导能力强，来源丰富，取材容易，能有效的弥补小儿肿瘤性骨缺损范围大，需要大量植骨，而自体骨可供移植用的骨量较少，取材困难的缺陷。     

Prevention of rejection of allogeneic bone marrow transplants by NK 1.1 antiserum

We have studied the effect of in vivo administration of NK 1.1 antiserum on two functions of natural killer (NK) cells: (A) in vitro cytotoxicity of B6 mice to T cell lymphoma, YAC-1, and (B) potential of B6 mice to reject allogeneic BALB/c bone marrow transplants. We demonstrated that a single i.v. injection of 0.4 ml of NK 1.1 antiserum significantly reduced in vitro NK cell cytotoxic potential and concomitantly prevented rejection of allogeneic bone marrow transplants. NK 1.1 antiserum was effective in diminishing both of these functions when injected 2, 6, or 24 hr before transplantation and NK cell assay, but it was ineffective when given 48 hr before transplantation or the NK cell test. As a specificity control, we investigated the effect of specific anti-T-cell-directed monoclonal antibodies, Thy 1.2 and Lyt 2.2, in the same systems. Neither of these antibodies exerted any effect on NK cell cytotoxicity to YAC-1 or rejection of allogeneic bone marrow transplants. These studies indicate that NK cells represent one of the major components of the mechanism of bone marrow graft rejection.     

Rapamycin-conditioned, alloantigen-pulsed myeloid dendritic cells present donor MHC class I/peptide via the semi-direct pathway and inhibit survival of antigen-specific CD8(+) T cells in vitro and in vivo

Dendritic cells (DC) are “professional” bone marrow-derived antigen (Ag)-presenting cells of interest both as therapeutic targets and potential cellular vaccines due to their ability to regulate innate and adaptive immunity. Harnessing the inherent tolerogenicity of DC is a promising and incompletely explored approach to the prevention of allograft rejection. Previously, we and others have reported the ability of pharmacologically-modified DC, that resist maturation, to inhibit CD4⁺ T cell responses and prolong allograft survival. Here we evaluated the ability of murine myeloid DC conditioned with the immunosuppressive pro-drug rapamycin (RAPA) to acquire and directly present alloAg to syngeneic CD8⁺ T cells. RAPA-conditioned DC (RAPA-DC) pulsed with allogeneic splenocyte lysate acquired and expressed donor MHC class I and enhanced the apoptotic death of directly-reactive donor Ag-specific CD8⁺ T cells in vitro. Moreover, following their adoptive transfer, they reduced the survival of these T cells in vivo. The ability of RAPA-DC to inhibit the survival of alloAg-specific CD8⁺ T cells provides a potential mechanism by which host-derived DC may act as negative regulators of T cell alloreactivity and support donor-specific unresponsiveness. Adoptive cell therapy with alloAg-pulsed RAPA-DC may offer an effective approach to suppression of alloimmunity, with reduced dependence on systemic immunosuppression.     

Allogeneic vascularized transplantation in cases of bone and joint defects

This paper presents preliminary results of allogeneic vascularized transplantations of three femoral diaphyses and four total human knee joints. Grafts were harvested from multi-organ-donors and immediately transplanted. Osteosyntheses were performed employing intramedullary nails. Vascular pedicles of the grafts were anastomosed in end-to-side technique. Immunosuppression mainly based on Cyclosporine and Azathioprine. Grafts' perfusion was demonstrated by DSA and Duplex-sonogramms, bone metabolism by SPECT-scintigraphy. Five months following transplantation osteotomies demonstrated consolidation in conventional X-rays. Biopsies of the grafted bone revealed intact osteocytes and arthroscopy demonstrated intact synovial, chondral and ligamentous structures. From the technical aspect vascularized transplantation of the femoral diaphyses and total knee joints is feasible. The main problems are of immunologic nature. Transplantations were performed respecting the ABO-compatibility but with a large HLA-mismatch. Acute and chronic rejection crises may damage the grafts. At least in synovial joints livelong immunosuppression of the recipients seems to be unavoidable.