Graft vs. host disease after liver transplantation: a new approach is needed.

Graft-vs.-host disease (GVHD) is a rare, serious complication of orthotopic liver transplantation (OLT). We have treated 5 patients to date with GVHD after OLT. A total of 78 patients worldwide have been reported to have experienced this complication. The means by which GVHD after OLT has been managed is guided by experience with the more common GVHD that occurs after stem cell transplantation. However, despite the use of various treatment modalities, the mortality of GVHD after OLT remains high. This case series and review of the literature demonstrates that successful resolution of GVHD after OLT cannot be expected with the use of those modalities that have been tried to date. It is imperative that new treatments be applied to GVHD after OLT in order to improve the prognosis of patients with this diagnosis.     

肝移植后发生移植物抗宿主病受者的骨髓象特征

目的 探讨原位肝移植术后发生移植物抗宿主病(GVHD)受者的骨髓象特征.方法 回顾性分析3例原位肝移植术后发生GVHD受者的临床资料.对3例受者的骨髓细胞学、血常规及血液生化等进行了检查,并对受者骨髓象及造血系统功能的改变进行了分析;采用聚合酶链(PCR)技术检测供、受者16个位点的短串联重复序列(STR),对1例受者的嵌合体状况进行了分析.结果 3例受者均出现发热、皮疹、腹泻等典型的GVHD临床表现;全血细胞进行性减少,骨髓增生低下,造血功能发生严重衰竭;血常规及血液生化检查显示,受者的白细胞、血小板和网织红细胞等大量减少;1例受者在移植术后第35天被证实存在供、受者问的D8S1179、D21S11两位点的嵌合体.最终1例受者经及时治疗后恢复造血功能,另2例受者死亡.结论 肝移植术后发生移植物抗宿主病可诱导受者骨髓造血功能衰竭.     

Role of donor leukocyte chimerism in establishing the etiology of neutropenia after liver transplantation.

BACKGROUND: The quantitation of donor leukocyte chimerism may aid in establishing the etiology of neutropenia after liver transplantation. METHODS: The incidence and clinical and laboratory characteristics of severe neutropenia were studied in adults who have undergone liver transplantation at our institution over the last 4 years. RESULTS: Severe neutropenia developed in 5 of 156 patients (3%). The clinical and pathological features were nonspecific. In two patients with a delayed diagnosis of graft-versus-host disease (GVHD), donor leukocytes comprised > or = 50% of the circulating peripheral blood mononuclear cells. In a third patient, an earlier diagnosis of GVHD was suspected on the basis of a donor leukocyte count of 3-10% in the peripheral blood. In contrast, donor leukocyte chimerism was < or = 0.01% in two patients with probable drug-induced neutropenia CONCLUSIONS: The determination of donor leukocyte chimerism has an important role in the investigation of neutropenia after liver transplantation, allowing early diagnosis and treatment of GVHD.     

非清髓性造血干细胞移植后移植物抗宿主病的临床观察

目的 观察非清髓性造血干细胞移植（NST）后移植物抗宿主病（GVHD）的发生情况。方法 将18例患者分为3组：A组为6例重型再生障碍性贫血（SAA）成人患者，行无关供者脐血造血干细胞移植；B组为5例SAA患者，行同胞供者骨髓联合外周血造血干细胞移植；C组为7例肿瘤性血液病患者，其中3例行同胞供者骨髓移植，4例行外周血造血干细胞移植。均采用以抗胸腺细胞球蛋白或抗淋巴细胞球蛋白为基础的预处理方案。A组和B组应用环孢素A（CsA）和甲泼尼龙预防GVHD，C组应用CsA和甲氨蝶呤预防GVHD。C组形成混合性嵌合体后行供者淋巴细胞输注（DLI）。结果 A组有4例形成并维持混合性嵌合体状态，1例死于真菌性败血症，1例自动出院。移植后早期，B组有3例供者型嵌合体占94％以上，并在短期内转变并维持完全供者嵌合体状态，获得无病存活，其中1例在移植后8个月发生慢性GVHD；另2例行供者千细胞输注后，1例6个月后死于继发性纵隔淋巴瘤，1例造血功能恢复。C组患者早期均形成混合性嵌合体，获得血液学部分缓解，患者DLI前无急性GVHD发生，1例于2次DLI后死于严重感染，1例失访；另5例分别经过4、3、7、5、4次DLI，全部转为完全供者型嵌合体，并获得血液学完全缓解，4例并发慢性GVHD，2例并发急性GVHD。结论 对于SAA患者，NST的临床效果较好，GVHD的发生率较低；而对于肿瘤性血液病，NST后患者的早期死亡率低，急性GVHD发生率下降，但慢性GVHD和感染的发生率较高。     

