STR基因位点检查在异基因造血干细胞移植中的应用

目的 探讨短串联重复序列(STR)基因位点检查在异基因造血干细胞移植中的应用。方法 采用PCR方法对4例异基因造血干细胞移植、1例非清髓造血干细胞移植的供者和受者移植前、后STR基因进行检测，了解造血干细胞植入情况。结果 4例异基因造血干细胞移植的受者移植后STR基因型与受者移植前STR基因型不同，与供者STR基因型完全相同，提示供者造血干细胞的植入；1例非清髓造血干细胞移植的受者移植后STR基因型表现为患者移植前STR基因型和供者STR基因型的嵌合状态。结论 STR基因位点检查可以用来判断异基因造血干细胞移植的植入情况。     

异基因骨髓移植和非清髓性干细胞移植嵌合形成过程的比较

目的 研究异基因骨髓移植（allo-BMT）和非清髓性干细胞移植（NST）两种移植方式在供体细胞嵌合状态的形成及转归上的差异,探讨早期供体细胞植入的关键因素。方法 对20例接受allo-BMT和18例NST的患者进行回顾性比较,研究两组患者疾病类型、干细胞来源、预处理方案和移植物抗宿主病预防方案。用复合扩增荧光标记STR-PCR结合毛细管电泳方法对移植后＋7、＋14、＋21d,＋1、＋3、＋6、＋9、＋12个月的嵌合体进行动态检测。结果 （1）NST组在受体年龄、单个核细胞（MNC）、CD34^＋及T细胞数量上均明显高于BMT组,造血重建方面,中性粒细胞绝对值恢复时间与BMT组无差别,但血小板恢复明显早于BMT组。（2）NST组患者供体细胞完全嵌合状态（FDC）的建立比BMT组早（1个月vs 3个月）,移植后早期（＋1个月）FDC比例亦明显高于BMT组（38．9％vs 20%）,而混合嵌合状态（MC）的发生率明显低于BMT组（61．2％ vs 80％）,移植1个月后各时间段两组在嵌合体形成上均无显著性差别。（3）氟达拉滨为基础的NST预处理方案与标准预处理方案相比并未延迟供体细胞的植入。（4）NST组慢性移植物抗宿主病的发生率明显高于BMT组（80％vs 50%,P〈0．01）,与NST组输入高剂量的CD34^＋细胞相关。结论 在供体细胞早期植入和嵌合体形成的过程中,移植物中造血干细胞和T细胞数量至关重要,并可能起决定性作用。     

OTI-010 Osiris Therapeutics/JCR Pharmaceuticals

Osiris Therapeutics is developing the donor-derived mesenchymal stem cell (MSC) therapy OTI-010, which repopulates the bone marrow stroma and thus supports engraftment of hematopoietic stem cells from the same donor. This stem cell therapy, which has been awarded Orphan Drug status, is currently in development for the potential enhancement of bone marrow transplants in cancer patients, for the prevention of graft versus host disease (GVHD), and for the treatment of Crohn's disease. Japanese licensee JCR Pharmaceuticals is investigating the therapy for the potential treatment of GVHD in patients undergoing bone marrow transplantation to treat leukemia. Phase II clinical trials in acute gastrointestinal GVHD and in adult and pediatric patients with treatment-refractory severe GVHD are currently underway.     

用PCR-VNTR法检测CML异基因外周血干细胞移植后动态嵌合状态的作用

目的明确移植后造血细胞来源及微小残留病,为临床治疗提供依据。方法对18例异基因外周血干细胞移植者,在移植后不同时间检测人类两个高度可变的串联重复序列(Viariable number tandem repeated sequences,VNTR),动态观察分子嵌合状态,并监测染色体核型及标志基因。结果不同引物供受者的检出率有明显差异。单用马利兰(Bu)预处理的混合嵌合(Mix chimerism,MC)的发生率在移植后+14天较Bu加环磷酰胺(CY)高预处理的。移植后6个月持续MC者复发率可达20%。1例完全嵌合(Complete chimerism,CC)患者移植后6个月重新出现受者带型,后复发。在指导临床治疗方面,VNTR优于染色体核型及标志基因。13例持续MC者减量或停用免疫抑制剂,11例(84%)转为CC。结论应用PCR-VNTR动态检测移植后嵌合状态,可以评估预处理方案、预测高危复发并指导移植后治疗。     

