Bone marrow chimerism and tolerance induced by single-dose cyclophosphamide

BACKGROUND: Establishment of hematopoietic chimerism is the most stable strategy for donor-specific tolerance. Safer pretreatment regimens are needed for clinical application. We evaluated the efficacy of a simple protocol using cyclophosphamide (CYP) on induction of chimerism and organ transplant tolerance across major histocompatibility complex (MHC) barriers in the rat. MATERIALS AND METHODS: Bone marrow cells from BN (RT1(n)) donors were infused to LEW (RT1(l)) recipients on day 0 after a single injection of CYP at various doses on day -1. Donor-derived hematopoietic chimerism was evaluated by flowcytometry. The recipients received BN or third party (BUF) heart allografts on day 100. RESULTS: While pretreatment with 200 mg/kg of CYP induced high levels of hematopoietic chimerism, six of eight recipients died of severe graft-versus-host-disease (GVHD). CYP at dose of 150 mg/kg induced 36.5 +/- 24.1% of donor-derived chimerism on day 10, and sustained macrochimerism was seen until day 100 without GVHD. Pretreatment with 100 mg/kg of CYP resulted in only transient chimerism (4.8 +/- 5.2%) which disappeared by day 20. In the recipients with 50 mg/kg of CYP, donor bone marrow cells were rapidly rejected and no chimerism was observed. The recipients with 150 mg/kg of CYP accepted BN heart allografts (>100 days x 5), while rejecting BUF allografts by day 12 (n = 4). BN heart allografts were rejected in the recipients with 100 (MST: 57 days, n = 5) and 50 mg/kg (MST: 7 days, n = 5) of CYP. CONCLUSIONS: A single dose of CYP can induce hematopoietic chimerism across MHC-barriers. The dose of 150 mg/kg seems to be optimal to induce organ transplant tolerance without developing GVHD.     

Construction of ectopic xenogeneic bone marrow structure associated with persistent multi-lineage mixed chimerism by engraftment of rat bone marrow plugs into mouse kidney capsules

Abstract: Poor bone marrow (BM) engraftment in a xenogeneic combination results at least in part from the limited engraftment capacity of BM-derived stromal cells, which support hematopoietic repopulation in a species-specific fashion. We attempted to construct a BM stromal microenvironment by engraftment of BM plug fragments into kidney capsules in a rat-to-mouse combination. BM plugs from F344/N Jcl- rnu/rnu (F344 nu ) rats were transplanted into the kidney capsules of C.B-17 scid/scid (C.B-17 scid ) mice treated with rabbit anti-asialo-GM1 serum to deplete natural killer (NK) cells and then with 3 Gy of whole body irradiation. As a conventional control, an equivalent amount of F344 nu bone marrow cells (BMCs) was intravenously injected into C.B-17 scid mice treated with a similar conditioning regimen. In both mouse recipients of rat BM plug engraftment in the kidney capsules and recipients of intravenous injection of rat BMC suspension, comparable extents of donor rat class I⁺ cells were persistently detected in the peripheral blood. However, the differentiation of rat-derived B cells in the mouse recipients of rat BM plugs was more rapid than that in the recipients of rat BMC suspension. In the late phase (10 weeks after BM transplantation), the percentage of rat-derived T cells (CD4⁺ cells) in the mouse recipients of rat BM plugs was significantly higher than that in the recipients of rat BMC suspension. At this time point, ectopic BM structure consisting of bone, mesenchymal cells, and hematopoietic progenitors was constructed in the kidney capsules of mice that received rat BM plugs. Most of the cells in the ectopic BM were derived from the donor rat. Thus, engraftment of BM plugs into the kidney capsules results in the construction of a donor-derived BM microenvironment, facilitating multilineage mixed xenogeneic chimerism.     

