Role of thymus in operational tolerance induction in limb allograft transplant model

BACKGROUND: In this study, we evaluated the role of host thymus in tolerance induction in composite tissue allografts (CTA) across major histocompatibility complex (MHC) barrier during a 7-day alphabeta- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. MATERIALS AND METHODS: A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1(1)) received vascularized hind-limb allografts from hybrid Lewis x Brown-Norway (F1), (LBN, RT1(1+n)) donors. Mixed lymphocyte reaction (MLR) and skin grafting assessed donor-specific tolerance in vitro and in vivo, respectively. Flow cytometry determined the efficacy of immunosuppressive protocols and the presence of donor-specific chimerism. Immunocytochemistry revealed the presence of donor-specific cells in the lymphoid organs of recipients. RESULTS: Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the alphabeta-TCR/CsA protocol, the MST of allografts was 51 days. For euthymic rats, the MST of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with alphabeta-TCR/CsA resulted in indefinite allografts survival (MST=370 days). MLR and skin grafting confirmed donor-specific tolerance in euthymic recipients. Flow cytometry showed stable chimerism in the euthymic rats and transient chimerism in thymectomized limb recipients. Immunoperoxidase staining revealed the persistence of donor-derived cells in the lymphoid tissues of euthymic recipients. CONCLUSION: We found that the presence of thymus was imperative for the induction of donor-specific tolerance in rat hind-limb composite tissue allografts using a alphabeta-TCR/CsA protocol.     

A new method of bone marrow transplantation leads to extension of skin allograft survival

Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT1(1))] and allogeneic [Lewis x Brown Norway (LBN --> F1, RT1(1+n)) to Lewis] rats under 35-day protocol of alphabeta T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with "crude" bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under alphabeta-TCR mAb protocol. The use of combined protocol of alphabeta-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P < .05). This new simple method of "crude" bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.     

Donor-specific tolerance in fully major histocompatibility major histocompatibility complex-mismatched limb allograft transplants under an anti-alphabeta T-cell receptor monoclonal antibody and cyclosporine A protocol

BACKGROUND: Recent studies have demonstrated that treatment with alphabeta-T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined alphabeta-TCR-CsA protocol. METHODS: The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of alphabeta-TCR and CsA or a combination of alphabeta-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. RESULTS: Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-TCR-CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (AxC Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. CONCLUSIONS: Combined therapy of alphabeta-TCR-CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.     

Extension of composite tissue allograft survival across major histocompatibility barrier under short course of anti-lymphocyte serum and cyclosporine a therapy

In this study, the authors investigated the effects of combined use of cyclosporine A (CsA) and anti-lymphocyte serum (ALS) on the survival of rat hindlimb allografts across a fully allogeneic major histocompatibility complex (MHC) barrier between Brown-Norway rats (BN, RT1 n) and Lewis rats (LEW, RT1 l). Thirty transplantations were performed in five groups of six rats each: Group 1 was the isograft control; Group 2 was the allograft control; Group 3 received ALS, Group 4 received CsA, and Group 5 received CsA and ALS. Treatment was started 2 hr before surgery and was then given for 21 days. Donor-derived chimerism was monitored by FACS analysis. Survival time was calculated as the number of post-transplant days until the first signs of rejection. The allografts in Group 2, Group 3, and Group 4 survived a mean of 5, 6, and 33 days, respectively. The longest mean survival time-51 days-was noted in Group 5 (p<0.05). Donor- derived chimerism peaked at 17 percent and fell to 0 percent at the time of rejection. A combined protocol of ALS/CsA extended survival of rat hindlimb allografts across a fully allogeneic MHC barrier.     

Development of donor-specific chimerism and tolerance in composite tissue allografts under alphabeta-T-cell receptor monoclonal antibody and cyclosporine a treatment protocols

Recently, we induced donor-specific tolerance to rat hindlimb allografts under a 35-day course of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCR mAb) and cyclosporine A (CsA). In this report, we investigated the role of shorter alphabeta-TCR/CsA protocols on tolerance induction. We performed 52 hindlimb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21-, 7-, and 5-day protocols of combined alphabeta-TCR/CsA treatment on tolerance induction. Donor-specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor-specific chimerism was assessed by flow cytometry. All transplants under 5, 7, and 21 days of combined alphabeta-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytometry revealed a high level of donor chimerism in the peripheral blood of long-term survivors. The extension of survival of limb allografts and allo-unresponsiveness were directly associated with the development of stable chimerism in the tolerant recipients.     