The use of growth factors in hematopoietic stem cell transplantation

Mobilized, peripheral blood stem cells (PBSC) are increasingly used for both autologous and allogeneic transplants. Granulocyte-colony-stimulating factor is the most widely used cytokine for mobilization. Several different mechanisms of stem cell mobilization have been proposed including protease-dependent and non-protease- dependent mechanisms. In autologous transplants, the addition of chemotherapy to mobilization can enhance the yield of PBSC collected but with substantial adverse effects, and not necessarily faster engraftment. In allogeneic transplants, the use of mobilized PBSC is associated with faster engraftment and donor chimerism compared to bone marrow. In the majority of studies, the rate of acute graft-versus-host disease (GVHD) has not been shown to be significantly higher with PBSC, but the rate of chronic GVHD appears to be increased. Several different strategies have been proposed for patients and donors who fail initial mobilization, including the use of novel agents. AMD3100 (Plerixafor) works by directly inhibiting the interaction between stromal cell-derived factor-1 and its receptor CXCR4, and mobilizes hematopoietic stem cells within hours. It is being studied alone or in conjunction with growth factors for PBSC mobilization in both autologous and allogeneic settings. Although the use of growth factors after PBSC transplantation results in faster neutrophil engraftment its impact on treatment-related mortality and survival does not appear significant. Here, we review the biology and methods of PBSC mobilization, the effect of growth factors on normal donors and the controversies of growth factor use in the post-transplant setting. We also review the data on novel agents for mobilization of stem cells.     

Induction of tolerance in organ recipients by hematopoietic stem cell transplantation

The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+lin-hematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism.     

Xenotransplantation: application of disease resistance

1. Organ transplantation is now clinically routine for patients with end-stage organ failure. One major limitation in transplantation is chronic rejection involving the loss of the graft despite the use of immunosuppressive agents. Haematopoietic stem cell (HSC) chimerism, achieved through bone marrow transplantation (BMT), induces donor-specific tolerance to transplanted organs and prevents chronic rejection. 2. A second major limitation to organ transplantation is the donor shortage. Xenotransplantation, the transplantation of organs between different species, would have the ability to increase the availability of donor organs. 3. Current immunosuppressive therapies do not prevent the rejection of xenografts. Therefore, the only reliable method for achieving donor-specific tolerance to xenografts may require HSC chimerism. 4. In order to justify the use of BMT to induce transplantation tolerance in patients with non-life-threatening diseases, the morbidity and mortality associated with current conditioning regimens must be addressed. 5. The use of partial conditioning regimens to promote engraftment of xenogeneic HSC and the development of donor-specific tolerance may eventually make xenotransplantation in humans a clinical reality. 6. Additional advantages of xenotransplantation are the ability to genetically engineer the donor xenograft and resistance of some xenografts to infection by human viruses because of the species specificity of most viruses. 7. The clinical application of disease resistance for HIV and hepatitis B virus is the focus of the present review.     

Allogeneic bone marrow transplantation: procedures and complications

Bone marrow transplantation (BMT) is discussed in terms of immunology, procedures, and complications and their treatment. Any patient with a disorder of the hematopoietic or immune system or a disease in which a transferable hematopoietic cell can supply a missing enzyme is a candidate for BMT. A priority in allogeneic BMT is the identification of a compatible donor through matching of human lymphocyte antigens (HLAs). The greater the disparity in HLAs, the greater the chance of rejection. The ideal donor is a monozygotic twin or an HLA-matched sibling, but only 30% of patients have such a donor. Before receiving the bone marrow infusion, patients must be conditioned to create space in the marrow for donor cells, suppress the immune system, and eradicate any tumor in patients with malignancies. Conditioning is achieved by the combination of total body irradiation and cyclophosphamide treatment; busulfan, etoposide, and cytarabine have also been used. For patients given unmanipulated marrow, the number of nucleated cells infused is about 3 X 10(8) per kilogram. Signs of engraftment are usually seen 14-21 days later. Toxic effects related to conditioning appear during this period and include infection, gastroenteritis, mucositis, and congestive heart failure. The most serious complication is graft-versus-host disease (GVHD), which can affect multiple organ systems. Prednisone, methylprednisolone, methotrexate, antithymocyte globulin, and cyclosporine have been used in an effort to prevent or treat GVHD. Bone marrow transplantation offers the chance of long-term survival to many patients with terminal disease, but associated morbidity and mortality rates remain high. Research is needed to address the problems of infection, leukemic relapse, and GVHD and the difficulty in obtaining and matching donors.     

