Cytotoxic function of umbilical cord blood natural killer cells: relevance to adoptive immunotherapy

Decreased graft-versus-host disease (GVHD), ease of accessibility, and sustained engraftment encourage the use of umbilical cord blood (UCB) as an alternative source to bone marrow for immune reconstitution in children with leukemia. Natural killer (NK) cells rapidly expand after stem cell transplantation and are important for regulating GVHD and providing graft-versus-leukemia (GVL) effects. This review highlights the phenotypic and functional differences between UCB NK cells and adult peripheral blood (APB) NK cells, and discusses the possible therapeutic benefit of using UCB NK cells for adoptive immunotherapy in leukemia. Alloreactive NK cells show potent cytotoxic activities against human leukocyte antigen (HLA)-nonidentical leukemic cells and reduce leukemia relapses. The higher numbers of NK progenitors in UCB makes it a convenient source for ex vivo expansion of UCB NK cells for posttransplant treatment. UCB NK cells readily respond to interleukin-15, which may greatly enhance their antitumor effect. Activation and expansion protocols for UCB NK cells are currently being developed.     

人脐血T、B淋巴细胞和NK细胞的免疫学表达特性

目的 探讨人脐血T、B淋巴细胞和NK细胞，以及T／NK细胞杀伤性抑制性受体（KIR）表达的免疫学特性及其意义。方法 应用流式细胞仪检测26例正常新生儿脐血的T、B淋巴细胞和NK细胞抗原标记，包括CD45RA、CD45RO、CD69、CD25、CD40L、CD40、CD10、CD20和CD16等抗原的表达，以及脐血T／NK细胞上KIR分子（CD158a和CD158b抗原）的表达，并与正常儿童外周血比较。结果 脐血T淋巴细胞中CD45RA^ 细胞表达高于外周血（P＜0．01），CD45RO^ 则明显低于外周血（P＜0．01）；脐血T细胞CD69表达极低；CD25在CD8^ 细胞亚群中几乎不表达；CD40L在脐血T细胞中表达低于外周血（P＜0．05）。脐血B淋巴细胞中不成熟亚群CD10^ ／CD19^ 比例增高，成熟B细胞表型CD19、CD20、CD40等抗原均明显高于正常外周血（P＜0．01）。脐血中T淋巴细胞和NK细胞均存在KIR分子表达；脐血和外周血中T淋巴细胞CD158分子表达低于NK细胞（P＜0．01），脐血T淋巴细胞亚群中CD158分子表达低于正常外周血；CD158几乎不表达于CD4^ T细胞，主要表达于CD8^ T细胞，且以CD158a^ 为主；脐血中NK细胞CD158分子表达高于T淋巴细胞（P＜0．05），但明显低于外周血NK细胞KIR的表达（P＜0．01）。结论 脐血T淋巴细胞包括原始和早期T细胞以及T细胞受体表达障碍，导致脐血T淋巴细胞免疫功能不成熟，可能是脐血移植（UCBT）后移植物抗宿主病（GVHD）发生率低和程度轻的重要原因之一。脐血B淋巴细胞免疫应答障碍可能缘于T淋巴细胞表型或功能的障碍。脐血T／NK细胞KIR的表达特性提示，KIR可能与UCBT中GVHD和移植物抗白血病（GVL）效应有关。     

Unrelated cord blood transplantation for severe congenital neutropenia: report of two cases with very different transplant courses

SCN is characterized by neutropenia, life-threatening infections, and progression to myelodysplastic syndrome/acute myelogenous leukemia. The only curative option is SCT, but few reports using UCB as a stem cell source exist. Here, we report two SCN patients transplanted with UCB. Patient 1 was transplanted at seven yr of age due to increasingly large injections of G-CSF (>100 microg/kg/day) and the risk of developing leukemia. He engrafted promptly and is clinically well and immune reconstituted >2 yr post-transplant. Patient 2 underwent UCB SCT at nine months of age for recurrent severe infections, despite high doses of G-CSF. He rejected his first graft, having 100% host cells on day +35, and immediately underwent a second UCB SCT. He engrafted but experienced late graft rejection six months after the second transplant. He received a third UCB SCT following a more immunosuppressive conditioning regimen. His course was complicated by HHV-6 viremia and gut GVHD, but he is now clinically well and has 99% donor engraftment >20 months post-transplant. We conclude that UCB is an acceptable stem cell source for SCN patients, but conditioning must be adequately immunosuppressive to ensure engraftment in patients without prior chemotherapy.     