Strategies to develop chimerism in vascularized skin allografts across MHC barrier

In this study, we investigated the effects of 7-day-protocols of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCRmAb), cyclosporine A (CsA), and tacrolimus (FK-506) immunosuppressive monotherapies, and their combinations on the survival of vascularized skin allografts (VSA). Forty-two transplantations of VSA across a strong MHC barrier were performed between ACI (RT1a) donors and Lewis (RT1(l)) recipients in seven groups. Isograft and allograft rejection controls received no treatment. Treatment groups received a 7-day protocol of alphabeta-TCRmAb, CsA, or FK-506 monotherapy, or a combination of alphabeta-TCRmAb/CsA and alphabeta-TCRmAb/FK-506. VSA transplants were evaluated on a daily basis. Donor-specific chimerism was determined by flow cytometry (FC). The combined protocols of alphabeta-TCRmAb/FK-506 and alphabeta-TCRmAb/CsA significantly prolonged VSA survivals compared to monotherapy groups ( P < 0.005). FC analysis revealed 15.82% of donor-specific chimerism on day 7 under the alphabeta-TCRmAb/CsA protocol and a gradual chimerism decline on day 63 posttransplant. The significant extension of VSA survival achieved under 7-day protocols of combined therapies was directly associated with the presence of donor-specific chimerism.     

Applications of bilateral vascularized femoral bone marrow transplantation for chimerism induction across the major histocompatibility (MHC) barrier: part II

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.     

Facial tissue allograft transplantation

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1(1)) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1(1+n)) in group 2 and from fully allogenic ACI(RT1(a)) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1(n) antigens, namely 3.39% CD4/RT1(n); 1.01% CD8/RT1(n) T-lymphocytes; and 3.54% CD45RA/RT1(n) B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1(a) and 4.59% of CD8/RT1(a) T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.     

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

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

Induction of donor-specific tolerance in rat hind-limb allografts under antilymphocyte serum and cyclosporine A protocol

Composite tissue allograft (CTA) transplantation became a clinical reality despite major side effects associated with the administration of chronic immunosuppression. Development of new treatment modalities eliminating life-long immunosuppression is essential for the future of CTA transplantation. In this study, combined use of cyclosporine A (CsA) and antilymphocyte serum (ALS) was tested for the potential to induce tolerance in the rat hind-limb allograft recipients across a major histocompatibility (MHC) barrier (Lewis-Brown-Norway [LBN, RT1(l+n)] to Lewis [LEW, RT1(l)] rats). Thirty transplantations were performed in 5 experimental groups. Animals received CsA and ALS 12 hours before surgery for 21 days thereafter. Although the allograft controls rejected their limbs at day 7 combined treatment of CsA and ALS resulted in indefinite survival (over 420 d) in all allograft recipients. Long-term survivors showed 35% to 42% of donor-specific chimerism in the peripheral blood. Clinical tolerance was confirmed by acceptance of the donor-specific skin grafts and immunocompetence was confirmed by rejection of the third-party grafts. Mixed lymphocyte reaction revealed suppressed response against donor-type antigens and increased response to third-party antigens. Donor-specific tolerance across MHC barrier was induced in CTA allografts under 21 days protocol of ALS/CsA.     

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.     

Results of vascularized joint allograft under immunosuppression with cyclosporine

The effects of cyclosporine (CsA), a strong immunosuppressive drug, on vascularized allogeneic joint transplantations were examined. An orthotopical transplant model of a vascularized knee joint allograft was developed using inbred DA and Lewis rats to investigate the fate of grafts following withdrawal of short-term immunosuppression compared to continuous immunosuppression with CsA. Five isograft controls acquired solid bone union at both femur and tibia sites within 4 weeks, and joint function as skeletal support was maintained until 25 weeks. Without immunosuppression, ten allografts were severely rejected within the first week, and joint destruction occurred immediately. Twenty-five short-term immunosuppressed rats acquired solid union, but, after withdrawal of immunosuppression, grafted joints showed gradual rejection and were destroyed due to pathological fractures or joint instability, although partial revascularization from the recipient occurred. Ten allografts under continuous immunosuppression at a dose of 10 mg/kg/day showed no rejection and remained viable for 12 weeks postoperatively, but thereafter all rats died. Death was considered to be a side effect of CsA. Fifteen animals, under continuous immunosuppression at a dose of 5 mg/kg/day, showed no rejection except in the bone marrow; the grafted joint function was not effected until 25 weeks. Continuous treatment with low and nontoxic doses (5 mg/kg/day) of CsA was necessary to maintain the functions of the grafted joint. © 1993 Wiley-Liss Inc.     

Development and maintenance of donor-specific chimerism in semi-allogenic and fully major histocompatibility complex mismatched facial allograft transplants

BACKGROUND: The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. MATERIAL AND METHODS: Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. RESULTS: Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. CONCLUSION: Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.     