荧光标记STR-PCR技术用于外周血干细胞移植监测

目的建立骨髓移植后监测的荧光标记STR-PCR检测方法,为白血病患者骨髓移植疗效评价提供可靠依据。方法建立荧光标记STR-PCR检测方法。于移植前、移植后7d至3个月不同时间采集供、受者血液提取DNA利用荧光标记STR-PCR方法进行分析。结果所检测的受者和供者均具有不同的DNA分型图。例1为HLA半相合母子,移植后监测结果仍为受者基因型,例2、3为HLA全相合的同胞,监测结果为完全供者基因型。结论荧光标记STR-PCR技术是一种准确、可靠的骨髓移植后监测方法。     

Post-transplant lymphoproliferative disorder in children: incidence, prognosis, and treatment options

Post-transplant lymphoproliferative disorder (PTLD) after solid organ or hematopoietic stem cell transplantation in children is a serious complication that has been responsible for high mortality rates over recent years. PTLDs are part of a clinically and histologically heterogeneous group of B-lymphocyte proliferations mostly induced by Epstein-Barr virus (EBV) in a context of immunosuppression. Major risk factors for PTLDs in solid organ transplantation are the EBV serostatus mismatch and the intensity, duration, and type of immunosuppression. T-cell depletion and the HLA-mismatched donor and recipient are the main risk factors following hematopoietic stem cell transplantation. For a long time, the only safe and effective therapeutic approach to PTLD was reduction of immunosuppression, with a risk of graft rejection. Based on a better knowledge of the pathophysiology and risk factors for PTLD, preventive and pre-emptive strategies have been recently proposed to control PTLD. New treatment modalities, such as anti-B-cell antibodies, cytokine inhibitor therapy, or anti-EBV cytotoxic T lymphocytes are promising and may improve the outcome of PTLD. These therapeutic approaches need to be further evaluated, especially in the context of pre-emptive strategies adapted to predictive markers of EBV-induced PTLD.     

ABO血型检测在异基因造血干细胞中的应用及移植后的输血

目的监测ABO血型不合的患者异基因造血干细胞移植前后ABO血型的变化,探索移植后的输血方案,为患者输注合适的血液成分提供依据。方法干细胞移植前检测患者ABO血型,植活后再次检测患者ABO血型抗原及抗体的变化,对输注血液成分的种类和数量进行统计学分析。结果 28例ABO血型不合的患者干细胞移植后35~193 d血型成功转变为供者血型,ABO主要不合、ABO次要不合及ABO主次要不合的受者输注红细胞、血浆和血小板的量和ABO相合组的输注量相比无统计学差异(P<0.05)。结论 ABO血型不合的干细胞移植后,患者血型成功转变为供者血型,相容性输血可以用于异基因造血干细胞移植并能够确保输血安全。     

Exposure of hematopoietic stem cells to ethylene oxide during processing represents a potential carcinogenic risk for transplant recipients

Stem cells for transplantation are obtained from bone marrow, umbilical cord blood, and peripheral blood. A rare complication of hematopoietic stem cell transplantation is donor cell-derived leukemia (DCL). The donors remain cancer free and the causes of these DCL are unknown. Stem cells must repopulate the bone marrow and then give rise to all hematopoietic cells for the rest of the transplant recipient's life. No procedure is acceptable that might introduce precancerous or cancerous mutations in cells performing such a critical function. Medical disposable sets consisting of bags, tubing sets and freezing containers are used to collect, purify and store stem cells. Sterilization of disposables with ethylene oxide is widespread, even though those sets unavoidably retain residual amounts of ethylene oxide which is a potent, direct-acting mutagen and clastogen that has been demonstrated to induce hematopoietic cancer in mice, rats and human beings. Potential exposure levels to ethylene oxide during processing under proposed US FDA guidelines for residual ethylene oxide would be biologically active and present a significant risk factor for DCL. For direct-acting mutagens, there is no recognized "no effect" dose using currently accepted cancer risk assessment models. The safety concerns with ethylene oxide can be eliminated by the use of alternative technologies including electron beam, gamma irradiation, or steam for the sterilization of all products used for stem cell processing and storage.     