Peripheral dendritic cell chimerism in allogeneic hematopoietic stem cell recipients

BACKGROUND: Relapse and graft-versus-host disease (GVHD) represent major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although leukocyte and T-cell chimerism analyses are performed routinely suggesting a predictive value on the patients outcome, little is known about chimerism of dendritic cells (DC) representing strong initiators of immune responses. METHODS: In this prospective study, peripheral DC1 (CD11c+) and DC2 (CD123+) chimerism was determined in hematopoetic stem cell recipients. DCs were isolated from peripheral blood by fluorescence activated cell sorting. Chimerism analyses were performed by fluorescent in situ hybridization or by polymerase chain reaction-based typing of short tandem repeats. RESULTS: At time of engraftment, DC chimerism analyses showed complete chimerism in 76.3% (DC1)/79.5% (DC2), mixed chimerism (MC) in 21.0% (DC1)/17.9% (DC2) and no chimerism in 2.7% (DC1)/2.6% (DC2) of the patients. Peripheral DC chimerism had no significant effect on relapse-free or overall survival. Although acute GVHD was observed more often in patients with MC for DC1/DC2 and chronic GVHD occurred more often in patients with MC for DC2, there was no statistically significant correlation. CONCLUSIONS: Although DCs as antigen presenting cells are supposed to have an impact on the induction of GVHD, there was no significant correlation between incidence of GVHD and DC chimerism after HSCT.     

Acute Graft Versus Host Disease Following Liver Transplantation: The Enemy Within

This article reviews acute graft vs. host disease (GVHD) as a complication of orthotopic liver transplantation (OLT). The incidence, presentation, clinical course and outcome of GVHD after OLT are summarized and the pathogenesis is discussed, drawing parallels with GVHD after allogeneic haematopoietic stem cell transplantation. Risk factors for GVHD after OLT are examined and the potential role of donor lymphocyte macrochimerism in the recipient peripheral blood as a diagnostic aid for GVHD is discussed. Finally, treatment of GVHD after OLT is reviewed with particular emphasis on the potential role of some of the newer biological agents.     

Acute graft-versus-host disease after liver transplantation: role of withdrawal of immunosuppression in therapeutic management.

Graft-versus-host disease (GVHD) after liver transplantation is rare but associated with a very high mortality (over 85%). Most treatments focus on increasing immunosuppression, addition of antibody preparations such as OKT3 and antithymocyte globulin to eliminate the donor lymphocytes, and supporting myelopoiesis by use of cytokines. However, the results are very poor. We reasoned that a better therapeutic approach would be to reduce the immunosuppression and allow the patient's immune system an opportunity to reject the allograft donor T cells. We tested this novel therapeutic approach in 3 patients diagnosed with GVHD. Two patients had rapid loss of donor T cell chimerism and resolution of their symptoms. The other patient continued to progress to severe GVHD and died. The patients who responded to withdrawal of immunosuppression had a later onset of symptoms and a lower level of donor CD3+ T cells at the start of treatment. We conclude that larger studies are needed to further evaluate these results and to determine what factors may affect the likelihood that a patient may respond to this approach.     

