Experimental study of vascularized tibiofibula graft in inbred rats: a preliminary report

An experimental study of vascularized tibiofibula grafts in inbred rats was performed. Roentgenologic and histologic changes of the grafted bone in the first seven postoperative weeks were especially investigated. After preliminary experiments on the vascular anatomy of the lower limbs of rats, tibiofibular vascularized grafts with femoral artery and vein were utilized in Fischer strain F-344 rats. The rate of bony union in the vascularized graft group was superior to that in the nonvascularized control groups. Fluorochrome-labeling studies of the grafted bone at the mid-diaphysis showed active periosteal new bone formation, following the vascularized graft. In contrast, normal tibial bone growth at the mid-diaphysis was mainly endosteal. However, both vascularized graft and normal bone demonstrated evidence of a "drift phenomenon" in the direction of growth. Since the life cycle of the rat is very short, compared with other laboratory animals, this experimental model may be useful in investigating the postoperative course of vascularized bone grafts with a short follow-up period.     

Immune reactivity after high-dose irradiation

Immune reactivity after total-body irradiation was investigated in rats using skin graft rejection as the indicator system. After sublethal irradiation with 10.5 Gy (approximately 50% lethality/6 weeks) the rejection of major histocompatibility complex allogeneic skin grafts was delayed significantly compared with nonirradiated control animals (28 versus 6.5 days). In contrast, skin grafts were rejected after 7.5 days in sublethally irradiated animals and 7 days in lethally irradiated animals if additional skin donor type alloantigens--namely, irradiated bone marrow cells--were given i.v. either simultaneously or with a delay of not more than 24 hr after the above conditioning regimen. These reactions were alloantigen-specific. They were observed in six different strain combinations with varying donors and recipients. Starting on day 2 after irradiation, i.v. injection of bone marrow gradually lost its effectivity and skin grafts were no longer rejected with uniform rapidity; skin donor marrow given on days 4 or 8 did not accelerate skin graft rejection at all. These data show that for approximately 1-2 days after high-dose total-body irradiation rats are still capable of starting a vigorous immune reaction against i.v.-injected alloantigens. The phenomenon of impaired rejection of skin grafted immediately after high-dose irradiation appears to result from the poor accessibility of skin graft alloantigens during the early postirradiation phase when vascularization of the grafted skin is insufficient.     

Prolonged improvement of total pancreatic allograft function by previous intrathymic bone marrow cell injection in rats

Donor-specific induction of tolerance was previously achieved in the diabetic rat by intrathymic injection of pancreatic islets. It allowed a secondary islet graft in any site without immunosuppression. Since total pancreatic graft in man is metabolically more proficient than islet graft, we attempted tolerance induction for total vascularized pancreas transplantation in diabetic BN recipient rats by an intrathymic bone marrow cell (BMC) injection from Lewis donor rats, associated to an antilymphocyte antibody (ALS) administration. Control groups consisted of isogenic grafts, allogenic grafts without tolerance induction and allogenic grafts with ALS alone. In all grafted groups, mean blood glucose and plasma insulin were normalised within 24 h. Graft rejection (clinically suggested by diabetes recurrence and later confirmed by histology) appeared at 18 +/- 2 postoperative days in the absence of intrathymic BMC injection and at 36 +/- 8 days in the group with BMC injection (p < 0.05). Intrathymic bone marrow graft was successful in delaying rejection in our study.     

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.     

Long-term survival of cardiac xenografts in fully xenogeneic (mouse --> rat) bone marrow chimeras