异基因造血干细胞移植治疗儿童白血病疗效观察

目的观察异基因造血干细胞移植治疗儿童白血病的疗效。方法在3例行异基因造血干细胞移植的白血病患儿中,2例行无血缘相关人类白细胞相关抗原（HLA）不全相合脐血造血干细胞移植,1例行同胞HLA全相合骨髓造血干细胞与外周血造血干细胞联合移植。移植后进行对症治疗及相关并发症的预防。结果 3例患儿均获造血重建,其中病例1在＋37d全血恢复正常;病例2在＋90d外周血供体嵌合率（STR）97.8%,脐血造血干细胞完全稳定植入。病例3在＋80d外周血STR：99.4%,造血干细胞完全稳定植入。结论异基因造血干细胞移植是一种治疗儿童白血病的较好方法,可以提高白血病患儿的长期生存率。     

Induction of transplantation tolerance via regulatory T cells

In the last two decades, graft survival has been greatly improved by the introduction of efficient immunosuppressive drugs. On the other hand, late graft loss caused by chronic rejection together with the side effects of long-term immunosuppression, remain major obstacles for successful transplantation. Operational tolerance, which is defined by the lack of acute and chronic rejection and indefinite graft survival with normal graft function in the absence of chronic immunosuppression, represents an attractive alternative. Several approaches have been explored to achieve transplantational tolerance, which is considered the "Holy Grail" of transplantation, including induction of central tolerance by establishing mixed chimerism through hematopoietic stem cell transplantation or induction of peripheral tolerance through modulation of allogeneic immune responses. Graft-specific alloreactive T cells, which largely mediate graft rejection, can be silenced through different mechanisms, including deletion, which may occur within the thymus or in the lymphoid organs; anergy, in which alloreactive T cells cannot adequately respond following restimulation with the specific antigen; and suppression, which may be mediated by direct interactions with regulatory T cells (Tregs) or by soluble factors produced by Tregs. This review attempts to summarize the most novel and successful strategies to achieve operational tolerance via induction of Tregs.     

Prevention of acute graft-versus-host-disease by Withaferin a via suppression of AKT/mTOR pathway

Acute Graft versus Host Disease (aGVHD) is a frequent and serious complication in patients receiving allogeneic bone marrow transplantation (allo-BMT) and often requires rigorous prophylaxis. The current treatment regimens for aGVHD are associated with several side effects which necessitates the development of novel interventions that prevent aGVHD without precluding graft-versus-tumor effects. In the present study, we show that treatment of donor graft with plant steroidal lactone Withaferin A (WA) prior to transplantation markedly reduced aGVHD mediated damage in target organs without compromising the graft-versus.-tumor activity of the transplanted lymphocytes. WA abrogated post-transplant cytokine storm associated with allo-activation of donor lymphocytes. This was attributed to the ability of WA to inhibit early signaling events in T-cell activation including lymphoblast formation and activation of AKT/mTOR pathway. Mortality and morbidity related to allo-transplantation was significantly reduced in recipients of WA treated donor splenocytes compared to recipient of vehicle treated donor splenocytes. Further, WA treatment did not have any effect on reconstitution of lymphoid and myeloid lineages in recipients, resulting in stable and complete donor chimerism. In agreement with previous reports showing the effectiveness of WA in a mouse model of partial chimerism, our data further establishes that WA is able to attenuate aGVHD in an MHC-mismatched high dose chemo-conditioned murine model without compromising engraftment. This study provides compelling scientific basis for possible application of WA for prevention and treatment of aGVHD in patients receiving allo-BMT.     