Graft Versus Host Disease in Intestinal Transplantation

Our aim was to analyze the clinical course and outcome of patients with graft vs. host disease (GVHD) after intestinal transplantation (ITx). All patients receiving ITx between May, 1990 and December, 2003 were retrospectively reviewed for evidence of GVHD. Two hundred and fifty patients underwent ITx during the study period. Graft vs. host disease was suspected clinically in 23 patients on the clinical basis of presentation such as skin rash, ulceration of oral mucosa, diarrhea, lymphadenopathy, or native liver dysfunction. Fourteen (eight children and six adults) patients (5.6% of total patient population) had GVHD confirmed by histopathological criteria including keratinocyte necrosis (n = 9), epithelial apoptosis of the native gastrointestinal tract (n = 4), and epithelial cell necrosis of oral mucosa (n = 1). Donor-cell tissue infiltration or extensive peripheral blood donor-cell chimerism was documented on seven occasions. The majority of cases of GVHD resolved with steroid administration and optimization of tacrolimus immunosuppression. The incidence of histologically proven GVHD after clinical intestinal transplantation is 6.5% (8/122) in children and 4.7% (6/128) in adults. Successful clinical management requires a high index of suspicion to minimize morbidity and mortality. Diagnostic and treatment strategies based on this experience are proposed.     

Graft-versus-host disease after liver transplantation: documentation by fluorescent in situ hybridisation and human leucocyte antigen typing

Graft-versus-host disease (GVHD) after liver transplantation is uncommon and the outcome is often fatal. A firm diagnosis of GVHD is difficult because the clinical triad of skin rash, marrow failure and diarrhoea can be indistinguishable from drug reaction or viral infection, and the presence of donor lymphocyte chimerism is not specific. We describe a case of severe GVHD in a female patient after liver transplantation from a male cadaveric donor. Skin biopsy showed characteristic changes of GVHD. Using Y-chromosome-specific fluorescent in situ hybridisation (FISH), male lymphocytes were demonstrated in 10% of marrow cells and in 90% of lymphocytes infiltrating the dermal epidermal junction. Donor human leucocyte antigens (HLAs) were detected in the peripheral blood, buccal mucosa and skin by polymerase chain reaction. The GVHD subsided with steroid and anti-thymocyte globulin, but recurred on tailing off of treatment. Despite maximum supportive therapy, including random donor leucocyte infusion, and marrow infusion from a HLA-identical sibling, the patient succumbed to sepsis. Our results showed the utility of combining morphological features with molecular techniques using FISH and HLA typing in confirming a diagnosis of GVHD.     

荧光原位杂交法检测造血干细胞移植后骨髓嵌合状态和微量残留病灶

目的 探讨荧光原位杂交（FISH）法在异基因造血干细胞移植（allo-HSCT）后检测供、受者骨髓嵌合状态和微量残留病灶（MRD）的作用。方法 2001年2月至2005年6月对83例患者进行allo-HSCT。对供者为异性的64例受者实施骨髓细胞性染色体着丝粒探针FISH法检测，评价供、受者骨髓嵌合状态及其动态改变；对供、受者性别相同而有特殊染色体异常的19例受者，应用相应的基因探针（BCR／ABL、AMIL1／ETO和MLL）对骨髓细胞行FISH法检测，评价微量残留病灶的发生。结果 64例异性allo-HSCT的受者中，50例供、受者骨髓嵌合度在99％以上；7例早期嵌合度偏低（96．2％～98．7％），在随访过程中逐渐上升至99％以上，移植后均未复发原病；另外7例嵌合度在随访过程中呈进行性下降，其中3例微量残留病灶在10％以上，均同时出现血液学复发；4例患者微量残留病灶在2％～5％之间，其中2例经快速停用免疫抑制剂后出现严重的移植物抗宿主病（GVHD）；1例在免疫抑制剂快速减量后骨髓嵌合度逐步上升至99．9％，目前仍处于完全缓解（CR）状态中；1例持续处于CR状态。19例供、受者性别相同的allo-HSCT受者中，16例移植后未检测到原来的异常核型；1例检测到10％的微量残留病灶，经免疫抑制剂减量4个月后降为1％，移植后1年仍在完全缓解中；2例分别在移植后1个月和4个月时出现原来的染色体异常。复查骨髓为原病复发，再次化疗未缓解。结论 应用FISH法检测allo-HSCT后骨髓嵌合状态和微量残留病灶，对判断植入、复发和指导早期干预性免疫治疗有重要意义。     