Induction of a transient graft vs. leukemia effect following unrelated cord blood transplantation

Umbilical cord blood (UCB) has become a frequent source of allogeneic hematopoietic stem cells for transplantation. Of theoretical concern is a potential decrease in the graft vs. leukemia (GvL) effect, given the lesser degree of graft vs. host disease (GvHD) with this donor source. We report a case of recurrent acute non-lymphoblastic leukemia (ANLL) following stem cell transplantation with unrelated mismatched UCB, which responded to the induction of GvHD. The response was documented both morphologically and by evaluation of chimeric engraftment by molecular DNA techniques. In addition, WT-1, a purported marker of minimal residual disease in acute leukemia, correlated with remission status in this patient. In summary, the GvL effect is seen with allogeneic UCB transplantation and has the potential to be induced along with GvHD.     

Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood

Endothelial progenitor cells (EPCs) are used for angiogenic therapies and as biomarkers of cardiovascular disease. Human umbilical cord blood (UCB) is a rich source of endothelial colony forming cells (ECFCs), which are EPCs with robust proliferative potential that may be useful for clinical vascular regeneration. Previous studies show that hematopoietic progenitor cells are increased in premature UCB compared with term controls. Based on this paradigm, we hypothesized that premature UCB would be an enriched source of ECFCs. Thirty-nine UCB samples were obtained from premature infants (24-37 wk gestational age (GA)) and term controls. ECFC colonies were enumerated, clonally isolated, and identified by expression of endothelial cell surface antigens and functional analysis. GA of 33-36 wk UCB yielded predominantly ECFC colonies at equivalent numbers to term infants. UCB from 24 to 28 wk GA infants had significantly fewer ECFCs. Surprisingly, 24-28 wk GA UCB yielded predominantly mesenchymal stem cell (MSC) colonies, capable of differentiating into adipocytes, chondrocytes, and osteocytes. MSCs were rarely identified in 37-40 wk GA UCB. These studies demonstrate that circulating MSCs and ECFCs appear at different GA in the human UCB, and that 24-28 wk GA UCB may be a novel source of MSCs for therapeutic use in human diseases.     

The regulatory function of umbilical cord blood CD4+ CD25+ T cells stimulated with anti-CD3/anti-CD28 and exogenous interleukin (IL)-2 or IL-15

The abundance of CD4⁺ CD25⁺ regulatory T cells in umbilical cord blood (UCB) might contribute to the decreased severity of graft-vs.-host disease (GVHD) for UCB transplantation. This study aims to characterize the phenotypes and suppressive function of UCB CD4⁺ CD25⁺ T cells under the influence of anti-CD3/anti-CD28 (CD3/CD28) and exogenous interleukin (IL)-2 or IL-15. Higher percentages of CD4⁺ CD25^high and FoxP3⁺ cells were detected in UCB compared to their adult counterparts. IL-15 was as effective as IL-2 in enhancing the proliferation of CD3/CD28 stimulated UCB CD4⁺ CD25⁺ T cells. Phenotypically, IL-2/IL-15-stimulated UCB CD4⁺ CD25⁺ T cells expressed higher level of CTLA-4, GITR, membrane bound transforming growth factor-β (mTGF-β), and especially Foxp-3 than controls. IL-2/IL-15-stimulated UCB CD4⁺ CD25⁺ T cells also produced much higher IL-10 and TGF-β than controls; while IL-2/IL-15-stimulated UCB CD4⁺ CD25⁻ T cells showed increased TGF-β, but not IL-10 production. IL-2/IL-15-cultured UCB CD4⁺ CD25⁺ T cells showed comparable suppressor activity on allogeneic adult CD4⁺ T-cell proliferation compared to controls, partly through a contact-dependent fashion. Taken together, IL-2/IL-15-stimulated UCB CD4⁺ CD25⁺ T cells show distinct regulatory T-cell phenotypic and functional features, and may be applied for the alleviation of GVHD severity following UCB transplantation.     