A comparative study of FK506 granules and capsules in renal transplant recipients

Nine renal transplant recipients in stable systemic condition on FK506 capsules were converted to FK506 granules in order to investigate the safety, efficacy, and pharmacokinetics of the granular formulation of FK506. The study period for the administration of FK506 granules was 4 weeks, and in principle, the oral dose was the same as that of the FK506 capsules. Renal graft function remained stable and no rejection signs were noticed while the patients were taking the granules. The area under the blood concentration-time curve (AUC), the maximum blood level (C_max), and the time to reach C_max (T_max) after FK506 capsules and FK506 granules were, respectively, 93.1 ± 66.4 and 97.0 ± 89.1 ng · h/ml (P = 0.81), 12.7 ± 7.1 and 15.2 ± 11.7 ng/ml (P = 0.39), and 2.0 ± 1.7 and 1.3 ± 0.6 h (P = 0.29). The mean trough blood level during FK506 medication was 4.25 ± 3.42 and 4.02 ± 3.83 ng/ml, respectively, for the capsules and the granules. FK506 granules, a new formulation, showed an efficacy comparable to that of the FK506 capsular formulation. Received: 28 July 1997 Received after revision: 25 November 1997 Accepted: 14 January 1998     

Comparison of short and long-term renal function in liver transplant patients receiving cyclosporin or FK 506

Abstract Long-term renal function was compared in 49 liver recipients [25 patients received cyclosporin (CyA) and 24 patients received FK 5061 followed for a period of 1 year. Creatinine (CR) and glome-rular filtration rate (GFR) pre-transplantation (pre-Tx) and at 1, 3, 5, and 12 months post-Tx were recorded, as well as incidences of hyperkalemia, post-Tx hypertension, and insulin-dependent diabetes mellitus (IDDM) in the two groups. At 1 year post-Tx, the mean Cr had risen from baseline by 56% and 60% in the FK and CyA groups, respectively; the mean GFR had dropped by 32% in FK patients and by 27 % in CyA patients. Acute nephrotoxicity occurred in 1/25 CyA patients (217 required dialysis) and 9/26 FK patients (7/9 required dialysis; 211 were switched to CyA). None remained on dialysis at 3 months. Renal insufficiency persisted at 1 year in 7/16 patients with early toxicity (CyA, 4; FK, 3) and in 3 of the remaining 36 pts ( P < 0.001). Hyperkalemia occurred in 4/25 CyA, and in 12/24 FK patients ( P < 0.025), post-Tx hypertension occurred in 15 CyA, and 7 FK patients ( P < 0.05), and IDDM occurred in 4 CyA and 7 FK patients ( P = ns). FK 506 and CyA, thus, exerted similar chronic renal effects. Although acute renal insufficiency improved upon dose reduction, renal impairment was permanent in some cases.     

Two-year follow-up study of the efficacy and safety of FK 506 in kidney transplant patients

Abstract We conducted a 2-year follow-up study of the efficacy and safety of FK 506 in 104 kidney transplant patients at 32 sites in Japan. The initial daily oral dose of FK 506 was 0.3 mg/kg, which was gradually reduced to 0.15 mg/kg by month 10 and remained stable thereafter. The mean trough level of FK 506 in whole blood and the mean serum creatinine level in year 2 were 7.9 ng/ml and 1.9 mg/dl, respectively. Patient and graft survival rates for all patients were 97% and 92%, respectively. Forty-ix patients (44%) experienced rejection episodes, and 84% of these episodes occurred within 3 months after transplantation. The principal adverse reactions to FK 506 therapy were hyperglycaemia, renal dysfunction and hyperkalaemia. Most of these events were dose-dependent, and disappeared or ameliorated following reduction of the FK 506 dose.     

