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.     

改进技术的大鼠全小肠移植术

目的建立并发症少、成活率高的大鼠异位全小肠移植模型。方法应用显微外科技术对１９６只Ｗｉｓｔａｒ大鼠施行异位全小肠移植。术前缩短禁食时间,补充能量,术中静脉输液;减少对供肠的机械和缺血性损伤;重建供肠血管采用腹主动脉－腹主动脉吻合以及门静脉－左肾静脉套管法吻合。结果热缺血时间≤４０±５分钟,吻合口无血栓形成及狭窄,９８只接受小肠移植大鼠的存活率为８８．７８％（８７／９８）。结论改进技术后的大鼠全小肠移植术具有并发症少,存活率高的特点,并且稳定、实用     

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

INTRODUCTION: Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. METHODS: Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1). the type of donor-specific cell augmentation and (2). the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. RESULTS: Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1alpha, IL-1beta, IFN-gamma, and TNF-alpha; liver expression mainly involved TNF-alpha. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1alpha, IL-1beta, IFN-gamma, as well as TNF-alpha, and liver expression of IL-1beta. CONCLUSIONS: (1). Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2). Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3). In our study, TNF-alpha appeared to be the cytokine most strongly associated with rejection.     

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation with and without donor-specific cell augmentation

BACKGROUND: Immunomodulatory strategies such as donor-specific bone marrow or blood transfusions have been used to promote engraftment after intestinal transplants. We previously showed that delivery of donor antigen via the portal vein can effectively reduce the rate of intestinal graft rejection. The purpose of our current study was to investigate the impact of donor-specific cell augmentation (blood versus bone marrow) via the portal vein on cytokine expression in intestinal grafts versus native livers. MATERIAL AND METHODS: We performed heterotopic small intestinal transplants between male Brown-Norway (donor) and female Lewis (recipient) rats. We studied 10 groups according to the type of donor-specific cell augmentation and the use and dose of immunosuppressive therapy. For cell augmentation, donor-specific blood or bone marrow was transfused via the donor portal vein immediately before graft implantation. For immunosuppression, tacrolimus was used post-transplant at a high or low dose. Control rats received neither immunosuppression nor cell augmentation. Tissue samples for histological assessment were obtained at designated time points. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta). Chimerism levels were determined using Q-PCR analysis. RESULTS: Without concurrent immunosuppression, neither portal donor-specific blood nor bone marrow transfusion reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood (versus bone marrow) transfusion. Irrespective of the type of donor-specific cell augmentation, severe rejection caused strong cytokine expression in the grafts of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha; in the native livers, mainly of TNF-alpha (with IFN-gamma showing hardly any increase). In general, rejection caused stronger cytokine expression in the grafts than in the native livers. Mild rejection correlated well with strong intragraft expression of IL-6, TNF-alpha, and TNF-beta (early rejection markers); severe rejection with IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha (late rejection markers).In addition to cell augmentation per se, the type of cell augmentation also had an impact on cytokine expression in both grafts and native livers. Cell-augmented (versus tacrolimus-treated) rats showed hardly any differences in intragraft cytokine expression, but the expression of almost all cytokines was significantly stronger in the native livers. With immunosuppression, bone marrow infusion increased intragraft cytokine expression of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF alpha, as well as liver cytokine expression of IL-1 beta, compared to blood transfusion. This finding reflected the more advanced rejection stages in the bone marrow infused group; different types of donor-specific cell augmentation had similar effects on liver cytokine expression. In the absence of myoablative therapy, chimerism levels were low, in both cell-augmented and non-cell-augmented groups. CONCLUSIONS: Rejection and donor-specific cell augmentation independently causes differences in intragraft versus native liver cytokine expression after intestinal transplants. Portal donor-specific blood transfusion, as compared with donor-specific bone marrow infusion, lowered the incidence of rejection and diminished intragraft cytokine up-regulation.     

