Peripheral donor leukocytes prolong survival of rat renal allografts.

BACKGROUND: The development of strategies to enhance the survival of transplanted organs and to potentially lower or even discontinue immunosuppressive therapy would represent a significant advancement in post-transplant patient care. METHODS: We studied the effect of pretransplant infusion of donor leukocytes alone or in combination with a short course of cyclosporine on the long-term outcome of a rat model of kidney allograft. RESULTS: A single intravenous infusion of donor peripheral blood leukocytes (100x10(6) cells) from Brown-Norway (BN) rats into major histocompatibility complex (MHC) incompatible Lewis recipients largely failed to prolong kidney allograft viability from the same donor transplanted 60, 40, or 30 days after cell infusion. A short course of cyclosporine (per se, unable to prolong graft survival) was started at the same day of donor leukocyte infusion, but instead was able to prolong the survival of the BN kidney transplant-performed 40 days later-but not of a Wistar Furth (WF) third party, with some animals even developing tolerance. A mixed lymphocyte reaction of host cells from long-term surviving rats to BN stimulator cells was significantly reduced as compared with controls. Donor BN DNA was detected in the peripheral blood of Lewis rats until day 40 after BN leukocyte infusion. Microchimerism persisted (60 to 70 days post-transplant) in most long-term graft recipients. Reducing the time interval between donor leukocyte infusion and subsequent kidney transplant to 10 days still prolonged graft survival. Donor peripheral blood mononuclear cells, but not polymorphonuclear cells, in the leukocyte preparation contributed to prolong kidney allograft survival. CONCLUSIONS: Pretransplant donor leukocyte infusion under the appropriate conditions can tip the immune balance toward improved graft acceptance. This result could be relevant to the achievement of donor-specific tolerance of the graft with the maintenance of an intact response to third-party antigens.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.     

The effects of different schedules of total-body irradiation in heterotopic vascularized bone transplantation. An experimental study in the Lewis rat

To evaluate the effects of irradiation on heterotopically placed vascularized knee isografts, a single dose of 10 Gy of total-body irradiation was given to Lewis donor rats. Irradiation was delivered either 2 or 6 days prior to harvesting or subsequent transplantation, and evaluated at 1, 2, and 4 weeks after grafting. Irradiation caused endothelial depopulation of the graft artery, although vascular pedicle patency was maintained throughout the study. Bone graft viability and mineralization were normal. Dramatic changes in the bone marrow were seen that included an increase of its fat content (P less than 0.001), and a concomitant decrease in bone marrow-derived immunocompetent cells. These changes were more prominent in recipients of grafts from day -6 irradiated donor rats. Total-body irradiation did not prejudice the use of vascularized bone grafts, and exhibited an associated immunosuppresant effect over the vascular endothelium and bone marrow. This may be a further rational conditioning procedure to avoid recipient manipulation in vascularized bone allotransplantation.     

Experimental study on vascularized bone allografts for reconstruction of massive bone defects

To study the healing mechanism of vascularized bone allografts under short-term as well as long-term immunosuppression with cyclosporin A, experimental vascularized intercalary bone allograft transplantation was carried out between inbred rats using the tibiofibula graft model. Bone scintigram and radiographs were used as an indicator for early detection of rejection after transplantation and bone union. In vascularized bone allografts under long-term immunosuppression with cyclosporin A, early bone union and continuous incorporation were similar to that observed in vascularized bone autograft transplantation. When administration of cyclosporin A was discontinued before completion of bone union, the graft was rejected and bone union was delayed. Apparent swelling on the operated limb associated with a decrease in bone scintigram uptake suggested the occurrence of rejection of the allograft. Vascularized bone allograft transplantation is useful for reconstruction of massive bone defects only if immunosuppressants are used and maintained at least until bone union is obtained. © 1994 Wiley-Liss, Inc.     

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.     

Vascularized bone allografts: an experimental study in dogs

This study attempted to re-establish vascularization to canine allograft bone by means of the introduction of a pedicle in the medullary canal with a capillary network. The experiment showed basically that the introduction of arteries and veins with a capillary network into the medullary canal of an allograft femur provided enough vascular supply to produce fibrous tissue and enough bone to cover preoperatively drilled holes, suggesting that the allograft "revives" with the introduction of a vascular supply.     

Prolonged survival of experimental extremity allografts: A new protocol with total body irradiation,granulocyte‐colony stimulation factor,and FK506

The induction of a high‐level of chimerism (macrochimerism) may be the most reliable strategy for achieving donor‐specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using total‐body irradiation (TBI) and granulocyte‐colony stimulation factor (G‐CSF) to induce high‐level chimerism following rat whole‐limb allotransplantation. In this study, we investigated whether the timing of TBI influenced the period of graft survival. In total, 50 whole‐limb allotransplants from LacZ transgenic rats to LEW rats were performed. TBI was performed at days 0 and 14, and G‐CSF was given for 4 days after TBI. FK506 was given for 28 days after transplant. Nontreated limb allografts were rejected after 4.2 days. The survival time was prolonged to 64 days in the FK506 monotherapy group. In the group receiving TBI at day 14, limb allograft survival was significantly prolonged to 81 days. In the group receiving TBI at day 0, 26% of recipients died but in the surviving recipients the grafts survived for longer than 1 year without lethal graft‐versus‐host disease (GVHD). Polymerase chain reaction (PCR) analysis revealed a high level of intrabone marrow chimerism in the recipient, thus demonstrating successful induction of macrochimerism. A new protocol of pretransplant TBI followed by treatment with G‐CSF and FK506 was found to induce a high level of chimerism and to significantly prolong the survival of limb allografts in recipients without lethal GVHD. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:457–461, 2010     