BACKGROUND: The shortage of human hearts remains a major barrier to the efficacy of heart transplantation for the treatment of end-stage heart disease. One potential solution to the supply problem would be the use of hearts from nonhuman donors (xenografts). We have established a model of mouse to rat xenogeneic bone marrow chimerism, and in this study we have hypothesized that such chimeric rats will accept both donor and recipient specific heart grafts while rejecting third-party mouse and rat grafts. We also investigated humoral responses in naive and chimeric rats with and without donor murine cardiac grafts. METHODS: Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 10(6) donor B10.BR mouse bone marrow cells intravenously. Peripheral blood of surviving rats (n = 18) was typed at 4 weeks and then monthly thereafter. Donor and recipient specific and third-party heterotopic heart transplantations were performed at 6 to 8 weeks after reconstitution with bone marrow. RESULTS: Multilineage bone marrow chimerism was produced in all experimental animals with complete replacement of recipient marrow by donor cells. Murine donor and rat recipient strain hearts transplanted in chimeric rats survived indefinitely. Third-party rat and mouse hearts were rejected, though at a slower rate than bone marrow matched naive controls. High levels of antimouse antibodies were detected in rats with rejected hearts. These antibodies were absent in chimeric animals with long-term surviving heart grafts. CONCLUSIONS: Long-term multilineage bone marrow chimerism can be produced in a mouse --> rat bone marrow transplant model. Long-term survival of donor specific and recipient specific vascularized cardiac grafts can be produced in these chimeric animals. These animals are clinically normal but show signs of subclinical immunosuppression regimen as they reject third-party hearts later than naive animals. Our results suggest that antibodies also play a significant role in concordant xenograft rejection, and induction of bone marrow chimerism can overcome this barrier.     

Inability of donor total body irradiation to prolong survival of vascularized bone allografts: experimental study in the rat

At the present time, the toxic side effects of recipient immunosuppression cannot be justified for human non-vital organ transplantation. Total body irradiation has proven effective in ablating various bone-marrow-derived and endothelial immunocompetent cellular populations, which are responsible for immune rejection against donor tissues. Irradiation at a dose of 10 Gy was given to donor rats six days prior to heterotopic transplantation of vascularized bone allografts to host animals. Another group of recipient rats also received a short-term (sixth to fourteenth day after grafting), low dose of cyclosporine. Total body irradiation was able merely to delay rejection of grafts across a strong histocompatibility barrier for one to two weeks, when compared to nonirradiated allografts. The combination of donor irradiation plus cyclosporine did not delay the immune response, and the rejection score was similar to that observed for control allografts. Consequently, allograft viability was quickly impaired, leading to irreversible bone damage. This study suggest that 10 Gy of donor total body irradiation delivered six days prior to grafting cannot circumvent the immune rejection in a vascularized allograft of bone across a strong histocompatibility barrier.     

Experimental vascularized bone transplantations

The postoperative hypertropy of the vascularized fibula graft is of particular interest. In order to clarify the etiology of the hypertrophy, we conducted experimental projects on vascularized bone grafts using fluorochrome bone labelings, his-tomorphometry and measurement of blood flow in rats and rabbits. In the murine vascularized tibio-fibula graft for bone defects of the tibia, where mechanical stress to the graft can be expected, cross-sectional bone growth was maintained with acceleration of new bone formation and an alteration of growth direction at 3 weeks after the transplantation. In vascularized bone grafts where no mechanical stress to the graft is expected, more bone resorption was seen than bone formation. In the murine tail bone graft to a bony defect of the femur, the cancellous bone gradually disappeared, and the trabeculae took on an architecture characteristic of the femur. This process slowly transforms the graft into a femur with the morphologic features of a long bone. These results suggested that remodeling of the graft corresponding with the changes in the dynamic environment was accompanied by adequate resorption. The cross-age transplantation of vascularized tibio-fibula grafts in rats showed that the younger the donor, the faster the hypertrophy. Age is one of the important factors affecting remodeling of the vascularized bone graft. The blood flow of the grafted bone in the rabbits increased immediately after the transplantation as a reaction to surgery. Thereafter the blood flow of the graft may depend on the biological demands for new bone formation in adapting to the biomechanical environment of the recipient site. © 1995 Wiley-Liss, Inc.     