Simplified techniques in rat heterotopic small bowel transplantation

AIM: Establish a simplified heterotopic small bowel transplantation (SBT) in the rat. METHODS: Ninety pairs of male Wistar rats were used as donors and recipients. The whole small intestine with a vascular pedicle composed of superior mesenteric artery (SMA) and portal vein (PV) was harvested as the graft. Revascularization was accomplished by end-to-side anastomosis between donor SMA and recipient infrarenal aorta and cuffed end-to-end anastomosis between donor PV and left renal vein of recipient. The distal end of graft was exteriorized to form an enterostoma. RESULTS: Average time of an operation was 130 minutes and the mean warm ischemia time of grafts was 30 minutes. The technical success rate of this model was 100% and 7-day survival was 95.6% (86/90). CONCLUSION: This simplified technique was effective and practical to improve the outcome of rat heterotopic SBT.     

Cyclosporine facilitates chimeric and inhibits nonchimeric tolerance after posttransplant total lymphoid irradiation

BACKGROUND: Previous studies showed that Lewis rats given posttransplant total lymphoid irradiation, antithymocyte globulin, and a single infusion of ACI peripheral blood or bone marrow cells develop tolerance to ACI heart allografts. METHODS: To determine the effects of cyclosporine on these tolerance induction protocols, groups of Lewis hosts, given either ACI blood or marrow infusions, were given a 60-day course of daily cyclosporine immediately after the cell infusion. RESULTS: Cyclosporine treatment was associated with uniform graft rejection in the groups given an ACI blood transfusion, and was associated with uniform graft acceptance in the groups given an ACI bone marrow infusion. Studies of donor-type T and B cell chimerism in the host blood showed that cyclosporine facilitated chimerism in the hosts given ACI bone marrow cells, and stable chimerism over a 300-day observation period was predicted by detectable chimerism by day 30. None of the hosts given ACI blood cells developed chimerism. CONCLUSION: Cyclosporine facilitated long-term graft acceptance in a tolerization protocol that induced mixed chimerism, but prevented long-term graft acceptance in a tolerization protocol that did not induce chimerism.     

大鼠小肠移植模型的改进

目的：在传统小肠移植模型基础上改进操作方法并试图建立一个操作简便、并发症少、稳定的大鼠小肠移植模型.方法：显微镜下切取供肠的范围包括近端空肠、门静脉及其带肠系膜上动脉的腹主动脉袖.动脉吻合采用供体带肠系膜上动脉的腹主动脉袖与受体的腹主动脉端侧吻合,静脉吻合采用供体的门静脉用袖套法与左肾静脉端端吻合,移植肠两端造瘘.结果：正式实验100次,手术成功率91%.供体手术控制在50 min以内,受体手术控制在80 min以内,手术时间约为150 min.结论：完全在显微镜下建立大鼠小肠移植模型并将手术方法加以改进能在手术视野清晰、术中操作定位准确、局部创伤小的基础上使手术时间缩短,并发症更少,存活率更高.     

输注供体骨髓细胞诱导大鼠小肠移植基因嵌合的研究

目的 探讨经门静脉途径输注供体骨髓细胞后,大鼠小肠移植中受体基因嵌合率的改变及可能机制.方法 建立大鼠异位节段小肠移植模型(供体为雄性BN,受体为雌性Lewis,各15只),并将其随机分为三组,对照组、FK506组、PV组(喂养FK506及经门静脉注射供体骨髓细胞组).应用原位荧光杂交定位检测以及实时定量聚合酶链反应技术分析供体雄性细胞的SRY基因在移植术后受体大鼠血液、肝脏及脾脏中的嵌合情况.结果 PV组受体雌性大鼠血液(3.03±0.16)%、肝脏(0.86±0.05)%、脾脏(4.08±0.12)%中供体SRY基因嵌合率均较FK506组(1.15±0.07)%、(0.21±0.02)%、(1.23±0.05)%、对照组(1.10±0.07)%、(0.22±0.02)%、(1.17±0.06)%明显增高(P＜0.01).结论 经门静脉系统输注供体骨髓细胞可建立稳定的混和嵌合体状态.     