Effects of vascularized bone graft on surrounding necrotic bone: an experimental study

Several difficult problems arise in the treatment of necrotic bone disorders such as femoral head necrosis or Kienb?ck's disease for which no definitive treatment as yet exists. The recent literature has recommended vascularized bone transfers as suitable for these disorders, but there have been few experimental studies done and resulting effects are still currently obscure. Using a rabbit model, the effects of vascularized bone graft on the surrounding necrotic bone were analyzed. Results suggest that the periosteal muscle cuff remains as fibrous or fatty tissue and interferes with the bony union between the graft and the host bone; the graft was found to be gradually resorbed. The main function of the procedure appears to be the provision of vessels into avascular bone.     

SEB combined with IL-1ra could prolong the survival of the rat allografts in high-risk corneal transplantation

PURPOSE: To determine whether the superantigen Staphylococcal enterotoxin B (SEB) combined with interleukin-1 receptor antagonist (IL-1ra) prolong allograft survival better than individual agents in high-risk corneal transplantation in a rat model. METHODS: Fisher 344 donor corneas were transplanted into Lewis recipients. High-risk transplantation meant that the transplants were sutured into the recipient beds with corneal neovascularization induced by placing three interrupted sutures in the host cornea. All of the recipients were divided in blinded fashion into four groups. Group I was injected with saline buffer. Group II was injected intraperitoneally with 0.2 mL SEB (75 microg/kg) at 4-day intervals on three occasions before transplantation. Group III was injected with 0.1 mL IL-1ra (1 mg/mL) subconjunctivally from the first day after transplantation for 2 weeks. Group IV received both SEB and IL-1ra. All transplants were evaluated for signs of rejection for 4 weeks after surgery. Ten days after transplantation, two recipients in each group were sacrificed for histopathological and immunological evaluation. RESULTS: The mean survival time of the allografts in the control group was 5.89 +/- 0.79 days; in SEB group, 10.70 +/- 2.52 days; in IL-1ra group, 8.25 +/- 0.71 days; in the SEB and IL-1ra group, 17.36 +/- 2.39 days. CD4+ and CD8+ lymphocyte infiltration into the allografts and the percentage of the lymphocytes in the spleen and mandibular lymphatic nodes was significantly decreased among the treated groups with dampened lymphocyte reactivity. The SEB plus IL-1ra combination group showed the strongest inhibition. CONCLUSION: SEB and IL-1ra are most effective in combination to treat high-risk corneal transplants.     

Prolonged survival of rat hepatic allografts after total-body irradiation of the donors.

The effect of total-body irradiation of the donor on hepatic allograft survival was studied in the rat, with ACI(RT1a) as the donor and LEW(RT1(1)) as the recipient. LEW recipients of ACI liver transplants experienced severe acute rejection, with a mean survival of only 10.2 +/- 0.3 days. The doses of irradiation were 450, 750, and 1000 rads administered 24 hr prior to harvesting or subsequent transplantation. TBI with a dose of 750 rads significantly prolonged the survival of the hepatic allograft to 30.3 +/- 1.7 days, without concomitant immunosuppression. However, neither 450 rads nor 1000 rads of TBI resulted in successful suppression of graft rejection. TBI appeared to have a beneficial effect on hepatic allograft survival and to have no deleterious effect on isograft survival, suggesting a possible modulation of the immunogenicity of the donor organ. Although the cause of this beneficial effect is not clear, TBI with a dose of 750 rads 24 hr prior to organ harvest seems to be optimal to eliminate-antigen presenting cells in the donor organs.     

High-level expression of viral interleukin-10 in cardiac allografts fails to prolong graft survival

BACKGROUND: Viral interleukin (vIL)-10, encoded in the Epstein-Barr virus genome, shares many of the anti-inflammatory properties of cellular IL-10 but is supposed to lack IL-10's immunostimulatory properties. Thus, vIL-10 is expected to offer superior immunosuppression. METHODS: We established transgenic mice (vIL-10 Tg) that express vIL-10 systemically and transplanted their hearts as vascularized allografts into unmodified major histocompatibility complex (MHC) full-mismatch or MHC class II-disparate mice. RESULTS: The vIL-10 Tg mice revealed high-level expression of vIL-10 in major organs including the heart. However, the heart grafts from the vIL-10 Tg mice failed to exhibit prolonged survival in combination with either the MHC full-mismatch or the class II-disparate mice. In the MHC class II-disparate mice, the vIL-10 Tg heart grafts showed severe CD8 T-cell infiltration and increased interferon (IFN)-gamma mRNA expression compared with non-Tg grafts. CONCLUSION: High level expression of vIL-10 in grafts can exacerbate immunological rejection in an allogenic transplantation model.     