Experimental study of vascularized bone grafts: hypertrophy of the grafted bone

The mechanism underlying hypertrophy of experimentally vascularized bone grafts was studied in 15-week-old rats. The segmental ulna was grafted to the tibial defect with an external fixator. In experiment 1, 24 rats were classified into four groups to evaluate conventional (non-vascularized), cuff (periosteum-encased, non-vascularized), and vascularized bone grafts, and vascularized segmental grafts with fracture. In experiment 2, 12 rats were classified into two groups according to the presence of mechanical loading. This involved vascularized bone grafts with external fixators, and vascularized bone grafts with external fixators removed after bone union. The bone dynamics of the grafts were investigated by several methods, including roentgenographic analysis, histologic studies, and fluorochrome labeling. In experiment 1, a slight bone formation was recognized in the conventional bone graft, while irregular bone formation with creeping substitution was observed in the cuff graft. The vascularized bone graft showed significant hypertrophy; hypertrophy of the vascularized bone with fracture was greater than that without fracture. In experiment 2, markedly circumferential bone formation was observed after removal of the external fixator, while slight new bone formation was observed during the late postoperative period in bone with an external fixator. These results suggest that hypertrophy can be promoted by artificial fracture of the grafted bone, and that mechanical loading is an important factor for remodeling of grafted bone.     

Successful allogeneic leg transplantation in rats in conjunction with intra-bone marrow injection of donor bone marrow cells

BACKGROUND: We have recently established a new method for bone marrow transplantation (BMT) in mice: bone marrow cells are directly injected into the intra-bone marrow (IBM) cavity. IBM-BMT induces persistent donor-specific tolerance and enhances the rapid recovery or reconstitution of the hematolymphoid system of donor origin without any signs of graft-versus-host disease (GVHD) or graft failure. Furthermore, the prior injection of fludarabine can reduce the irradiation dose to the sublethal level (4.5 Gy x 2). Therefore, we hypothesize that IBM-BMT plus fludarabine is applicable to allogeneic leg transplantation in rats. METHODS: Brown Norway (BN; RT1An) rats were injected intravenously with 50 mg/kg of fludarabine phosphate, followed by sublethal fractionated irradiation (4.5 Gy x 2) 1 day before IBM-BMT. The hind limbs from Fischer 344 (F344; RT1Al) rats were transplanted on day 0, and bone marrow cells (3 x 10(7) cells/50 microL) obtained from the donor F344 rats were injected into the bone marrow cavity of the left tibias of the recipient BN rats. RESULTS: The hematolymphoid cells in the recipient BN rats were completely reconstituted by the cells of the donor F344 rats. The limbs transplanted from the donor F344 rats were accepted for >1 year without any clinical signs of rejection (10 of 10). The lymphocytes of the BN rats showed tolerance to both donor-type and recipient-type major histocompatibility complex determinants in mixed lymphocyte reaction, but showed a significant response to the third-party major histocompatibility complex determinants. CONCLUSIONS: Using a combination of the injection of fludarabine, low-dose irradiation, and IBM-BMT, we have succeeded in allogeneic limb transplantation without using any immunosuppressants after the operation. This strategy would be applicable to the transplantation of other vascularized organs in humans.     

Simultaneous protection against allograft rejection and graft-versus-host disease after total lymphoid irradiation: role of natural killer T cells

BACKGROUND: The use of combined organ and bone marrow transplantation has been studied extensively in rodent models to induce immune tolerance to organ grafts. However, bone marrow transplants with mature donor T cells can induce graft-versus-host disease even in human leukocyte antigen-matched humans. We determined whether total lymphoid irradiation can simultaneously protect against graft-versus-host disease while facilitating tolerance. METHODS: To more closely model clinical studies, we added mature donor T cells to bone marrow grafts combined with heart grafts, and compared murine graft and host survival after conditioning with nonmyeloablative total body or total lymphoid irradiation and depletive anti-T-cell antibodies. RESULTS: Conditioning with total lymphoid irradiation protected hosts against both graft-versus-host disease and organ graft rejection. Although nonmyeloblative total body irradiation prevented organ graft rejection, all hosts succumbed to lethal graft-versus host disease. Induction of tolerance with total lymphoid irradiation and anti-T-cell antibodies was dependent on the presence of regulatory host natural killer T cells, and expression of CD1d on donor marrow but not heart graft cells. CONCLUSION: Conditioning with total lymphoid irradiation and anti-T-cell antibodies prevented host-versus-donor and donor-versus-host alloimmune responses. Tolerance required host natural killer T-cell recognition of CD1d on donor marrow cells.     