Chimerism induction in vascularized bone marrow transplants augmented with bone marrow cells

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 alphabeta-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.     

Induction of tolerance to hind limb allografts in rats receiving cyclosporine A and antilymphocyte serum: effect of duration of the treatment

BACKGROUND: This study assessed the ability of antilymphocyte serum (ALS) and cyclosporine A (CsA) to induce tolerance for hind limb composite tissue allograft in rats without chronic immunosuppression. METHODS: Hind limb transplantations were performed in Lewis-Brown-Norway (LBN, RT1(1+n)) and Lewis (LEW, RT1(1)) rats. Treatment consisted of ALS only (0.4 mL/kg), CsA only (16 mg/kg), and a combination of CsA and ALS, and it was administered 12 hr before surgery at three different intervals (7, 14, and 21 days). Long-term survivors were tested for tolerance by standard skin grafting from the recipient (LEW), the donor (LBN), and the third party (ACI, RT1 ) 60 days after cessation of the treatment and by mixed lymphocyte reaction at 100 days. T-cell lines were analyzed with flow cytometry. RESULTS: Single use of ALS in all treatment intervals did not prolong allograft survival. Single use of CsA extended survival up to 23 days in the 21-day protocol group. CsA and ALS caused indefinite survival in two of six rats in the 14-day protocol and in all six rats in the 21-day protocol (>420 days). The six long-term survivors in the 21-day protocol accepted the skin grafts from the donor (LBN) and the recipient (LEW) and rejected third-party grafts (ACI). Tolerant animals showed a donor-specific hematopoietic chimerism of 35% to 42% in the peripheral blood. Mixed lymphocyte reaction assay demonstrated tolerance to the host and donor alloantigens and increased response to the third party. CONCLUSIONS: Administration of CsA and ALS for 21 days induced donor-specific tolerance in the recipients of the rat hind limb composite tissue allografts. The mechanism of tolerance should be investigated further.     

The effect of A2A adenosine receptor agonist on composite tissue allotransplant survival: an in vivo preliminary study

BACKGROUND: In vitro experimental studies showed that A2a receptor agonists reduce allostimulatory functions of the dendritic cells through modulation of surface expression of the costimulatory molecules and down-regulation of cytokines. Similar suppressive effects on dendritic cells have also been observed with Cyclosporine A (CsA). In this study we sought to explore if combined use of these two drugs in vivo could prolong the survival of composite tissue allografts across MHC barriers. MATERIALS AND METHODS: A total of 24 hindlimb transplantations were performed across a major MHC barrier from Brown Norway rats (BN; RT1n) to Lewis rats (Lew; RT1l) In the control group, either isografts (group 1, n = 2) or allografts (group 2, n = 3) were performed and no treatment was given. In the experimental group three kinds of treatment protocols were used: (1) A2a adenosine receptor agonist alone (group 3, n = 6); (2) CsA alone (group 4, n = 7); and (3) A2a adenosine receptor agonist + CsA combined treatment (group 5, n = 6). Mean survival times of each group as well as cytokine levels including IL-2, IL-4, IL-10, INF-gamma, and TNF-alpha were analyzed by ELISA. RESULTS: Isografts survived indefinitely. The mean survival times for allograft groups (group 2 to 5) were 9.8 +/- 1.3, 10.5 +/- 1.0, 29.8 +/- 1.7, and 22 +/- 1.4 days, respectively. Statistically, there was no difference between the allograft control group and the A2a adenosine receptor agonist treated group (P = 0.35). However, survival of the allografts in the CsA-treated group was significantly higher than the A2a adenosine receptor agonist treated (P < 00001) and combined CsA + A2a adenosine receptor agonist treated group (P < 0.0001). In vivo A2a adenosine receptor agonist treatment alone increased the levels of IL-2, INF-gamma, and TNF-alpha, which are important cytokines for induction of allotransplant rejection. However A2a adenosine receptor agonist in combination with CsA significantly reduced the levels of suppressor cytokines IL-4 and IL-10. CONCLUSION: As opposed to the previous in vitro studies, the results from this in vivo study showed that A2a adenosine receptor agonist treatment does not prolong composite tissue allograft survival. Compared to CsA treatment alone, the allotransplant survival was even shorter with the combined treatment. Treatment with A2a adenosine receptor agonist possibly promotes the differentiation of alloreactive CD4 cells predominantly into T-helper 1 phenotype. As a result, the levels of stimulatory cytokines contributing to allograft rejection are increased and suppressor cytokines are reduced, leading to accelerated allograft rejection.     

