Evidence for graft-versus-host disease in total-lymphoid-irradiated recipients of allogeneic bone marrow and skin grafts

It has been demonstrated that tolerance to skin allografts can be induced across the strong murine histocompatibility barrier H2^b → H2^d by pretreatment of the recipients with total lymphoid irradiation, TLI, and donor bone marrow, BM. It has been claimed that tolerance can be achieved in this system without concomitant graft-versus-host disease, GVHD. In the present study, designed to confirm these findings, BALB/c^d recipients were pretreated with 3400 R fractionated TLI and 3–5 × 10⁷ donor C57BL/6J^b BM cells (experimental) or medium (control). The mice received C57BL/6J^b skin grafts 1–6 days following bone marrow and/or medium infusion. All but one of the TLI plus BM group (n = 24) died within 50 days (mean 33.4 ± 2.7 days) with progressive weight loss, diarrhea, and ruffled fur but only one animal rejected its graft. A lower mortality, i.e., 36%, was observed in controls which received TLI without BM (n = 18). The controls which did not survive died earlier (21.8 ± 5.1 days) than the BM group (P ≤ 0.05) and died without progressive weight loss. These results suggest that significant GVHD may occur in tolerant mice pretreated with TLI and allogeneic BM.     

乙肝肝移植受体术后骨髓CD34+细胞和PBMCs内乙肝病毒DNA的同步检测

目的 肝移植术后乙肝复发严重影响预后,HBV感染的病毒来源值得研究.采用PCR技术可检测到术后病人外周血单个核细胞(PBMCs)中HBV DNA的存在,然而PBMCs的寿命有限,其中的病毒来源仍不清楚.该研究对此问题做了初步的探索.方法 采集13例乙肝肝移植术后受体外周血和骨髓标本,运用Ficoll密度梯度离心法结合免疫磁性分离法(MACS)分别从外周血和骨髓标本中分离出PBMCs和骨髓来源CD34+细胞即造血干祖细胞,利用实时荧光定量PCR法检测细胞中的HBV DNA,同时检测血清HBV标志物和血清HBV DNA.结果 13例病人术前血清HBV DNA阳性8例,阴性5例.术后平均37个月的随访期内(16～77个月)血清中HBV DNA,HBsAg和HBeAg检测结果全为阴性;13例PBMCs中均检测到HBV DNA的存在,阳性率为100%.PBMCs中DNA含量对数平均值为3.40±0.85;13例骨髓来源CD34+细胞中都检测到HBV DNA的存在.阳性率也是100%.CD34+细胞中DNA含量对数平均值为3.30±0.58.病人PBMCs和CD34'细胞内DNA含量对数值均数差异无统计学意义(P>0.05).同一病人PBMCs与CD34+细胞HBV DNA同时为阳性.术前血清HBV DNA检测阳性和阴性病人的PBMC内DNA含量对数值均数差异无统计学意义(P＞0.05);术前血清HBV DNA检测阳性和阴性病人的CD34+细胞内DNA含量对数值均数差异也无统计学意义(P＞0.05).结论 现有预防措施下,乙肝肝移植受体虽然外周血清中不能检测到HBV DNA和HBV抗原成分的存在,但术后骨髓CD34+细胞和PBMCs中皆长期存在HBV DNA.含有HBV DNA的骨髓来源CD34+细胞可能是含有HBV DNA的PBMCs的来源,也可能是肝移植术后PMBCs内HBV DNA长期存在的原因,及以后导致HBV复发的潜在危险因素.然而,现在尚无有效方法可以完全清除这些残存病毒,术后长期的抗病毒药物预防措施是必要的.     