腹部器官簇移植治疗终末期肝病合并胰岛素依赖的2型糖尿病五例

目的 总结了改良的腹部器官簇移植(MCT)治疗终末期肝病合并胰岛素依赖的2型糖尿病(T2DM)患者的经验.方法 回顾性分析了单中心连续实施的5例MCT的资料.5例全部为男性,原发病为乙型肝炎后肝硬化合并T2DM 3例,乙型肝炎后肝硬化合并T2DM 1例,原发性肝癌(HCC)合并T2DM 1例.受者切除患肝后,原位植入包括肝脏、胰腺、部分十二指肠的器官簇,器官簇来源于同一个供者.胆道和胰腺外引流采用供者十二指肠与受者上段空肠Roux-en-Y吻合或者侧侧吻合方式.术后采用巴利昔单抗诱导,他克莫司(Tac)+吗替麦考酚酯(MMF)+皮质激素联合应用预防排斥反应,之后过渡至单用Tac维持治疗.结果 5例手术均顺利,患者于术后早期(8～27 d)完全脱离胰岛素治疗,并且血糖水平维持正常.其中3例术前C肽水平严重降低,术后早期明显升高,并维持于正常水平.1例术后发生移植物抗宿主病,并死亡,其他4例顺利恢复,术后分别随访22、15、5和4个月,均存活,并已恢复正常生活,移植物功能正常,血糖水平正常.结论 腹部器官簇移植是治疗终末期肝病合并胰岛素依赖的2型糖尿病的有效手段.

Abstract：

Objective Modified upper abdominal cluster transplantation (MCT), which was inspired by the classical cluster transplant technique, has been proven more effective and feasible in the treatment of patients with end stage liver diseases associated with insulin-dependent diabetes mellitus (DM) than orthotopic liver transplantation (OLT) alone. In this study, we summarized our experience with MCT in 5 consecutive patients suffering from end stage liver diseases associated with insulin-dependent type 2 DM in our single center.Methods Five patients with hepatitis B-related chronic liver cirrhosis and insulin dependent type 2 DM received MCT in our single center. The biliary and exocrine pancreatic drainage reconstructions were achieved by a Roux-en-Y duodenojejunostomy or a side-to-side duodenojejunostomy. A quadruple immunosuppressive regimen based on tacrolimus including Basiliximab induction, mycophenolate mofetil (MMF) and steroids was used in the early stage post-transplant, and then converted to tacrolimus monotherapy.Results All of the patients experienced an uneventful post-operative recovery. They were rendered independent from insulin therapy shortly after transplantation. The fasting glucose and glycosylated hemoglobin levels were within normal range. In addition, the fasting C-peptide value was increased from much lower than the normal range pre-transplant to within normal range post-transplant and maintained stable since then. However, the third patient suffered from graft verse host disease (GVHD) 20 days post-operatively and died from severe infection on the post-operative 47 days. The other 4 patients had returned to work and a normal lifestyle over 22, 15, 5 and 4 months of follow-up.Conclusion MCT is an effective method in treating patients suffering from end stage liver diseases combined with insulin-dependent type 2 DM. Whether a cluster graft would increase the risk of GVHD needs further investigation.     

Non-myeloablative allogeneic stem cell transplantation for metastatic renal cell carcinoma

Between 1999 and 2004, 11 patients with metastatic renal cell carcinoma (RCC) underwent non-myeloablative stem cell transplantation (NST) with conditioning using fludarabine-based regimens in two institutions of Korea. Among 11 patients, only one patient showed partial response (response rate: 9%), three showed stable disease, and six progressive disease. Three patients developed acute graft-versus-host disease (GVHD), and among them, one developed grade III acute GVHD which caused early death at day 60 after transplantation, and this patient showed partial response at day 30. Six patients developed chronic GVHD, three limited, and three extensive GVHD, respectively. Survival after one yr was 18% in transplanted patients. Median overall survival for entire cohort was 4.3 months. Eight patients died from progressive disease and three (27%) from treatment-related mortality. Only one patient survived 51.2 months after NST with slowly progressive disease. This patient received donor lymphocyte infusion three times after NST and achieved complete donor chimerism. NST does not lead to durable response and prolonged overall survival in the majority of patients with RCC in our series.     