Clinical experience with mixed chimerism to induce transplantation tolerance

Lymphohematopoietic chimerism was first shown to be associated with donor‐specific allograft tolerance more than 60 years ago. However, early clinical experience with bone marrow transplantation soon revealed that conventional, myeloablative approaches were far too toxic and the risk of graft‐versus‐host disease too great to justify using this technology for the purpose of organ allograft tolerance induction in the absence of malignant disease. In this review, we discuss a step‐wise approach that has been applied by several centers to establish less toxic approaches to using hematopoietic cell transplantation (HCT) for tolerance induction. These steps include (i) feasibility and efficacy data for tolerance induction in large animal models; (ii) safety data in clinical trials for patients with hematologic malignancies; and (iii) pilot trials of combined HCT and kidney transplantation for tolerance induction. Thus far, only one published trial conducted at the Massachusetts General Hospital in Boston has achieved long‐term acceptance of human leukocyte antigen‐mismatched kidney allografts without chronic immunosuppressive therapy. Alternative protocols have been successful in large animals, but long‐term organ allograft tolerance has not been reported in patients. Thus, proof‐of‐principle that nonmyeloablative induction of mixed chimerism can be used intentionally to induce organ allograft tolerance has now been achieved. Directions for further research to make this approach applicable for a broader patient population are discussed.     

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells

Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RT1ⁿ) rat heart grafts in 6 out of 8 Lewis (LEW, RT1^l) rat recipients. LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1^c) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism. Received: 7 August 1996 Received after revision: 12 February 1997 Accepted: 17 February 1997     

Long‐term small bowel allograft function induced by short‐term FK 506 application is associated with split tolerance

Abstract Functional long‐term allograft survival after experimental small bowel transplantation (SBT) is limited by chronic rejection. Initial application of high‐dose FK 506 has been shown to induce stable long‐term graft function. In order to examine whether this long‐term function is associated with donor‐specific tolerance, we analyzed the functional status of recipient T cells in vivo and in vitro. One‐step orthotopic SBT was performed in the allogeneic Brown Norway (BN)‐to‐Lewis rat strain combination. FK 506 was given daily at a dose of 2 mg/kg from days 0‐5 in the rejection model and from days 0‐9 in the long‐term functional model. Mean survival time in the rejection model was 98 ± 2.8 days. Histological examination of these small bowel allografts disclosed signs of chronic rejection. In contrast, all animals of the long‐term functional model survived long term (> 250 days) without clinical signs of chronic rejection. The latter model, furthermore, produced evidence of donor‐specific tolerance. Whereas heterotopic Dark Agouti (DA) hearts were rejected regularly within 7 days, BN hearts survived indefinitely (> 70 days). In vitro, mixed leukocyte reactivity of CD4 + T cells was similarly strong against donor (BN) antigens as against third‐party (DA) antigens. The split tolerance revealed by our in vivo and in vitro results enabled acceptance of both the small bowel allograft without signs of chronic rejection and of donor‐specific heart allografts.     

FK506预处理下骨髓移植诱导同种异体小鼠皮肤移植耐受的实验研究

目的探讨短期大剂量FK506作为宏嵌合诱导供体特异性耐受中,骨髓移植前对受体预处理方法的可行性及临床实用性. 方法 100只雄性C57BL/6和60只雌性BALB/C小鼠分别作为皮肤移植的供体和受体,雄性ICR小鼠15只作为无关第三品系用以检测移植耐受状态的特异性.将60只受体小鼠随机分为5组,即无处理对照组、单纯FK506组、单纯骨髓细胞移植(BMT)组、实验组(FK506 BMT)和无关供体对照组,每组12只.FK506诱导及维持方案是皮肤移植前对受体小鼠先给予大剂量FK506腹腔注射(3 mg/kg×2 d),移植当天尾静脉输注2×107个骨髓细胞,再以小剂量FK506(0.5 mg/kg×7 d)短期维持治疗.观察皮肤移植存活时间、对第三方皮肤的排斥反应及对供体鼠的单向混合淋巴细胞反应,并用多聚酶链式反应(PCR)检测嵌合体的形成. 结果常规剂量的骨髓输注或短期FK506治疗并不能延长移植物的存活时间,也没有宏嵌合形成.实验组皮肤移植物存活时间(24.0±1.5)d比无处理对照组(9.6±1.1)d、单纯FK506组(10.5±1.6)d、单纯骨髓细胞移植组(10.3±1.5)d、无关供体对照组(9.8±1.1)d明显延长(P<0.05).混合淋巴细胞反应实验组供者特异性抑制率80.55%±14.10%明显高于单纯FK506组38.65%±12.43%及单纯骨髓细胞移植组35.41%±8.99%(P<0.05),实验组宏嵌合呈阳性. 结论采用短期大剂量FK506这一温和的非照射预处理方法,可获得一定程度的免疫耐受,延长移植物存活.移植前输注供体骨髓细胞能够促进宏嵌合的形成及移植物的存活.     