Cord blood revelations: the importance of being a first born girl, big, on time and to a young mother!

Umbilical cord blood (UCB) has become an alternative source for providing haematopoietic stem/progenitor cells as well as non-haematopoietic stem cells, compared to the conventional sources of bone marrow (BM) and peripheral blood (PB). Although UCB has many advantages over BM and PB there are still limitations to its use in the clinical setting, principally cell numbers. Thus, this study aimed to characterise components that comprise UCB samples and the physiological factors that affect them: (i) gender, (ii) obstetric history, (iii) infant birth weight, (iv) gestation stage and (v) mother's age. Our results show that UCB total nucleated cell (TNC) and haematopoietic stem cell (CD45+/CD34+) content is significantly affected by the baby's birth weight, mother's age at delivery, mother's obstetric history, and gestational stage at due date, all with p values<0.0001. The only parameter not found to be significant was gender, although results did suggest that female infants provide greater stem cell numbers than their male counterparts. Other UCB cellular sub-types affected were T-cells, dendritic cells and B-cells. In conclusion, this study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking UCB units for transplantation.     

Recent advances in bone marrow transplantation

The major barriers to successful bone marrow transplantation (BMT) are graft-versus-host disease (GVHD), infection, rejection and relapse. The combination of methotrexate and cyclosporin is significantly better than either alone in controlling GVHD. Removal of T cells from donor marrow prior to BMT has also decreased GVHD significantly, but a 5-10% rejection rate occurs and an increased relapse risk is being reported by some centres. Cyclosporin is valuable in the treatment of both acute and chronic GVHD. Interstitial pneumonitis due to cytomegalovirus (CMV) is a major cause of mortality. Protection can be provided with CMV hyperimmune globulin and also by the avoidance of blood donors who are CMV antibody positive. Fractionated total body irradiation is associated with decreased toxicity compared to single dose. There is a 75% 4 year disease-free survival following BMT for acute non-lymphoblastic leukemia in first remission, a 50% survival for acute lymphoblastic leukemia in second remission and an 88% survival for chronic myeloid leukemia in chronic phase. BMT for beta-thalassaemia major in young patients without organ dysfunction cures 80% of patients and identical results are achieved for severe aplastic anaemia when BMT is undertaken prior to blood product transfusion.     

非血缘相关脐血移植治疗儿童高危白血病的临床观察

目的：非血缘脐血具有快速寻求、容易得到和HLA配型不严格的特点，该文进行了非血缘相关脐血移植（UD-UCBT）治疗儿童恶性白血病的研究并探讨其疗效问题。方法：对6例难治性白血病患儿，包括3例急性淋巴细胞白血病（2例高危CR1，1例标危CR2），2例幼年慢性粒单细胞白血病（1例缓解期，1例加速期）和1例急性髓系白血病（AML- M5，CR1）进行了非血缘相关脐血移植，HLA高分辨1例全相合，1例5个位点相合，1例4个位点相合，3例3个位点相合。预处理选用白消安/环磷酰胺/ATG或全身放疗/环磷酰胺/ATG为主方案。于 0 d 回输脐血，有核细胞中位数为8.51×107/kg，CD34+细胞中位数为1.81×105/kg。预防移植物抗宿主病（GVHD）采用环孢霉素A、甲基泼尼松龙和骁悉或CD25单抗。结果：中性粒细胞绝对值（ANC）≥0.5×109/L和PLT≥20×109/L的中位天数分别是+13 d、+30 d，移植证据均为供者型。4例出现Ⅰ~Ⅲ度GVHD，均控制。随访中位时间12个月，未发生慢性GVHD，现存活4例血型均转为供者型，无复发。结论：脐血提供快速有效的造血干细胞，为治疗儿童白血病提供良好时机，非血缘相关脐血移植能耐受HLA多个位点不相合。急性GVHD发生率也较高，存在移植物抗白血病作用。     

