Abstract 18

Leukemia, 1997

The role of presensitizing murine recipients with donor spleen matched at multiple minor histocompatibility loci. These cells prior to T cell-depleted or-repleted H-2 compatible allodonor-host combinations differ with respect to the Gpi-1 geneic bone marrow transplantation (BMT) was investigated at phenotype which allows determination of the level of donor two different doses of total body irradiation (TBI). Recipients blood chimerism as described previously. 6 Recipient mice that were presensitized with 2 ؋ 10 7 irradiated donor spleen were irradiated using a 60 Co source in a Gammatron-3 Unit cells at 1 week before a sublethal dose of 6 Gy TBI and BMT showed no evidence of donor blood chimerism while unsensit-(Siemens, Germany) at a high-dose rate between 40 and ized recipients showed about 80% donor engraftment as 50 cGy/min. Presensitization was performed by intravenous determined by blood Gpi phenotyping. After raising the TBI injection of 2 × 10 7 lethally irradiated (20 Gy) donor spleen dose to 9.5 Gy an increase in mortality from marrow failure was cells 1 week prior to irradiation and BMT. observed in presensitized animals. No significant engraftment-Bone marrow was depleted of T cells by monoclonal antipromoting effect of up to 2 ؋ 10 6 T cells (20% of total marrow body (anti-Thy1.2) and complement treatment according to a dose) was seen either in presensitized or unsensitized mice. It can be concluded that presensitized recipients are more sus-method previously described. 7 This procedure reduced T cell ceptible to acute marrow rejection and that T cells added to the frequencies from 1.45 ± 0.27% (s.e.m.) in normal marrow to bone marrow did not influence the level of donor engraftment 0.18 ± 0.05% as determined using FITC-conjugated anti-Thy1 in these recipients.

Biology of Blood and Marrow Transplantation, 2005

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/ resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unitgranulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2 b ) mice were lethally irradiated and transplanted with various doses of BALB/c (H2 d ) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.

Cellular & Molecular Immunology

The role of the vascularized bone marrow component as a continuous source of donor-derived hematopoietic stem cells that facilitate tolerance induction of vascularized composite allografts is not completely understood. In this study, vascularized composite tissue allograft transplantation outcomes between recipients receiving either conventional bone marrow transplantation (CBMT) or vascularized bone marrow (VBM) transplantation from Balb/c (H2d) to C57BL/6 (H2b) mice were compared. Either high- or low-dose CBMT (1.5 × 108 or 3 × 107 bone marrow cells, respectively) was applied. In addition, recipients were treated with costimulation blockade (1 mg anti-CD154 and 0.5 mg CTLA4Ig on postoperative days 0 and 2, respectively) and short-term rapamycin (3 mg/kg/day for the first posttransplant week and then every other day for another 3 weeks). Similar to high-dose conventional bone marrow transplantation, 5/6 animals in the vascularized bone marrow group demonstrated long-term allograft ...

Microsurgery, 2007

Composite tissue allografts (CTAs) are currently accepted in the clinic; however, long-term immunosuppression is still needed for allograft survival. The presence of donor-specific chimerism may induce tolerance. Thirty-six vascularized bone marrow transplantation (VBMT) allotransplantation were performed across MHC barrier under short-term protocol of 7-day ab-TCRmAb and Cyclosporin A therapy to determine the efficacy of VBMT alone and VBMT augmented with donor bone marrow transplantation (BMT) in chimerism induction. Flow cytometry analysis revealed that VBMT supported with donor BMT directly into the bone resulted in chimerism augmentation and maintenance compared to VBMT. In vivo and in vitro tolerance testing showed prolonged survival of donor skin graft up to 35 days and moderate reactivity in MLR assay that suggests only tolerance induction. Transplantation of vascularized bone without chronic immunosuppression provides a substantial source of bone marrow cells, leading to the development of stable donor-specific chimerism. V V C 2007 Wiley-Liss, Inc. Microsurgery

Transplantation Proceedings, 2006

We proposed to evaluate differences between recipient's immune response to vascularized skin and combined vascularized skin/bone allografts, under a 7-day ␣␤-TCR plus cyclosporine (CsA) treatment protocol.

Archives of Orthopaedic and Trauma Surgery, 1997

Matching of donors and recipients for tissue antigens is vitally important for successful transplantation of essentially all organs and tissues, the major exception being bone. The importance of tissue-typing for the healing of bone allografts remains, however, a controversial issue as development of both humoral and cell-mediated immunity against the grafted bone has been observed in some experimental systems. In the present study, we compared the healing patterns of frozen antigen-mismatched allografts, frozen antigen-matched allografts (syngeneic grafts), and flesh cortical bone autografts in an experimental rat model. Histomorphometry of the graft-host interface revealed that new bone formation started significantly earlier in autografts than in allografts or syngeneic grafts. By 2 weeks, the level of new bone formation in the syngeneic grafts had reached that in autografts. Antigenmismatched allografts, however, continued to exhibit a retarded formation of new bone throughout the union process. These histomorphometric observations were confirmed by molecular biologic analyses for the mRNA lev-Ies of type I collagen, which increased earlier and reached a higher level in autografts than in allografis. Use of syngeneic grafts resulted in a longer persistence of type I collagen mRNA expression in the healing tissue than in antigen-mismatched allografts. No apparent differences were seen between allografts and autografts in the expression of type III collagen. No cartilage-specific type II collagen mRNA was observed, indicating that antigen-mismatching or preservation by freezing did not alter the basic mechanism of the interface healing process, although it did slow down the beginning of the process. The experiments suggest that a major antigen mismatch between donor and recipient affects the temporal gene expression of extracellular bone matrix and delays new bone formation at the graft-host interface of cortical bone allografts.