Establishment of chimerism in donor liver with recipient-type bone marrow cells prior to liver transplantation produces marked suppression of allograft rejection in rats

Abstract In this study, we investigated whether establishment of chimerism in donor liver with recipient-type bone marrow cells (BMCs) prior to liver transplantation could prolong the liver allograft survival. Donor female ACI rats were inoculated with recipient-type BMCs of male LEW rats via the portal vein, with or without irradiation as cytoablation, followed by intramuscular administration of FK506 for 5 days. At 1–2 months later, livers were harvested and transplanted into naive female LEW rats. No immunosuppressants were used. Chimerism in donor rats was confirmed by primers specific for the sex determinant Y chromosome of rats. With livers from rats pretreated with recipient-type BMCs, survival of liver allografts was significantly extended, irrespective of irradiation. These results showed that modification of the donor liver by intraportal injection of recipient-type BMCs and concomitant administration of FK506 prior to liver transplantation prolonged liver allograft survival in rats.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells: Possible implications for intrauterine bone marrow transplantation across major histocompatibility barriers

Abstract. Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Delayed administration of FK 506 is sufficient to suppress acute rejection changes after aortal transplantation in rats

Arterial allografts are used now-a-days as a modality in the treatment of vascular prosthesis infections. Prolonged administration of immunosuppressive drugs seemed to be essential for long-time patency rates of alloarterial vascular reconstructions. Nevertheless, the use of immunosuppressives if there exist an acute infection is controversial. The experimental work described herein studied effects of a delayed low-dose FK 506 administration on the development of acute rejection changes 30 days after aortal transplantation in rats. The response of the recipient's immune system to aortal wall antigens of the donor in the field of no immunosuppression resulted in an intimal proliferation and its infiltration by immunocompetent cells of the recipient, necrosis of medial smooth muscle cells, including deposition of immunoglobulins, and a massive adventitial infiltration of CD4 and CD8 positive cells. On the other hand, all the principal histological signs of rejection listed above were suppressed by FK 506 administration, no matter whether the immunosuppressive was administered on day 0 or day 7 after the transplantation.     

Bone mass and mineral metabolism in liver transplant patients treated with FK506 or cyclosporine A

The purpose of this study was to compare the effects of Cyclosporine A (CyA) and FK506 on bone mass and mineral metabolism in liver transplantation (LT) patients. A prospective study was performed on 18 male patients who underwent LT treated with CyA, and 7 LT patients who received FK506. Bone mineral density (BMD) of the lumbar spine and proximal femur (DPX-L) was measured before and at 6, 12, and 24 months after transplantation. Moreover, intact parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) levels were determined at the same time. The cumulative dose of glucocorticoids was calculated in all patients. At 6 months, lumbar BMD decreased 5.2 ± 1.2 % (P=0.0005) and 2.9 ± 2.1 % (p=ns) in CyA and FK506 groups, respectively. Lumbar BMD reached baseline values at 1 year in the FK506 group and 2 years after LT in the CyA group. Moreover, significant intergroup differences in femoral neck BMD changes after 2 years of transplant were observed (CyA: −5.2 ± 1.97 versus FK506: +1.55 ± 2.2 %;P=0.039). In the first year posttransplant both groups showed a marked increase in PTH and 25OHD levels. The mean cumulative dose of glucocorticoids was higher in the CyA group (CyA group 11.06 ± 0.46 g versus FK 506 group 6.71 ± 0.42 g;P<0.001), and multiple linear regression analysis showed a negative correlation between BMD changes at the lumbar spine and mean cumulative dose of glucocorticoids (P=0.022). In conclusion, our data suggest that after liver transplantation treatment with FK506 shows a more favorable long-term effect on bone mass evolution than CyA therapy. These differences seem to be associated with the lower dose of glucocorticoids used in the FK506 group.     