Synergistic effects of CTLA-4Ig and sirolimus on orthotopic lung-allograft survival and histology

BACKGROUND AND AIMS: The combination of CTLA-4Ig with sirolimus can promote indefinite survival in allograft models for which CTLA-4Ig monotherapy is ineffective. We sought to determine whether a limited course of CTLA-4Ig and sirolimus would alter survival of rat orthotopic single-lung transplantations. METHODS: Left lungs of Brown Norway rats were transplanted into four groups of Lewis recipients (n=6 per group): group 1, no treatment; group 2, mCTLA-4Ig (250 microg/day for 4 days); group 3, sirolimus (3 mg/kg per day for 14 days); group 4, combined therapy with sirolimus and mCTLA-4Ig. Graft survival was determined by daily radiologic examination. Histologic grading of rejection and immunohistochemical staining for T and B lymphocytes were carried out at the time of radiologic graft loss. RESULTS: Rejection of lung allografts in group 1 occurred at a median of 6.5 days. Neither sirolimus nor mCTLA-4Ig monotherapy resulted in significant prolongation of graft survival (median 9.5 and 8.0 days, respectively). Graft survival in group 4 was significantly prolonged compared with all other groups (median 29.5 days), and a significant reduction in histologic grade of rejection was observed following combination therapy compared with all other groups. Infiltration by CD8+ve T cells at the time of rejection was proportionately greater than CD4+ve T-cell infiltration for groups 1, 2, and 3 but not for the combined-therapy group. CONCLUSIONS: A brief course of combined mCTLA-4Ig and sirolimus prolongs graft survival, reduces severity of rejection, and attenuates CD8+ve T-cell infiltration of fully major histocompatibility complex mismatched lung allografts.     

Intraosseus transplantation of donor-derived hematopoietic stem and progenitor cells induces donor-specific chimerism and extends composite tissue allograft survival

We investigated the effect of the intraosseous allotransplantation of the donor-derived hematopoietic stem cells (HSC) CD90+ on chimerism induction and survival of rat hind limb transplants. Eighteen rat hind limb transplantations were performed between Lewis-Brown-Norway and Lewis rats in three groups. Isograft and allograft rejection controls received no treatment. In the experimental group, 0.8 to 1.2 x 10(6) of separated and purified CD90+ HSC cells were transplanted intramedullary into the bone marrow cavity of the recipient's tibia during opposite hind limb transplantation, without immunosuppressive therapy. Transplants from isograft group survived indefinitely. Allograft controls rejected transplants on day 7 posttransplant. The injection of separated and purified CD90+ cells of the donor origin extended survival of the transplanted limbs up to 15 days in group III. We introduced a novel method of transplantation of the CD90+ cells of the donor origin into the recipient's bone marrow cavity. This technique resulted in extended allograft survival, without immunosuppressive therapy.     

Evidence that cyclosporine prevents rejection and recurrent diabetes in pancreatic transplants in the BB rat

Cyclosporine prevents the development of diabetes in spontaneously diabetic BB rats and NOD mice. However, islet transplants have been shown to be subject to immunologic destruction in hosts treated with CsA and anti-CD4 antibody or those rendered tolerant to donor antigens. This study determines whether the minimum dose of CsA necessary to prevent rejection of pancreatic transplants will also prevent recurrent diabetes in pancreas transplants in BB rats. Lewis recipients promptly reject BN (n = 13, MST 9.2 +/- 0.7 days) and Fisher (n = 6, MST = 11.3 +/- 0.8 days) pancreatic transplants. Treatment with CsA 5 mg/kg/day 0-50 days posttransplant and 2 mg/kg/day 51-100 days (low-dose CsA) produced indefinite survival of BN (n = 5) but not Fisher (n = 4) allografts. Fisher allografts were uniformly successful if maintained on 5 mg/kg/day (n = 4). Treatment with CsA 5 mg/kg/day for 14 days had no deleterious effect on glucose tolerance in Lewis isografts (control [K = 1.81 +/- 0.24, n = 8] vs. CsA treated [K = 1.76 +/- 0.20, n = 8, P = NS]). Low-dose CsA ensured permanent survival of MHC-compatible WF (n = 7, MST greater than 115 days) and MHC-incompatible BN (n = 9, MST greater than 117 days, P = NS) pancreatic transplants in spontaneously diabetic BB/Wor hosts. Three of 11 recipients surviving greater than 100 days enjoyed seemingly permanent acceptance of their allografts after discontinuation of CsA. Immunocompetence was demonstrated by rejection of their pancreas transplants when challenged with donor skin. Vascularized pancreatic allografts treated with CsA appear to be less vulnerable to recurrent diabetes than are islet transplants. Low-dose CsA protects vascularized pancreatic allografts in BB rats from both rejection and recurrent diabetes.