Impact of graft length on surgical damage after intestinal transplantation in rats

BACKGROUND: Intestinal grafts greatly affect nutrition and immunology in the host. The growth of the recipient and incidence of graft-versus-host disease depend on graft length. A larger graft may affect the host immune system, but little is known about how the length of the intestinal graft severely affects surgical intervention. We developed a cervical small bowel transplantation (SBT) rat model that minimized technical variations using a cuff method and studied the effects of graft length on surgical damage in SBT. MATERIALS AND METHODS: We transplanted a whole (70 cm) or partial (15 cm) intestine into a syngeneic rat combination of LEW (MHC haplotype: RT1(l)) to LEW and evaluated changes in perioperative hemodynamics and the endogenous endotoxin level. Natural killer (NK) cell activity in the peripheral blood and the immunologic response of the recipient spleen were also studied. RESULTS: In the whole SBT model, body weight loss was more severe than in the segmental SBT model; the rats in the former model often died, while all in the latter survived indefinitely. The systemic blood pressure markedly decreased in the whole SBT group immediately after reperfusion. The proliferative activity of splenic lymphocytes stimulated by concanavalin A was also more severely inhibited in the former model than in the latter postoperatively. NK cell activity in the whole SBT rats declined more severely than the segmental SBT rats 3 days postoperatively. CONCLUSION: The longer graft severely induced surgical intervention; and influenced host immunosuppression, resulting in the higher mortality in rats undergoing whole SBT.     

Treatment of short gut syndrome with early living related small bowel transplantation

AIM: To investigate the results of treating short bowel syndrome with an early living related small bowel transplantation (SBT). METHODS: A 17-year-old boy with a 20-cm-long residual intestine due to necrotic volvulus received an early living related SBT from his mother. Donor-specific blood transfusion was performed for 8 weeks before transplantation, each time for 50 mL every week. Cytomegalovirus status in both donor and recipient was negative. A 160-cm distal ileal segment was removed from the donor. The graft ilecolic artery and vein were anastomosed to the recipient's infrarenal aorta and caval vein. The proximal end of the graft was anastomosed end-to-end to the residual recipient jejunum; the distal anastomosis, between the distal end of the graft and transverse colon. An ileostomy was also performed. Immunosuppression, infection prophylaxis, and antithrombotic and nutrition support were given postoperatively. RESULTS: The donor had an uneventful recovery. No technical complications were observed. The recipient was alive and well at 31 weeks after the operation. No graft rejection or infection was observed. He was off TPN 8 weeks after the operation and took low-fat food. The D-xylose test in the recipient was almost normal. CONCLUSIONS: Early living related small intestine transplantation is a good treatment for short bowel syndrome.     

大鼠异位节段小肠移植模型建立技术的改进

目的：建立大鼠异位节段小肠移植模型。方法：对40例（80只）Wistar大鼠施行异位小肠移植,供受体术前抗生素灌胃,改变供受体术式,减少受体手术时间、手术损伤及供肠缺血时间;采用腹主动脉-肠系膜上动脉吻合以及左肾静脉-门静脉单套管吻合,血管吻合方法采用单纯间断吻合,重建供肠血管;移植小肠双造口,静脉补液通路采用股静脉。结果：肠缺血时间≤35min,吻合口无狭窄,40例大鼠接受小肠移植,建模成功35例。结论：改进小肠移植技术中的多个细节后,降低了大鼠小肠移植术的难度。     

小鼠异位小肠移植模型的技术改进

目的 探讨小鼠异位小肠移植的技术要点并进行相应改进,为小肠移植的研究提供可靠的动物模型.方法 53只Balb/C小鼠的节段小肠异位移植于C57BI/6小鼠腹腔.适当缩短切取小肠及血管蒂门静脉的长度;应用缝针法建立受体腹主动脉前壁的椭圆形切口,穿刺法建立下腔静脉前壁切口;两点定位连续缝合法吻合血管;近端结扎封闭,远端与受...     

Effect of FTY720 and ex vivo graft irradiation in rat small bowel transplantation: apoptosis of crypt cells and lymphocytes

OBJECTIVE: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 and ex vivo graft irradiation during rejection. MATERIALS AND METHODS: Orthotopic small bowel transplantations (SBT) were performed from Brown Norway (BN) rats to Lewis (LEW) rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations: untreated allograft, allograft with FTY720, allograft with irradiation, and allograft with FTY720+irradiation. Cryostat sections were prepared from the grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/FasL. RESULTS: The graft survival was prolonged in the FTY720-treated groups. In the FTY720-treated group, the number of ISEL-positive enterocytes was significantly down-regulated on days 3, 5, and 7 compared with the untreated allograft group. The number of ISEL-positive mononuclear cells was also significantly down-regulated compared with the untreated allograft group. The FTY720 the radiation and the FTY720+irradiation treated groups showed significantly down-regulated numbers of Fas/FasL-positive enterocytes on day 7 compared with the untreated allograft group. Fas/FasL-positive mononuclear cells were also significantly down-regulated in the allograft compared with the untreated allograft group. CONCLUSIONS: FTY720 and ex vivo graft irradiation prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes, and also prolonged small bowel allograft survival. Combination FTY720 and ex vivo graft irradiation did not affect graft survival and apoptotic cell expression compared with the FTY720 only group. These findings suggest that FTY720 may prevent both rejection-associated and sepsis-induced apoptosis during the late phase of small bowel graft rejection.     