Repetitive DNA polymorphisms in following chimerism after allogeneic bone marrow transplantation

Information about the chimeric status of patients is of great importance in comparison of different conditioning and prophylactic regimens as well as for the post-bone marrow transplantation (BMT) therapies. In some cases, mixed chimerism (MC) can also be predictive of relapse. Analysis of the short tandem repeats (STR) loci by polymerase chain reaction (PCR) is a choice method for this purpose. In this study, we monitored 15 patients after BMT. Twelve of them underwent classical-conditioning regimen while the remaining three patients were subjected to non-myeloablative conditioning (minitransplantation). Evaluation of chimerism was performed using five STR and one variable number of tandem repeats (VNTR) locus. Four additional loci were PCR-amplified in cases of minitransplantation. Samples were analyzed by electrophoresis in an ALFexpress sequencer. MC was detected in seven cases of which it was predictive of relapse for two patients, who suffered from acute lymphocytic leukemia (ALL). The PCR-STR method proved to be a fast and relatively simple method, while the tested STR loci showed a high level of informativeness.     

FK506和抗淋巴细胞血清诱导兔异体膝关节移植耐受的实验研究

目的　探讨短期使用FK5 0 6和抗淋巴细胞血清 (ALS)诱导兔同种异体膝关节移植嵌合耐受的作用。　方法　雄性大耳白兔作为供体 ,雌性新西兰白兔作为受体 ,施行同种异体异位膝关节移植 ,设单独使用ALS( 1ml/kg)组、单独使用FK5 0 6( 0 5mg/kg)组、联合使用FK5 0 6和ALS组及对照组。FK5 0 6和ALS在移植术前 1d开始使用 ,ALS连续使用 3d ,FK5 0 6连续使用 15d。观察移植物的存活时间 ,检测受体内供体血细胞嵌合率 ,进行组织切片检查。　结果　对照组平均存活 ( 13 6± 0 8)d ,单独使用ALS组为 ( 17 4± 1 8)d ,单独使用FK5 0 6组为 ( 2 3 8± 1 5 )d ,联合使用FK5 0 6和ALS组为 ( 4 0 4±2 9)d。联合使用FK5 0 6和ALS组的供体血细胞嵌合率停药后 3周稳定在 10 %以上。　结论　短期使用FK5 0 6和ALS可显著延长兔同种异体膝关节移植的存活时间 ,可维持 3周的嵌合耐受。     

The superior effect of the combination of FK 506 and deoxyspergualin on rat cardiac allograft survival

In this present study, the effects of FK 506 and 15-deoxyspergualin (DSG), with respect to dose, timing, and combination, were investigated in an ACI-to-LEW rat cardiac allograft model. FK 506 was adminstered intramuscularly for 14 days starting on day 0 after grafting, while DSG was given intraperitoneally for 7 days starting on day 0,4, or 7 after transplantation. FK 506 or DSG monotherapy prolonged cardiac allograft survival in dose-dependent manners, and the minimum effective dose for overcoming rejection was 0.1 mg/kg per day in the case of FK 506 and 1.0 mg/kg per day for DSG. The graft survival rate was higher with administration of DSG starting on day 4 on day 0 after transplantation. A low dosage of FK 506 strating on day 0, in combination with DSG starting on day 0 or day 4 (but not on day 7), had a synergistic effect in prolonging allograft survival for 14.0±3.3 days and 25.4±8.2 days, respectively. The most effective combination treatment schedule for prolongation of allograft survival was FK 506 starting on day 0 and DSG starting on day 4 after transplantation.     