体外扩增脐血间充质干细胞的生物学特性和

目的从脐血中分离单个核细胞(MNCs),体外培养扩增间充质干细胞(MSCs)并研究其生物学特性和诱导分化潜能.方法取35份足月顺产新生儿脐血,密度梯度离心法分离出其中的MNCs,采用含胎牛血清的DMEM培养基体外培养扩增MSCs.显微镜下观察MSCs的形态、细胞化学染色,流式细胞仪测定MSCs的细胞免疫表型,体外诱导分化实验检测MSCs分化成骨细胞、脂肪细胞和神经细胞的能力.结果从12份脐血中可培养出MSCs,形态上与从其他来源的MSCs类似,可传至20代而无形态上的变化.细胞化学染色示碱性磷酸酶(ALP)阴性,非特异性酯酶--α-丁酸萘酚酯酶(alpha-naphthol butyric acid esterase,NBE)阳性.其表达CD29、CD44和CD105,特别是人类MSCs标记SH-2 和 SH-3 阳性,而CD3、CD14、CD19、CD34和CD45阴性,说明它们并非来自造血细胞.这些MSCs在适当诱导分化剂的作用下,2周左右可以诱导分化形成骨细胞, 20天左右可以诱导分化形成脂肪细胞.脐血MSCs预诱导12 h后,胞体发生收缩,细胞边缘有细的突起.正式诱导5 h后大多数细胞呈现典型的神经元样.结论胎儿脐血MNCs可分离培养出MSCs.这些MSCs具有与其他来源MSCs类似的表型及分化潜能.     

人脐血间充质干细胞对脐血CD+34细胞体外扩增作用的研究

目的 探讨含人脐血来源的间充质干细胞(MSCs)体系在体外对脐血造血干细胞(HSCs)扩增作用。方法 (1)用含人脐血MSCs及不同造血生长因子(HGFs)组合的无血清扩增体系对人脐血CD34^ 细胞进行体外扩增。(2)于扩增前及扩增后第6、12天分别用双色流式细胞仪动态检测HSCs表面抗原标记：CD34^ 、CD34^ CD38^-、CD34^ CD3^ 、CD34^ CD19^ 、CD34^ 和CD34^ CD41^ 。细胞的含量。(3)按本实验室方法行体外半固体培养,观察扩增前后脐血细胞粒-单核细胞集落形成单位(CFU-GM)、爆式红系集落形成单位(BFU-E)、混合集落形成单位(CFU-Mix)及高增殖集落形成单位(CFU-HPP)集落形成情况。结果(1)含人脐血MSCs体系对脐血CD34^ CD38^ 细胞的扩增倍数在第6天和第12大分别为159和437倍。该业群百分比在单纯因子组扩增第12天时为1．98％,而在含脐血MSCs组为9．98%,明显高于扩增前。(2)集落培养表明,含脐血MSCs组扩增第12天与扩增第6天相比,其CFU-Mix和CFU-HPP的扩增倍数增加,而单纯因子组这两种集落的扩增倍数下降。(3)随扩增天数的增加,两组扩增体系中CD34^ CD3^ 和CD34^ CD41a^ 细胞均明显增加,而CD34^ CD19^ 和CD34^ CD3^ 细胞均明显减少。两组相比,含脐血MSCs组差异更显著。结论 (1)含脐血MSCs体系不仅能扩增更原始的造血干／祖细胞(HSPC),且具有在短期内(12d)保持HSCs不耗竭。(2)含脐血MSCs体系对脐血CD34^ 细胞向定向祖细胞的扩增,主要为髓系及巨核系祖细胞,而对其向淋巴系祖细胞的扩增具有抑制作用。     

Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children

BACKGROUND: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). METHODS: Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. RESULTS: Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. CONCLUSIONS: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .     