Organ donor or graft pretreatment to prolong allograft survival: lessons learned in the murine model

Permanent donor-specific tolerance to allografts is the goal of transplantation research. Currently, morbid immunosuppressive therapy is used to mitigate rejection initiated in part by Ia-bearing interstitial graft dendritic or antigen-presenting cells (APCs) that are thought to migrate into the host after transplantation. We hypothesized that donor or organ immune modulation directed against graft APCs might influence graft immunogenicity and promote prolonged graft acceptance in histoincompatible hosts in the absence of immunosuppressive therapy. Haplotype-specific monoclonal antibodies (mAb), mAb specific to graft APC, adhesion or costimulatory molecules and anti-LFA-1-Ricin and anti-Iak-Ricin immunoconjugates (IC) were prepared and administered in varying doses and time intervals to donor C3H/HeJ (H-2k) mice. Thereafter, their spleens and hearts were removed at varying time intervals and used either as stimulator cells in one-way mixed lymphocyte reaction or transplanted into naive Balb/c (H-2d) recipients, respectively. Explanted C3H hearts were pretreated with anti-Iak mAb on the Langendorf apparatus. Hearts were also used from major histocompatibility complex (MHC) class I, MHC class II, and MHC class I- and II-deficient "knockout" mice. Splenocytes exposed to at least 500 microg of anti-Iak mAb in vivo for more than 4 hr were able to inhibit the mixed lymphocyte reaction to almost background levels, but only after incubation with rabbit complement in vitro. Similarly pretreated cardiac allografts (both in vivo or explanted and pretreated on the Langendorf apparatus) did not experience prolonged survival in nonimmunosuppressed Balb/C recipients when compared with control solutions, regardless of the concomitant use of complement. Splenocytes from immunoconjugate pretreated donors inhibited the mixed lymphocyte reaction completely without the use of complement; however, hearts from these donors also did not experience prolonged survival nor donor hearts exposed to mAb specific for graft APC, adhesion or costimulatory molecules. Only hearts from MHC class I and class II "knockout" mice survived significantly longer than controls. We conclude that donor or graft pretreatment with haplotype-specific anti-Ia mAb, haplotype-specific immunoconjugates, or mAb directed against graft APC, adhesion or costimulation molecules have little efficacy in promoting acceptance of cardiac allografts in nonimmunosuppressed recipients. The enhanced survival of hearts from MHC class I- and class II-deficient donors suggest that novel methods to effect the immunogenicity of the graft will be required if long-term allograft acceptance is to be achieved in the absence of host immunosuppression.     

Histological characteristics of acute rejection in vascularized allografts of bone

Using a genetically defined rat model for the heterotopic transplantation of a vascularized knee in the rat, histological and histochemical studies of acute rejection in vascularized allografts of bone were carried out. The graft consisted of the knee joint with the distal end of the femur, the proximal part of the tibia, the cartilaginous growth plates, the articular cartilage, and a minimum cuff of muscle, which was transferred to a location under the abdominal skin. A total of 160 transplants, including vascularized and non-vascularized isografts, vascularized and non-vascularized allografts that were transplanted across a strong histocompatibility barrier, and vascularized allografts of bone that were transplanted across a weak histocompatibility barrier, were studied by light microscopy at intervals for as long as twelve weeks after transplantation. Vascularized allografts of bone that were transplanted across a strong histocompatibility barrier showed evidence of rapid rejection, similar to that after transplantation of allografts of visceral organs. This was manifested at one week by necrosis of osteocytes, cessation of microcirculatory flow, massive extravasation of red cells, and deposition of fibrin in the marrow. The large vessels demonstrated changes that were characteristic of vascular rejection. Allografts that were transplanted across a weak histocompatibility barrier showed a more gradual, less intense process of rejection that allowed observation of the evolution of the process. In these grafts, the osteoblasts and marrow in the primary spongiosa of the metaphysis were early targets of rejection, as indicated by necrosis of osteoblasts, extravasation of red blood cells, and deposition of fibrin in the marrow spaces. Loss of osteoblasts from the surfaces of osteoid as well as from bone on spicules of calcified cartilage resulted in the cessation of new-bone formation. Calcification of the longitudinal septa between the lowermost hypertrophic chondrocytes was decreased. However, the proliferation and maturation of chondrocytes in the zone of proliferating chondrocytes and in the upper hypertrophic zone continued and resulted in the formation of a thickened growth plate. The loss of osteocytes in other areas of the graft occurred later and only in the areas where the microcirculation had been lost. These data suggest that ischemic damage, which is probably secondary to an immune-related vascular compromise, is a significant factor in the failure of grafts. In the grafts that were transplanted across a weak histocompatibility barrier, the growth of new bone and revascularization by the host occurred by twelve weeks.