Vascularization, cellular behavior, and union of vascularized bone grafts: experimental study in the rabbit

The rabbit fibula was used as the experimental model in this study of the biological changes in nonvascularized and vascularized bone grafts (N = 72). Radiological study showed that the time required for union was about the same for both types of graft. Histological study with tetracycline as a marker showed that there were cellular changes in these two types of graft: moderate hematopoietic and osteocytic loss associated with an increase in bone porosity in vascularized grafts and virtually total osteocytic renewal and marrow necrosis in nonvascularized bone grafts. Quantitative study of vascularization by flow measurement using labeled microspheres showed a brief initial phase of hypervascularization in vascularized grafts, while revascularization took place early in nonvascularized grafts and was followed by hyperemia from the first to the third month. Calcium uptake curves for the two types of graft were quite closely superimposable upon blood flow rate curves. However, the comparative results presented in this study may be tempered by the fact that all grafts were applied to a recipient bed of excellent quality, consisting of well-vascularized muscles.     

Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft

BACKGROUND: An important component of a composite tissue limb allograft (CTA) is the vascularized bone marrow and bone marrow stroma, which when transplanted could create immediate marrow space and engraftment. We have previously demonstrated that tolerance to musculoskeletal allografts can be achieved with a 12-day course of cyclosporine without the presence of long-term peripheral donor cell chimerism. The objective of this study was to determine the fate of the donor bone marrow after transplantation of a limb allograft in a miniature swine model. METHODS: CTAs from donor swine were heterotopically transplanted into six MHC-matched, minor-antigen-mismatched recipients, and a 12-day course of cyclosporine was given. Previous animals transplanted without cyclosporine rejected their grafts in less than 42 days. A non-MHC-linked marker, pig allelic antigen (PAA), was used to distinguish host and donor cells. Three PAA- animals received PAA+ CTAs, and three PAA+ animals received PAA- CTAs. Bone marrow was harvested from the donor limb grafts and the recipient and analyzed by flow cytometry and histology. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine and evaluated for the presence of donor cells by flow cytometry. RESULTS: All animals receiving cyclosporine demonstrated permanent tolerance to their allografts. Donor bone marrow cells were present in all grafts at the time of transplantation and during the immediate postoperative period. By 48 weeks, donor cells were no longer detectable within the marrow space of the allograft. In long-term animals host bone marrow cells replaced donor cells in the graft marrow space. No evidence of donor cell engraftment was found in recipient animals. CONCLUSION: This study demonstrates that in long-term tolerant recipients of musculoskeletal allografts there is no evidence of persistent donor bone marrow cells in the hematopoietic tissues of the graft or the host. Rather, the recipient's bone marrow cells and lymphocytes repopulate the donor marrow space of the graft.     

Tolerance induction following allogeneic vascularized bone marrow transplantation – the possible role of microchimerism

We have noticed that bone marrow transplanted in a vascularized limb graft, providing a continuous supply of donor bone marrow cells (BMC), may prolong the survival time of a skin graft from the same donor. The question arises whether the microchimerism raised plays a role in the prolonged survival of skin allografts. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection process of transplanted skin. Brown Norway (BN) rats served as donors and Lewis rats as recipients of VBMTx and free skin flap allografts. A hind limb was transplanted, followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node, and bone marrow with the monoclonal antibody OX27 directed against MHC class I polymorphic RT1 on BN cells and quantitatively analyzed in a FACStar. In the VBMTx group, the free skin flap survived 70 days after weaning off cyclosporine A (CsA). An intravenous infusion of BMC in suspension equivalent to that grafted in the hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after weaning off CsA but not in recipients of i. v. suspension BMC grafting.     