Successful engraftment without radiation after fludarabine-based regimen in Fanconi anemia patients undergoing genotypically identical donor hematopoietic cell transplantation

BACKGROUND: To potentially reduce late effects of malignancy, chronic graft-versus-host disease (GVHD), endocrinopathy, and infertility in patients with Fanconi anemia (FA) undergoing HLA-matched related donor hematopoietic cell transplantation (HCT), we developed a regimen using fludarabine (FLU), cyclophosphamide (CY), and anti-thymocyte globulin (ATG) followed by infusion of T-cell depleted (TCD) bone marrow (BM) or unmanipulated umbilical cord blood (UCB). GVHD prophylaxis consisted of cyclosporine and short course methylprednisolone. PROCEDURE: Between April 2000 and June 2003, 11 patients (10 aplastic anemia (AA), 1 myelodysplastic syndrome (MDS)) underwent HCT using this regimen. Stem cell sources were BM and UCB in eight and three patients, respectively. RESULTS: All patients demonstrated primary engraftment. Median days to neutrophil and platelet engraftment were 11 days (range 9-21) and 38 days (range 19-381), respectively. No patient developed GVHD after primary HCT. The patient with MDS relapsed with AML and a maternal donor recipient experienced secondary graft failure. For the nine FA patients with AA who underwent HLA-identical sibling donor HCT, the Kaplan-Meier estimates of overall survival and event-free survival (EFS) at 2 years are 100% and 82%, respectively, at a median follow-up of 2.9 years (range 1.9-4.8). CONCLUSIONS: In summary, a FLU-based, non-irradiation approach is effective for FA patients with AA undergoing HLA-identical sibling donor HCT.     

人脐血及脐带间充质干细胞的分离培养及表面标记分析

目的探讨从人脐带血及脐带分离和培养间充质干细胞(MSCs)的方法,并分析MSCs的表面标记。方法人脐带血按常规方法制备单个核细胞,利用MSCs贴壁生长的特性,经培养、换液、传代纯化MSCs;分离脐带华尔通胶(Wharton’s jelly),采用组织块贴壁法获得脐带MSCs并传代。将传代的MSCs冻存,1个月后再复苏,观察复苏后MSCs的生长情况。利用FACScan流式细胞仪检测脐带血及脐带细胞表面抗原。结果经过传代后,贴壁细胞形态趋于同一。人脐血及脐带MSCs体外生长形态相似,类似成纤维细胞,可以稳定增殖和传代。经冷冻保存,复苏后仍能较好生长。人脐血及脐带来源的MSCs表面标记CD29、CD44、CD59高表达,而表面标记CD14、CD33、CD34和CD45低表达。结论人脐带血及脐带均可分离出MSCs,在体外能扩增纯化及冻存复苏,为组织工程提供丰富的细胞来源。     

急性白血病患儿CD4~+CD25~+调节性T细胞和NK细胞免疫作用探讨

目的观察外周血CD4+CD25+调节性T细胞(CD4+CD25+Treg)及自然杀伤细胞(na-ture killer cell,NK)在白血病患儿及非白血病患儿的不同,了解白血病患儿的免疫状态,探讨CD4+CD25+Treg细胞及NK细胞在小儿急性白血病肿瘤免疫中的意义。方法以流式细胞术检测急性白血病初诊患儿及非白血病患儿各30例的外周血CD4+CD25+Treg细胞、NK细胞的数量及比例。结果外周血CD4+CD25+CD127-细胞占CD4+T细胞的比例白血病组为(11.45±1.41)%,显著高于对照组为(6.98±1.09)%(P<0.05)。而NK细胞数量白血病组为(5.13±2.97)%,显著低于对照组为(15.06±3.91)%(P<0.05)。结论 (1)急性白血病患儿外周血CD4+CD25+Treg细胞数量升高,NK细胞数量降低,表明急性白血病患儿NK细胞免疫功能处于抑制状态。CD4+CD25+Treg细胞可能在白血病的发生、发展中起一定作用。(2)通过检测CD4+CD25+CD127-T细胞可较好的反映CD4+CD25+Treg细胞的比例,简便可行、重复性好、检测结果准确、可靠。     