Effect of posttransplantation administration of peripheral blood lymphocytes in skin-grafted mice treated with antilymphocyte serum or antilymphocyte serum plus bone marrow

Posttransplantation administration of donor-specific bone marrow cells to ALS-treated allograft recipients produces graft survival that is greater than that produced by ALS treatment alone. We have now studied the effect of peripheral blood lymphocytes (PBLs) in a mouse skin allograft model using this protocol (C3H skin grafted to B6AF1 mice, day 0; i.p. ALS on days -1 and +2; i.v. bone marrow days +6 or +7). When PBLs are injected in place of bone marrow cells, graft survival is extended beyond that noted for control mice given ALS only. The timing and specificity of this phenomenon suggest that it resembles the effect produced by posttransplant bone marrow administration and not that associated with pretransplant blood transfusion. The PBLs active in graft prolongation are Thy-1-negative and display a density in Percoll gradients similar to that of the active marrow cells. In contrast, when PBLs are injected in combination with bone marrow cells, the length of graft survival is shortened in comparison with that produced by bone marrow alone. The cells associated with this partial abrogation of the effectiveness of bone marrow appear to be mature T cells; this abrogation cannot be produced by PBLs treated with anti-Thy-1 plus complement or by thymocytes, but it is a property of lymph node cells enriched for T cells by nylon-wool fractionation. This study suggests that clinical application of this posttransplantation induction of specific allograft unresponsiveness can be facilitated by the use of peripheral blood lymphocytes rather than marrow, sparing a living organ donor from having to undergo bone marrow harvest. Additionally, the data indicate that contamination of marrow with blood lymphocytes should be minimized. However, the density gradient fractionation method that we have found to be effective in preparing the graft prolonging bone marrow cells simultaneously removes the PBLs deleterious to graft survival.     

Comparison of chimeric acid and non-chimeric tolerance using posttransplant total lymphoid irradiation: cytokine expression and chronic rejection.

BACKGROUND: Previous studies showed that an intravenous infusion of donor blood cells facilitates tolerance to ACI heart allografts in Lewis rat hosts given posttransplant total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG). The object of the current study was to compare tolerance induction using donor cells that do or do not induce chimerism. METHODS: Normal peripheral blood mononuclear cells (PBMC), granulocyte colony-stimulating factor (G-CSF)-mobilized PBMC, and bone marrow (BM) cells from ACI donors were tested for their capacity to prolong ACI heart allograft survival in Lewis hosts. Chimerism, anti-donor cell reactivity, and cytokine gene expression in grafts were determined. RESULTS: Intravenous injections of equal numbers of all three donor cells markedly prolonged graft survival (median: >164 to >175 days) as compared to uninjected controls (median: 53 days). Chimerism among T and B cells in the blood was determined by immunofluorescent staining in hosts bearing long-term (> 150 days) grafts. Although no chimerism was detected in hosts given normal or G-CSF-mobilized PBMC, chimerism was detected at variable levels in all hosts given BM cells. Vigorous anti-donor reactivity in the mixed leukocyte reaction was present only in non-chimeric hosts. Long-term grafts from hosts given normal ACI PBMC developed chronic rejection, but those from hosts given ACI BM cells did not. The latter hosts showed the lowest levels of intragraft cytokine mRNA. CONCLUSIONS: Chimeric tolerance is more robust than non-chimeric tolerance in the model of posttransplant TLI, ATG, and donor cell infusion, and is associated with less chronic rejection.     