Microsurgery, 2007

Composite tissue allotransplantation holds a great potential for providing increased knowledge of anatomy and microsurgical experience for life-enhancing reconstructions. Many transplant cases around the world have made this a clinical reality at the present time. Composite tissue allotransplants contain multiple tissue types, including bone, muscle, vessels, nerves, skin, and immune cells and bear a huge antigenic load. Although immunosuppressive drugs are applied successfully to prevent allograft rejection, their side effects pose a barrier to worldwide use. 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 clinical and experimental models are presented in two major categories: solid organ and composite tissue transplantation.

Journal of Orthopaedic Research, 2005

Although cell traffic from the graft into the recipient and from the recipient into the graft had been noticed in allogeneic organ transplantation, little is known following whole-limb allografting. This study was conducted to define cell migration between donor and recipient. Sixty-seven vascularized hind limb allotransplantations were performed in rat sex-mismatched pairs and the recipient animals were treated with FK506 immunosuppression. The ratio of donor and recipient cells was evaluated by semi-quantitative PCR using the specific primers of the Y-chromosome. Allografted limbs had no rejection episode until the final assessment. The male recipient cells were detected in female limb grafts not at 1 week but at 48 weeks after transplantation. The male donor cells were detected in the humerus and tibia in the female recipient but not in the gastrocnemius muscle and leg skin. Our results demonstrated that recipient-derived cells gradually migrated into the grafted bone, muscle and skin cells with the duration of time. Donor-derived cells migrated into the healthy bones but not into the healthy muscle and skin. Because active regeneration occurs in the grafted limb to compensate graft damage secondary to ischemia and operative intervention, recipient-derived cells may mediate a muscular and dermo-epidermal renewal.

Journal of Reconstructive Microsurgery, 2002

Background: Composite tissue allotransplantation holds promise in reconstructive surgery, but its application is limited by the need for long-term immunosuppression. The objective of this study was to investigate the feasibility of low-dose cyclosporine and vascularized bone allotransplantation in prolonging the survival of vascularized adipose tissue allograft. Methods: In the adipose tissue allograft model, adipose tissue allografts based on superficial epigastric vessels from Lewis-Brown-Norway rats were allotransplanted into Lewis rats. In the adipose tissue and bone marrow allograft model, combined vascularized bone marrow and adipose tissues were allografted from Brown Norway rats into Lewis rats. The graft survival, the onset and progression of rejection, and the effects of cyclosporine at different dosages and treatment durations were recorded. A rejection grading system was created based on gross observation and was correlated with histologic examinations. Results: Even at a low dose of 2 mg/kg/day, cyclosporine continued to provide effective allograft protection. Tolerance was not observed in either model. Adipose tissue survival after discontinuation of cyclosporine was independent of treatment duration. The inclusion of vascularized bone to the adipose tissue allograft provided an additional protective effect. This effect was synergistic with concomitant use of immunosuppressant. Conclusions: Adipose tissue allotransplantation is a potential reconstructive option that requires only minimal use of immunosuppressants. Its survival can be further prolonged with simultaneous bone marrow allotransplantation. (Plast.

Bone Marrow Transplantation, 2000

Conventional allogeneic stem cell transplantation is a valuable approach to therapy for many hematologic malignancies. However, high-dose conditioning regimens designed both to control the malignancy and to prevent graft rejection are associated with a high incidence of acute and long-term side-effects. This has largely precluded the use of allografting for patients older than 55 years or for younger patients with certain preexisting organ damage. In order to manage the sideeffects, transplants have traditionally been delivered in highly specialized hospital wards or intensive care settings. Thus, an important goal is to develop safer allografting procedures that can be extended to older patients or patients with pre-existing organ dysfunction who are currently excluded from consideration for transplant. Recent observations have shown that donor lymphocyte infusions (DLI) can eradicate some malignancies that relapse after conventional allografting. These observations confirmed earlier evidence in favor of a graft-versus-leukemia effect based on the association of graft-versus-host disease (GVHD) with a lower likelihood of relapse of malignancy after allografting. Given the potential efficacy of DLI as the sole modality for eradication of malignancy, new strategies for allografting can incorporate the concept of less intensive conditioning therapy which is given with the sole aim of facilitating allogeneic engraftment. Recent pre-clinical studies in a canine model have shown that conditioning regimens for allografting can be markedly reduced in intensity yet still achieve the goal of engraftment. This review briefly summarizes the initial translational clinical studies, using a minimally myelosuppressive-conditioning regimen based on low dose total body irradiation (TBI) or fludarabine alone or in combination with other drugs followed by a short course of immunosuppression with post-grafting cyclosporine and methotrexate or mycophenolate mofetil. Bone Marrow Transplantation (2000) 25, 345-350.