65例中国非亲缘性脐血造血干细胞移植的回顾研究

目的1998—2004年,广州脐血库向国内25家移植中心提供非亲缘性脐血治疗恶性及非恶性肿瘤性疾病患者65例,对其进行回顾性分析,探讨与非亲缘性脐血移植密切相关的影响因素。方法脐带血均采自广州市妇婴医院正常分娩的足月顺产产妇,脐血分离、冻存、复温方法参照纽约脐血库的标准及相关文献;选择脐血的依据是HLA相合程度及有核细胞数量。结果65例接受非亲缘性脐血移植的病例中,白血病49例,遗传缺陷性疾病16例;男性42例,女性23例;受者中位年龄10岁(1~33岁);中位体重27kg(10~67kg);HLA-A、B,DRB1匹配全相合9例,1个位点不合43例,2个位点不合13例;总有核细胞(TNC)中位输入量5.7×107/kg[(1.84~20.00)×107/kg]。移植后植入50例有核细胞总数(ANC>500/μl),植入的中位时间为17.5d(7~44d),植入率77%;41例受体发生GVHD,发生率为63%。30例长期存活(无病生存时间>365d),无病存活率为61%。结论非亲缘性脐血移植具有良好的临床应用前景。     

脐血造血细胞含量与白血病脐血移植疗效分析

目的分析4711份库存脐血造血细胞含量及探讨脐血造血细胞含量与白血病脐血移植疗效的关系。方法分析4711例库存脐血总有核细胞数(TNC)和CD34+细胞数分布情况,探讨不同的造血细胞输入量、供受者HLA不相合数、受者性别、年龄、体重和疾病类型间植入率和生存率的差异。结果 4711例库存脐血TNC和CD34+细胞中位数分别为1.14×109/kg和4.06×106/kg,按3.7×107/kg有效TNC输入量计算,93.2%脐血可供体重50 kg以下受者移植。89例白血病患者移植后植入75例,植入率为84.3%。中性粒细胞绝对值≥0.5×109/L、血小板≥20×109/L和≥50×109/L的时间分别为移植后17、34和46 d。75例植入病例中,长期无病存活47例,死亡26例,2例复发;急性移植物抗宿主病(GVHD)Ⅰ～Ⅱ度、Ⅲ～Ⅳ度和慢性GVHD发生率分别为54.7%、20.0%、9.3%。影响移植植入率的因素包括受者年龄、TNC和CD34+细胞输入量;影响生存率的因素包括受者年龄、体重和输入CD34+细胞数。结论在无法找到HLA全相合骨髓供者时,可选择脐血作为替代骨髓的造血干细胞来源治疗儿童与成人白血病,TNC和CD34+细胞数仍是选择脐血移植物的参考指标。     

Susceptibility to Fas and tumor necrosis factor-α receptor mediated apoptosis of anti-CD3/anti-CD28-activated umbilical cord blood T cells