HSCT联合不同剂量内皮祖细胞输注对小鼠造血重建的影响

目的 探讨异基因造血干细胞移植联合不同剂量内皮祖细胞(EPC)输注对移植后造血重建的影响.方法 以C57BL/6小鼠为供鼠,Balb/c小鼠为受鼠,进行HSCT,输注骨髓单个核细胞数量为5×106个/只.仅进行HSCT者为单纯骨髓细胞移植组;行HSCT的同时经尾静脉输注供者骨髓单个核细胞诱导培养的EPC的受鼠为EPC联合移植组,EPC的输注量分别为5×104、1×105、5× 105和1×106个/只.另设正常对照组和致死量照射组.观察小鼠的存活率、造血重建情况及骨髓微环境的变化.结果 各EPC联合移植组小鼠存活时间长于单纯骨髓移植组,5×105 EPC联合移植组至观察结束时存活率为100％,高于其他各组(P＜0.05).移植后10和15 d,5×105 EPC联合移植组外周血白细胞数量高于其他组(P＜0.05).移植后15 d,5×105 EPC联合移植组外周血血小板数量高于其他组(P＜0.05).5×105 EPC联合移植组造血组织增生程度也好于其他组.5×105 EPC联合移植组骨髓内HSC比例为(1.06±0.03)％,高于其他各组(P＜0.05).结论 小鼠异基因骨髓移植中联合输注5×105 EPC能够有效促进造血重建,提高小鼠存活率.     

Risk of viral transmission via bone marrow progenitor cells versus umbilical cord blood hematopoietic stem cells in bone marrow transplantation

Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.     

内皮祖细胞抑制小鼠异基因骨髓移植后肝小静脉血栓形成的作用

目的 探讨内皮祖细胞(EPC)抑制异基因骨髓移植后肝小静脉血栓形成的作用.方法 采用随机数字表法将Balb/c小鼠分为3组,单纯骨髓移植组的小鼠在全身照射后经尾静脉输注C57BL/6小鼠的骨髓单个核细胞5×106/只 ;联合EPC移植组则在单纯骨髓移植的基础上同时输注C57BL/6小鼠的EPC 5×105/只 ;正常对照组的小鼠不作任何处理.分别于移植后第0、5、10、15和20天时,计算各组小鼠的肝脏指数,光镜下观察肝小静脉血栓形成情况以及肝细胞及血管内皮损伤情况,电镜下观察肝小静脉、肝血窦内皮、肝细胞损伤及血小板黏附情况,检测外周血活化血小板的比例及肿瘤坏死因子α(TNF-α)浓度的变化.结果 移植后第0、5、10、15和20天时,单纯骨髓移植和联合EPC移植组的活化血小板比例、肝脏指数和外周血TNF-α浓度均呈现上升趋势,至第15天时达到高峰,随后下降,但仍明显高于正常对照组(P＜0.05) ;各时间点联合EPC移植组的上述指标均明显低于单纯骨髓移植组,差异均有统计学意义(P＜0.05).与单纯骨髓移植组比较,联合EPC移植组的血小板黏附减少,肝小静脉血栓形成较少,肝细胞水肿和坏死程度均较轻,且肝脏损伤修复较快.结论 小鼠骨髓移植时联合输注EPC能显著抑制肝小静脉血栓形成,明显减轻肝脏损伤.     

肢体缺血大鼠骨髓内皮祖细胞的动员与组织血管新生的相关性研究

目的　探讨肢体缺血因素对骨髓内皮祖细胞 (EPC)的动员及肢体血管新生的影响。方法　切除、结扎单侧股总、股浅动脉建立大鼠后肢缺血模型 ,另设对照组。 1周后通过细胞培养 ,观察外周血EPC数量的变化 ,4周后检测缺血组织微血管密度 (MVD )及血管内皮生长因子(VEGF)的表达情况。结果　实验组外周血EPC (4 8.6± 7.5 )个 /mm2 较对照组 (2 3 .1± 4.3 )个 /mm2 明显增加 (P <0 .0 1) ;组织MVD (2 2 4.84± 5 .87)n/mm2 显著高于对照组 (13 6.3 0± 4.5 2 )n/mm2 (P <0 .0 1) ;实验组缺血组织VEGF表达明显增强 (吸光度值A :0 .162± 0 .0 18) ,对照组仅少量表达 (A :0 .0 5 5± 0 .0 0 6) ;外周血EPC与组织MVD、VEGF吸光度之间有非常显著相关 (P <0 .0 1)。结论　肢体缺血通过动员骨髓EPC ,促进缺血组织的新血管生成 ,改善局部供血 ,达到部分代偿目的。     

淋巴因子激活的骨髓细胞对小鼠同种异体骨髓移植的影响

以致死性剂量照射的成年小鼠为受鼠进行同种异体骨髓移植（Ａｌｌｏ－ＢＭＴ），实验组小鼠观察期间平均存活天数较对照组明显延长（Ｐ＜０．００１），应用肿瘤移植及单向混合淋巴细胞培养（ＭＬＣ）二项指标证实Ａｌｌｏ－ＢＭＴ的部分植活。实验结果表明，淋巴因子激活的骨髓细胞（ＬＡＢＭＣ）可有效预防致死性移植物抗宿主病（ＧＶＨＤ），且不影响供髓植活。     