Tolerance induction following allogeneic vascularized bone marrow transplantation — the possible role of microchimerism

Abstract We have noticed that bone marrow transplanted in a vascularized limb graft, providing a continuous supply of donor bone marrow cells (BMC), may prolong the survival time of a skin graft from the same donor. The question arises whether the microchimerism raised plays a role in the prolonged survival of skin allografts. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection process of transplanted skin. Brown Norway (BN) rats served as donors and Lewis rats as recipients of VBMTx and free skin flap allografts. A hind limb was transplanted, followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node, and bone marrow with the monoclonal antibody OX27 directed against MHC class I polymorphic RT1 on BN cells and quantitatively analyzed in a FACStar. In the VBMTx group, the free skin flap survived 70 days after weaning off cyclosporine A (CsA). An intravenous infusion of BMC in suspension equivalent to that grafted in the hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after weaning off CsA but not in recipients of i. v. suspension BMC grafting.     

Generation of donor hematolymphoid cells after rat-limb composite grafting

BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.     

Pre-formed vascularized bone grafts using polyethelyne chambers.

Previous work has shown that corticocancellous bone chips placed in a titanium chamber with an arteriovenous vascular pedicle will result in a pre-formed vascularized bone graft. The present study was designed to determine whether these grafts can be transferred as an island or free vascularized bone graft, and to examine the material properties of these grafts. Thirty-two male, New Zealand white rabbits were divided into four groups based on the time of sacrifice following the initial chamber implantation. Injected molded cylindrical polyethelyne chambers, measuring 22 mm long and with an 8-mm inner diameter, were used. Corticocancellous bone chips were placed in the chambers and each chamber was implanted in the mid-thigh, with saphenous vessels running through the chamber. The chambers were implanted into the right and left thigh of each animal. To test the hypothesis of the possibility of transferring this graft as an island or free vascularized graft, ligation of the distal vascular pedicle on one side was achieved at re-exploration at 3, 6, 9, and 12 weeks. The contralateral side served as a control in which the vascular pedicle was not ligated. In the controls, bony bridging between the corticocancellous bone chips was observed after 7 weeks. A solid bone graft was present within the chamber by 10 weeks. However, histomorphometric evaluation indicated significant bone resorption. By 13 weeks, resorption progressed to the point where only small islands of bone remained. Ligation of the distal vessels resulted in thrombosis of the vessels within the chamber. Necrosis of newly-formed bone was observed in the area adjacent to the vascular thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A partial conditioning approach to achieve mixed chimerism in the rat: depletion of host natural killer cells significantly reduces the amount of total body irradiation required for engraftment.

BACKGROUND: Mixed allogeneic bone marrow chimerism induces tolerance to solid organ grafts. Although we previously reported that partially ablative conditioning with 700 cGy of total body irradiation (TBI) is sufficient to allow for bone marrow engraftment in mice, we determined that a minimum of 1000 cGy was required in the rat. Because T cells and NK cells are critical in bone marrow graft rejection, our purpose was to examine whether targeting of radioresistant NK cells and/or T cells in the recipient hematopoietic microenvironment would reduce the TBI dose required for engraftment of allogeneic rat bone marrow. METHODS: Wistar Furth rats received either anti-NK3.2.3 monoclonal antibodies on days -3 and -2, anti-lymphocyte serum on day -5, a combination of both or no pretreatment. TBI was performed on day 0 and rats were reconstituted with 100x10(6) T cell-depleted bone marrow cells from ACI donors. RESULTS: Engraftment of T cell-depleted rat bone marrow was readily achieved in animals conditioned with 1000 cGy TBI alone (12/12) and the level of donor chimerism averaged 89%. At 900 cGy TBI alone only one of eight recipients engrafted. In striking contrast, 11 of 12 animals pretreated with anti-NK monoclonal antibodies and irradiated with 900 cGy showed donor chimerism at a mean level of 41%. No further enhancement of bone marrow engraftment could be achieved when recipients were pretreated with antilymphocyte serum alone or antilymphocyte serum plus anti-NK monoclonal antibodies. Mixed allogeneic chimeras exhibited stable multilineage chimerism and donor-specific tolerance to subsequent cardiac allografts. CONCLUSION: Specific targeting of radioresistant host NK cells allows for a significant reduction of the TBI dose required for allogeneic bone marrow engraftment.     