Decreased severity of graft-versus-host disease after mismatched umbilical cord blood (UCB) transplantation may be attributed in part to the increased propensity to apoptosis of UCB T cells following activation. Interleukin (IL)-15, a pleiotropic cytokine that is essential for T-cell proliferation and survival, may serve as promising immunomodulative therapy post-CB transplantation for its anti-apoptotic effect. This study aimed to determine the kinetics of Fas or tumor necrosis factor-α receptor (TNFR) mediated caspase-3 expression and apoptosis of anti-CD3/anti-CD28 activated UCB T cells in the influence of IL-15. Activated caspase-3 expression was analyzed by Western blotting and the percentage of apoptotic cells was determined by annexin-V/propidium iodide (PI) flow cytometric staining. Significant expression of Fas and TNFR2 was detected on anti-CD3/anti-CD28 pre-activated UCB T cells. These cells were susceptible to anti-Fas but not TNF-α-induced apoptosis. Kinetic study shows that caspase-3 expression became evident at 6th–8th h following anti-Fas stimulation, while early apoptotic cells with annexin-V⁺/PI⁻ expression appeared at 12th–16th h. IL-15, though successful in decreasing apoptosis in pre-activated UCB T cells, failed to completely prevent Fas-mediated caspase-3 expression and apoptosis of CB T cells. The pre-activated UCB and adult peripheral blood T cells behaved similarly with regard to death receptor expression, caspase-3 expression and apoptosis upon Fas-engagement. Although IL-15 promotes overall activated UCB T-cell survival, it did not particularly prevent Fas-mediated activation-induced cell death.     

Hematopoietic stem-cell transplantation using unrelated cord-blood versus matched sibling marrow in pediatric bone marrow failure syndrome: one center's experience

Hematopoietic stem-cell transplantation (HSCT) is an effective mode of therapy in pediatrics for the treatment of both malignant and non-malignant disorders. We compared the course of children transplanted with unrelated umbilical cord blood (UCB) to those transplanted with allogeneic sibling bone marrow (BM) for bone marrow failure syndromes. Thirteen patients with a median age of 6.3 years were transplanted for the following diseases between April 1992 and November 1997: myelodysplastic syndromes, aplastic anemia, Diamond-Blackfan anemia, myelofibrosis, paroxysmal nocturnal hemoglobinuria, osteopetrosis and dyskeratosis congenita. The stem cell source was BM in ten patients and UCB in three. We retrospectively examined the conditioning regimens, stem cell source and dose, days to engraftment, survival and complication rate to see whether there was a significant advantage in using one source over the other. The median time to an absolute neutrophil count > 500 per microL was 25 days for UCB patients and 16 days for BM patients. The median time to a platelet count > 20,000 per microL was 55 days for UCB patients and 22 days for BM patients. The 100-day mortality was 66% in UCB patients and 20% in BM patients. The overall mortality rates were 66% and 40%, respectively. Three patients died prior to engraftment. Seven patients (54%) were still alive as of May 1999 with a median follow-up of 1574 days post-transplant. The patients transplanted with BM had faster engraftment and lower rates of graft-versus-host disease, 100-day mortality and overall mortality. HLA-matched sibling BM is preferred as a source but transplantation using unrelated UCB is still an option in treating pediatric bone marrow failure syndromes.     

甲氧基聚乙二醇修饰对移植物抗白血病的作用

目的探讨甲氧基聚乙二醇(mPEG)修饰供者小鼠骨髓移植物单个核细胞表面抗原对移植物抗白血病作用(graft versusleukemiaeffect,GVL)的影响。方法小鼠随机分为A、B、C、D四组。A组单纯接受60Coγ照射;B、C、D组每只小鼠腹腔接种1×106L615肿瘤细胞制成白血病模型;全部照射后,C、D组分别移植mPEG未修饰和修饰的骨髓、脾细胞悬液。观察小鼠一般反应、外周血涂片L615细胞计数、组织病理及生存时间。结果A组全部死于造血衰竭;B组死于白血病;C组全部出现明显移植物抗宿主病(GVHD)表现并死亡;D组部分小鼠(4/15)长期存活,11/15死于白血病,平均生存24.2天,长于其他组(P<0.05),生存率27%,高于其他组(P<0.05),两者差异有统计学意义。结论小鼠移植mPEG修饰骨髓移植物后保留了一定GVL作用,并且减轻GVHD。