低氧作用下肾小管上皮细胞对大鼠骨髓内皮祖细胞的影响

肾小管周毛细血管丢失和低氧是小管间质损伤及纤维化的共同通路,因此,重建间质毛细血管,改善间质低氧是治疗慢性肾脏病(CKD)的又一重要途经~([1]).内皮祖细胞(EPC)具有强大的成血管能力~([2]).研究肾小管上皮细胞(RTEC)与EPC间的相互作用,对于重建小管周毛细血管具有重要意义.本课题拟在体外研究低氧作用下的RTEC对EPC的归巢、成血管能力的影响和可能的机制.     

NUP98-HOXA10hd-expanded hematopoietic stem cells efficiently reconstitute bone marrow of mismatched recipients and induce tolerance

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98- HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.     

Regeneration of TdT+, pre-B, and B cells in bone marrow after allogeneic bone marrow transplantation

In 15 children transplanted with allogeneic bone marrow for acute leukemia and in complete remission, regeneration of the early stages of the B cell system was studied. Bone marrow aspirates taken before and longitudinally after BMT were investigated for pre-B and B cells by immunofluorescence techniques; in some cases, TdT+ cells were also determined. Normal values were derived from bone marrow samples taken from 23 healthy individuals who served as bone marrow donors. In normal bone marrow, B cells outnumber pre-B cells and the latter are more numerous than TdT+ cells. Before BMT, the numbers of BM pre-B were outside the normal range in all cases; B cell numbers were abnormal in most of the 11 patients studied, probably due to the antileukemic remission induction/consolidation therapy. After BMT, two distinct patterns of regeneration of the B cell system were observed. In 9 patients, TdT+ cells were considerably increased early after BMT. This was followed by a rise in pre-B cells, with values well above the normal range, and resulting in ratios of TdT+:pre-B cells and of pre-B cells:B cells that were transiently greater than 1. In the other 6 patients, the regeneration of TdT+ cells varied and the reconstitution of the pre-B cells was more gradual than in the first group, with pre-B-to-B cell ratios less than 1 during the whole observation period. The only consistent difference between the patients of the two groups, possibly relevant to the regeneration of the B cell lineage, was the duration of corticosteroid therapy, which was much longer in the 6 patients with slow-pace reconstitution. The pace of regeneration of the B cell system in the bone marrow was correlated with the recovery of the humoral immunity, as indicated by a significant increase in specific antibody titers after the second vaccination with diphtheria-tetanus-poliomyelitis vaccine in 7 of 9 patients in the rapid-pace group, versus 2 of 6 patients in the slow-pace group.     

Quantitative and qualitative differences in thrombopoietin-dependent hematopoietic progenitor development between cord blood and bone marrow

BACKGROUND: To assess the clinical application of thrombopoietin (TPO) for thrombocytopenia of patients receiving cord blood (CB) or bone marrow (BM) transplants, we examined whether various types of hematopoietic progenitors including megakaryocyte (MK) progenitors from CB and BM exerted different proliferative and differentiative potential in the presence of TPO. METHODS: The development of MK, granulocyte-macrophage, and erythroid/mixed erythroid (E/Mix) progenitors in a serum-deprived liquid culture medium supplemented with TPO was compared between CD34+ CB and BM cells. RESULTS: The CD34+ CB cells generated 30-fold more MKs than the CD34+ BM cells, but the CB-derived MKs were more immature. A single-cell culture study showed that CB CD34+CD38- cells as well as CD34+CD38+ cells proliferated in response to TPO, whereas the two subpopulations of CD34+ BM cells showed little multiplication. In short-term liquid cultures containing CD34+ CB or BM cells, TPO significantly increased the absolute numbers of various types of colony-forming cells, compared with the input values. In particular, MK progenitors and E/Mix progenitors in CB were amplified to a substantially greater extent than in BM. The superior response of CD34+ CB cells to TPO observed in this study may be due in part to the use of cryopreserved cells. CONCLUSIONS: Our results suggest that TPO alone cannot only stimulate megakaryocytopoiesis but also increase the numbers of various types of hematopoietic progenitors, and that quantitative and qualitative differences in TPO-dependent hematopoietic progenitor development exist between CB and BM.     