Incidence of viral and fungal complications after utilization of alternative donor sources in hematopoietic cell transplantation

Allogeneic hematopoietic cell transplantation (HCT) remains the only curable option for adult patients with hematologic malignancies. According to guidelines published by the American Society for Transplantation and Cellular Therapy, allogeneic HCT should be offered to all intermediate- and high-risk patients with acute leukemia. While matched-related donor (MRD) grafts continue to be the preferred stem cell source for allogeneic HCT, studies comparing MRD grafts to matched-unrelated donor (MUD) grafts showed comparable outcomes in patients with acute leukemia. Unfortunately, for those without a suitable matched-related graft, the probability of finding a suitable matched-unrelated donor varies significantly depending on racial and ethnic background. With allogeneic HCT procedures increasing year after year due to the increased availability of suitable donors, each of these alternative donor sources merits special clinical considerations, specifically with regard to infections. Infections remain a significant cause of morbidity and mortality after allogeneic transplant, especially in those receiving alternative donor grafts. Due to the high-risk nature associated with these donor grafts, it is important to understand the true risk of developing infectious complications. While there are a multitude of infections that have been described in patients post-allogeneic HCT, this review seeks to focus on the incidence of cytomegalovirus (CMV) and invasive fungal infections (IFI) in adult patients receiving alternative donor source transplantation for hematologic malignancies.     

Negative inotropic effect of selective AT2 receptor stimulation and its modulation by the endocardial endothelium

Kidney transplantation represents the therapy of choice for many patients with end-stage renal disease. However, the success of renal engraftment is hindered by a number of factors, the most important of which being adverse effects of systemic immunosuppressive therapy, chronic transplant dysfunction and a severe shortage of donor kidneys. Gene therapy approaches may provide valuable strategies in each of these areas. First, gene therapy holds the potential of local therapy, thus circumventing systemic side effects of chronic immunosuppression. Second, chronic transplant dysfunction may be addressed by innovative strategies to induce local immune tolerance, immune suppression and additional graft protecting mechanisms. Third, gene therapy may be instrumental in increasing the quality of the grafts by limiting ischemia-reperfusion injury, especially in non-heart beating donors, thereby expanding the donor pool. In this article, we give an overview of the current state of gene therapy in experimental models of kidney transplantation.     

Current and future approaches to treat graft failure after allogeneic hematopoietic stem cell transplantation

Introduction: One significant obstacle to the success of allogeneic hematopoietic stem cell transplantation (HSCT) is represented by graft failure, defined as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Graft failure mediated by host immune cells attacking donor stem cells is named graft rejection. Factors associated with graft failure include HLA disparity in the donor/recipient pair, underlying disease, viral infections, type of conditioning regimen and stem cell source employed.

Areas covered: In this article, the experts summarize current approaches to treat graft failure/rejection after HSCT, and they discuss new strategies of graft manipulation and immune therapy of particular interest for preventing/treating this complication.

Expert opinion: A limited array of options is available to treat graft failure. The experts believe that re-transplantation from another donor or the same donor (if there is no evidence of immunologically mediated graft failure) is the treatment of choice for patients with primary graft failure or acute graft rejection. The experts think that strategies based on innovative approaches of graft manipulation, new agents or cellular therapies could render in the future graft failure a much less relevant problem for HSCT recipients.     

Embryonic stem cell-derived hematopoietic stem cells: challenges in development, differentiation, and immunogenicity

Embryonic stem cells (ESC) can potentially be manipulated in vitro to differentiate into cells and tissues of all three germ layers. This pluripotent feature is being exploited to use ESC-derived tissues as therapies for degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, inefficient production of ESC-derived tissues before transplantation, inability of ESC-derived tissues to integrate well into the adult microenvironments due to developmental stage incompatibility, or active immune rejection of the ESC-derived graft are all potential challenges to successful ESC-derived therapies. One way to induce immunological tolerance to allogeneic tissues is via the establishment of mixed hematopoietic chimerism in which the host and donor cells are educated to recognize each other as "self". Proof of principle that in vitro cultured ESC-derived hematopoietic progenitors can be transplanted and induce immunological tolerance to allogeneic tissues exists in mouse models. In this review, we discuss the challenges to in vitro development of a bona fide ESC-derived hematopoietic stem cell and their differentiation fate in vivo, and provide suggestions to predict the immunogenicity of specific ESC-derived hematopoietic populations before transplantation that could be used to prevent their rejection after transplantation into an adult host.