Long-lasting donor passenger leukocytes after hepatic and intestinal transplantation in rats

BACKGROUND: Donor passenger leukocytes (DPLs) that migrate after organ transplantation stimulate the recipient immune system and normally cause rejection and graft vs. host reaction. However, DPLs also contribute to the unresponsiveness to the donor organ. The quantity and quality of these migrating cells are considered dependent on individual transplanted organs. We compared the DPLs of the liver, which might contain somatic stem cells, with those of intestinal grafts that have highly immunogenetic cells. To study DPLs over a long period, we used green fluorescent protein (GFP) transgenic (Tg) rats developed by us as donors. METHODS: We performed orthotopic liver transplantation (OLT) and small bowel transplantation (SBT) from GFP Tg rats to wild recipients. A short course of tacrolimus (0.64 mg/kg, intramuscularly) was used to prevent antigenicity of the GFP. The fate of the DPLs in the peripheral blood and the recipient bone marrow was monitored by flow cytometry. Using long-surviving recipients, the GFP(+) cells in the graft and various host immunologic organs were measured and characterized by immunohistochemical staining. RESULTS: In both groups, the numbers of the GFP(+) cells in the peripheral blood increased transiently and then gradually decreased to undetectable levels. While no GFP(+) cells were identified in the long-surviving-recipient bone marrow, there were a few GFP(+) cells in the graft liver, graft mesenteric lymph nodes and the recipient spleen. These cells showed major histocompatibility complex (MHC) class II antigen. There was no significant difference in the migration patterns of the GFP(+) cells in the OLT and SBT rats. CONCLUSIONS: In both the OLT and SBT groups, the DPLs migrated transiently in the recipient peripheral blood. A small numbers of MHC class II-positive DPLs were present at the graft site and in the host spleen, but not in the bone marrow. There were no significant differences in the migration patterns of the DPLs between the OLT and SBT rats over the long term.     

CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts

BACKGROUND: CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. METHODS: Small bowels of Brown-Norway rats (RT1n) were heterotopically transplanted into Lewis rats (RT1l). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. RESULTS: Long-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1b) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 microg/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 microg/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Thl to Th2 cytokine production.     

Rejection following donor or recipient preoperative treatment with FTY720 in rat small bowel transplantation

BACKGROUND: Severe rejection of small bowel transplantation (SBTx) has been ascribed to abundant lymphoid tissues in the small intestine without well-established evidence. However, the role of donor lymphocytes in rejection is still unclear. The novel immunosuppressant, FTY720, is reported to transfer peripheral blood lymphocytes (PBLs) to lymphoid tissues such as mesenteric lymph nodes (MLNs) and Peyer patches (PP). In the present study, the number of donor lymphocytes in the graft was increased by FTY720, and the influence on rejection was studied in a rat model. Furthermore, the number of the PBL of recipient was decreased by FTY720 before SBTx and the effect on rejection was examined. MATERIALS AND METHODS: Orthotopic total SBTx was performed in Brown-Norway and Lewis rats. In the donor pretreatment study, FTY720 was administrated to donor rats 24 h prior to harvesting to increase the number of graft lymphocytes (FTY donor-pretreated group). In contrast, MLNs were surgically removed from the grafts to decrease the number of graft lymphocytes (MLN-resected group). In the recipient pretreatment study, FTY720 was administrated to recipient rats 24 h before SBTx to decrease recipient PBL (FTY group). In contrast, a subclinical dose of cyclosporine A (CsA) was administrated after SBTx (CsA group). Rats were administrated preoperative FTY720 combined with post-SBTx CsA (FTY+CsA group). Graft survival, pathology, lymphocyte count, and subtype were examined. RESULTS: In the donor pretreatment study, pretreatment with FTY720 did not enhance graft rejection. MLN resection did not prolong graft survival. In the recipient pretreatment study, FTY720 caused a significant reduction in the number of infiltrating lymphocytes in the graft, as well as the percentage of recipient CD4+ and CD25+ cells within the graft. FTY720 and CsA synergistically prolonged graft survival. CONCLUSION: SBTx rejection correlated with the number of recipient PBL, and not with the number of donor lymphocytes transplanted together with the graft. The pretreatment of the recipient with FTY720 was effective in the case of combined use of the low-dose postoperative CsA.     

Effect of a New Immunosuppressive Agent,FTY720, on Survival of Islet Allografts

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.     

Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts

BACKGROUND: We investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts. METHODS: Four- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 x 10(7) cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe). RESULTS: At 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells. CONCLUSIONS: Intrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.