口服他克莫司血药浓度-时间曲线下面积

目的　探讨口服他克莫司 (Tacrolimus ,FK5 0 6 )的药代动力学规律 ,寻求临床上准确反映早期药物浓度时间曲线下面积 (AUC)的监测方法。　方法　 16例肾移植受者首剂口服 0 .0 75mg/kgFK5 0 6后 ,采用ELISA法测定服药后 0 .5、1.0、1.5、2 .0、3.0、5 .0、8.0、12 .0h时间点血药浓度 ,采用 3p87药代动力学计算程序将测得的FK5 0 6浓度自动拟合计算出AUC ,并分别将各时间点血药浓度与AUC进行相关性检验 ,计算相关系数。　结果　AUC变化范围为 4 4 .4 0～ 15 8.0 1μg·h-1·L-1,平均 ( 92 .2 3± 34.97) μg·h-1·L-1,个体间AUC可相差 4倍 ;血药谷浓度Cmin与AUC之间的相关性有显著差异 (P <0 .0 0 1,rmin=0 .6 5 0 )。　结论　首剂口服同一剂量FK5 0 6后 ,个体间药物浓度时间曲线下面积差异很大。Cmin能准确反映首剂口服FK5 0 6后的AUC ,临床上可通过监测Cmin达到FK5 0 6早期剂量的个体化     

FK506和供体特异性输血在大鼠异位心脏移植中的作用

目的探讨FK506和供体特异性输血在大鼠异位心脏移植中的作用。方法利用大鼠异位心脏移植模型以了解在移植当天或移植术后第4日进行供者特异性输注（DST）的基础上,应用FK506能否延长移植物的存活。结果在移植当天行DST或单独用FK5061mg／kg连续10天,可将移植心中位存活时间从对照组的5天分别有效延长至7天和11天,而FK506与DST联合应用时并不产生增强效应。结论FK506和DST单独应用时虽均能延长大鼠同种移植心存活,但是它们没有协同作用。     

TLSFJM联合小剂量FK506促进大鼠小肠移植耐受

目的 探讨TLSFJM(JM急性T淋巴细胞白血病细胞分泌的抑制因子)作为抑制因子对小肠移植急性排斥反应的抑制效应及其机理,并与FK506的抑制效应特点和副作用进行比较分析.方法 雄性BN和LEW大鼠分别作为供、受体行小肠移植,共分5组 小肠移植组(SBT组)、大剂量FK506 [0.5 mg/(kg·d)]组、小剂量FK506 [0.25 mg/(kg·d)]组、TLSFJM [10 U/(kg·d)]组和FK506 [0.25 mg/(kg·d)] TLSFJM [10 U/(kg·d)]组.FK506和TLSFJM分别以肌肉或腹腔注射方式给药.在不同时间段分别观察各组动物体重、生存时间及肝、肾功能,组织病理学检查排斥反应发生情况.结果 TLSFJM应用7 d,对移植大鼠肝、肾功能无损害.TLSFJM 小剂量FK506不但避免了大剂量FK506对肝功能的损害,而且显著延长了受体的生存时间.但单独应用TLSFJM仍有一定程度排斥反应出现,不能明显延长受体的生存时间.结论 TLSFJM作为一种有效的移植免疫抑制因子,联合小剂量FK506应用能延长宿主和移植肠的生存时间,延缓排斥反应的发生,减轻排斥反应的强度,避免大剂量FK506治疗带来的并发症.TLSFJM有望成为一种新型、高效、低毒的免疫抑制剂.