Liver transplantation followed by adjuvant nonmyeloablative hemopoietic stem cell transplantation for advanced primary liver cancer in humans

BACKGROUND: Tumor recurrence after orthotopic liver transplantation (OLT) in patients with advanced primary liver cancer is common. To achieve an adjuvant graft-versus-tumor effect, the authors investigated whether transplantation of allogeneic peripheral blood stem cells (PSCT) after OLT can induce sustained complete donor chimerism. METHODS: Five patients with advanced primary liver cancer were included in the trial. None of the patients had signs of extrahepatic tumor before OLT. However, overall, the extent of surgery, as judged by morphologic examination of the explanted liver, was considered inadequate. A nonmyeloablative preparative regimen of fludarabine combined with total-body irradiation or cyclophosphamide preceded the allogeneic PSCT, which was then performed 16 to 135 days after OLT with human leukocyte antigen-matched donors. Mixed chimerism was monitored weekly by polymerase chain reaction of variable number tandem repeats after PSCT. RESULTS: In two patients, no engraftment of donor cells was seen, whereas one rejected the cells 2 months after PSCT. In two of the patients, a stable mixed donor chimerism was established. A mild transient graft-versus-host reaction was also noted in two patients. Three of the patients died of progressive disease 7 to 9 months after OLT. The other two are presently alive without recurrence at a follow-up of 26 and 10 months, respectively. CONCLUSIONS: These data suggest that PSCT after OLT is feasible, with low transplant-related morbidity. The rate of nonengraftment or rejection of the transplanted stem cells in this group of patients was three of five. An augmented pretreatment to prevent donor T-cell rejection seems to be necessary in this setting.     

Incidence of acute and chronic graft-versus-host disease and donor T-cell chimerism after small bowel or combined organ transplantation

Purpose

Graft-versus-host disease (GVHD) after organ transplantation is a rare but life-threatening complication with very high mortality.

Methods

A retrospective review was performed of all patients undergoing small bowel or combined organ transplantation at a single institution during 2003 to 2009. Patients with donor T-cell chimerism were analyzed in detail for development of GVHD.

Results

Thirty-two patients were included in the study. Of 32 patients, 11 (34%) had donor T-cell chimerism (range, 0%-53%) studies performed; 7 (64%) of those 11 patients demonstrated clinical features of GVHD. All patients who demonstrated GVHD had detectable donor T-cell chimerism. All patients with GVHD presented with skin involvement. Graft-versus-host disease responded to increased immune suppression therapy. Mortality was 43% (3/7) among patients with GVHD and was caused by multiorgan failure and sepsis in all cases.

Conclusion

Acute and chronic GVHD were observed frequently after combined solid organ transplantation and were associated with significant mortality and morbidity. Alloreactive donor T cells cotransplanted with the organ likely play a role in the pathophysiology because levels of donor-derived T-cell chimerism correlated with the clinical course of GVHD.     

非清髓性外周血干细胞移植后白血病复发的防治

目的探讨非清髓性造血干细胞移植后白血病复发的防治。方法7例合并有其它系统疾病的白血病患者接受非清髓性外周血干细胞移植,移植后采用环孢素A及短程甲氨蝶呤预防移植物抗宿主病(GVHD),造血细胞植入后,动态检测骨髓单个核细胞嵌合体的变化,根据嵌合体状态,采用供者淋巴细胞输注(DLI),并配合环孢素A用量的调整来防治白血病复发。结果7例患者移植后造血功能得到恢复,3例移植前合并的疾病有所加重,经处理缓解,移植早期相关并发症少而轻。移植后有3例嵌合体由混合嵌合体(MC)转为完全供者型嵌合体(CC),再转为MC,伴Ph染色体阳性,DLI5~6次后Ph染色体转为阴性,嵌合体状态又转为CC,其中2例发生广泛皮肤慢性GVHD;3例的嵌合体持续为CC,无复发,其中2例接受了1次DLI,此2例在环孢素A减量过程中发生GVHD;1例的嵌合体持续为MC,虽经3次DLI,但无效,患者死于白血病复发。结论非清髓性造血干细胞移植后应动态监测嵌合体状态,采用供者淋巴细胞输注,并配合环孢素A用量的调整,对白血病的复发有一定的效果。     

Abatacept Use in Graft-Versus-Host Disease After Orthotopic Liver Transplantation: A Case Report

Background

Graft-versus-host disease (GVHD) is a rare, serious, fatal disease that occurs after orthotopic liver transplantation (OLT).