Tolerance induction permits the development of graft-versus-host disease: donor-mediated attack following small bowel transplantation in mixed chimeras.

The induction of tolerance to organ allografts would eliminate acute and chronic rejection as well as the need for nonspecific immunosuppression. We have shown that tolerance induced through the creation of mixed allogeneic bone marrow chimeras allows for the long-term engraftment of cardiac and small bowel allografts across strong multiple major histocompatibility barriers. The possibility that tolerance might render the host susceptible to graft-versus-host disease (GVHD) has not been investigated in this or other models of tolerance. To test this possibility chimeras were created by transplantation of T-cell depleted ACI and Lewis bone marrow into lethally irradiated Lewis rats. Chimerism was determined post bone marrow transplant (BMTx) by flow cytometry of lymphocytes from reconstituted animals. ACI/Lew chimeras (ALC), Lewis/ACI F1 (LACF1), and Lewis (LEW) rats all received heterotopic ACI vascularized small bowel grafts. A second group of chimeras received small bowel grafts from ACI rats pretreated with low dose irradiation to eliminate T-cells from the graft. LEW-->LEW small bowel isografts were also performed. Animals were examined for evidence of GVHD by clinical signs and histologic examination of biopsied tissues. GVHD was quantified using the popliteal lymph node enlargement assay. All LACF1 rats developed severe lethal GVHD following ACI small bowel transplant. Bone marrow chimeras, ALC (n = 6), developed fatal GVHD in a similar fashion after receiving a small bowel transplant. LEW-->LEW isografts and chimeras receiving bowel from irradiated ACI rats survived long term without GVHD while ACI-->LEW allogeneic transplants all underwent acute rejection. GVHD or its absence was confirmed histologically. Popliteal lymph node enlargement indices reflected the presence of GVHD in the chimeras (1.87) and LACF1 (5.4) receiving allografts, but not in isografts or chimeras receiving irradiated allogeneic transplants. Analysis of cytokines in the tongues of rats undergoing GVHD showed elaboration of Th1 type proinflammatory cytokines which was not seen in isografted rats or rats receiving preirradiated small bowel. These results demonstrate that tolerance induction through mixed chimerism results in susceptibility to small bowel induced GVHD. Preirradiating the donor bowel prior to SBTx can prevent GVHD.     

Rapamycin-conditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion

Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-gamma and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.     

Experimental vascularized bone allografting

Presented here is a compendium of studies investigating the fate of vascularized bone allografts. The first set of experiments employ the posterior rib graft in two canine models. The rib-to-mandible model was used to evaluate the rejection phenomena of vascularized bone allografts in an outbred dog model. This ascertained the time course of rejection and histological characteristics of the grafts. Immunosuppression of the graft recipients was attempted with azathioprine and cyclosporine. The results demonstrated that azathioprine was not an effective immunosuppressant, whereas cyclosporine resulted in survival of cortical osteons. The use of the vascularized rib allograft, with and without azathioprine, to bridge the defect in the dog femur was met with failure. Further studies employed a genetically defined rat model to determine the effect of different histocompatibilities on the survival of vascularized knee allografts. Grafts were transplanted from Lewis rats to syngeneic Lewis rats as isografts and to Fischer-344 rats (F-344) and Brown-Norway rats (BN) as allografts. Grafts across a major histocompatibility barrier to BN were rejected by 7 days, whereas grafts across a weak histocompatibility barrier to F-344 were rejected more slowly. The use of cyclosporine in this model abrogated the rejection response when administered to both groups continuously. However, a short course of cyclosporine was effective in preventing rejection in the F-344 animals. Efforts to induce tolerance by blood transfusions, from the donor strain or from a third-party donor, were not effective in preventing rejection.