Case Report

We treated a 60-year-old man who underwent OLT owing to familial amyloidosis. The patient developed fever on postoperative day 16. The fever was persistent and did not respond to antibiotic therapy. Cultures and radiologic studies were done and excluded infection as a potential cause. On postoperative day 26, a skin rash appeared on his chest, accompanied by diarrhea and persistent fever. The rash spread all over the trunk, neck, and arms, but spared the palms of his hands and soles of his feet. In the meantime, his blood cell count revealed pancytopenia. Skin biopsy was done and showed interface lymphocytic infiltrate that are largely centered on the dermal–epidermal junction, is consistent with GVHD (this pattern of rash distribution is unique and different from the rash of GVHD after hematopoietic stem cell transplant, which is confined to palms of the hands and soles of the feet; Fig 1). The diagnosis was confirmed by colonoscopy and multiple forceps biopsies, which revealed extensive crypt loss. After hematology consultation, the patient was treated by withdrawal of all immunosuppressive therapy coupled with abatacept infusion. Abatacept is a chimeric protein that inhibits T-lymphocytes and is approved by the US Food and Drug Administration for the treatment of rheumatoid arthritis. Interestingly, after second dose of abatacept the patient showed marked clinical and laboratory improvement. The patient was discharged after 47 days in a stable condition.

Conclusion

Because of the lack of a consensus for treatment of these patients, we report our experience with a male patient who had post-OLT GVHD and showed a marked improvement in response to abatacept.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.     

Nonmyeloablative haploidentical stem-cell transplantation using anti-CD2 monoclonal antibody (MEDI-507)-based conditioning for refractory hematologic malignancies

We initiated a clinical trial of nonmyeloablative haploidentical stem-cell transplantation (SCT) using MEDI-507, an immunoglobulin-G1 monoclonal anti-CD2 antibody. The trial was based on a preclinical major histocompatibility complex-mismatched bone marrow transplant model in which graft-versus-host disease (GVHD) was prevented and mixed chimerism as a platform for adoptive cellular immunotherapy was reliably induced. Twelve patients (three cohorts of four patients each) received cyclophosphamide, MEDI-507, and haploidentical unmanipulated bone marrow (n=8) or ex vivo T-cell-depleted peripheral blood stem cells (n=4) for chemorefractory hematologic malignancy. A two-dose regimen and schedule modifications of MEDI-507 were undertaken because of graft loss in the first cohort of four patients and GVHD in the second cohort. With ex vivo T-cell-depleted peripheral blood SCT, mixed chimerism occurred in all four patients without GVHD. Two patients, however, subsequently lost their grafts. Nonmyeloablative preparative therapy with MEDI-507 and haploidentical SCT have led to the reliable induction of at least transient mixed chimerism as a potential platform for adoptive cellular immunotherapy.     

The role of cyclophosphamide and granulocyte colony-stimulation factor in achieving high-level chimerism in allotransplanted limbs.

The establishment of a high-level of chimerism may be the most stable strategy for donor-specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Seventy-three whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP was injected at day 2, and G-CSF was given from day 0 to 3. Nontreated limb allografts were rejected after 4.2 days. In FK506-treated group for 28 days, the survival time was prolonged to 64 days. In the group treated with CYP/G-CSF, limb allografts were rejected after 5.4 days and 5 of 15 recipients showed acute lethal graft-versus-host disease (GVHD). Polymerase chain reaction (PCR) study showed a high level of chimerism even within 1 week after transplantation. Fourteen of 30 recipients given CYP/G-CSF/FK506 died within 2 weeks. The limb survival was significantly prolonged, however, with three grafts surviving more than 300 days. Seven recipients (24%) showed chronic GVHD. A high-level of chimerism was maintained when limb allografts were not rejected by recipients. Limb allografting could function as a vascularized carrier for bone marrow transplantation, provide a continuous source of donor cells and contribute to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolonged the survival of limb allografts but frequently caused chronic GVHD in the recipients.