Deficient T cell granulocyte-macrophage colony stimulating factor production in allogeneic bone marrow transplant recipients

The proliferation and differentiation of donor hematopoietic progenitor cells in bone marrow transplantation (BMT) recipients is influenced by hematopoietic growth factors, which could derive from either T cells or adherent stromal bone marrow cells, or both. In this study of 20 BMT recipients, we asked whether T lymphocytes arising from donor bone marrow grafts were able to express normal levels of granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA, and to secrete normal levels of soluble GM-CSF in response to the mitogen phytohemagglutinin. We have found that T cells obtained up to 18 months following BMT express little or no PHA-induced GM-CSF message. T cell GM-CSF secretion in response to PHA is also reduced or absent. This T cell GM-CSF defect was observed in all patients studied, whether or not donor bone marrows had undergone T cell depletion. This defect likely reflects a broader deficit in mitogen-induced lymphokine production. This defect likely contributes to BMT recipients' blunted responses to infections, and contributes to graft failure in T cell-depleted transplants.     

Endothelial cell-derived microparticles in allogeneic hematopoietic stem cell recipients

BACKGROUND: Alterations of microparticles derived from different cell types are described in a number of diseases associated with inflammation and hemostatic disorders. METHODS: In this prospective study, we firstly analyzed endothelial cell derived microparticles (EMP) in 19 hematopoietic stem cell recipients. Cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha (TNF-alpha) served as positive controls. EMP were analyzed by fluorescent activated cell sorting (FACS), detecting the particels via expression of CD62 (E-selectin) and anionic phospholipids binding to annexin V. RESULTS: EMP were not significantly influenced by conditioning regimens with non-myeloablative chemotherapy and 4 Gy total body irradiation (TBI) or by myeloablative regimens containing 12 Gy TBI. During acute graft versus host disease (aGVHD), significantly higher levels of EMP were detected than in patients without aGVHD (18.5/microl s=10.1 vs. 14.6/microl SD = 11.5; P = 0.004) while infectious complications did not alter EMP levels significantly. Immunosuppressive therapy with corticosteroids tendentially elevated EMP levels. HUVEC treated with TNF-alpha 1 ng/ml, 10 ng/ml and 100 ng/ml released significantly more EMP than unstimulated cultures (30.0/microl ss = 13.6 vs. 126.8/microl SD = 66.9, P = 0.032 / vs. 683.3/microl SD = 349.9; P = 0.03 / vs. 489.3 s = 184.4; P = 0.013). CONCLUSIONS: Elevation of EMP during aGVHD might express severe endothelial cell injury within this complication after hematopoietic stem cell transplantation and might serve as a diagnostic test for early differentiation of aGVHD from other transplanted related complications.     

诱导大鼠骨髓内皮祖细胞成内皮细胞的体外增殖规律的观察

目的　观察大鼠骨髓内皮祖细胞 (EPCs)体外诱导成内皮细胞的生长增殖规律。方法　密度梯度离心得到Wistar大鼠骨髓单个核细胞 ;以 10 μg/L血管内皮细胞生长因子 (VEGF)和2 μg/L碱性成纤维细胞生长因子 (bFGF)体外诱导 ,通过细胞形态、免疫荧光法观察内皮细胞表型 ;原代细胞克隆形成率、细胞计数和流式细胞术检测VEGFR 2、VE 钙黏蛋白 (cadherin)阳性表达率观察体外增殖能力与表型。结果　诱导后细胞呈铺路石样形态。免疫荧光法发现诱导细胞表达小鼠抗大鼠Ⅷ因子 (vWF)、VEGFR 2、VE cadherin和CD3 1。原代克隆形成率为 6.66± 0 .75 ;细胞随代次增加增殖能力逐渐下降 ;VEGFR 2和VE cadherin在P2与P5中的阳性率差异无显著性 (P >0 .0 5 )。结论　骨髓EPCs在体外可诱导为成内皮细胞 ,并可保持相对稳定的诱导阳性率。     

Enhancement of fractionated bone marrow transplantation on hematopoietic cell homing in a mouse allogeneic model

BACKGROUND: The conventional method of bone mar row transplantation (BMT) requires a large quantity of bone marrow cells (BMC) transplanted by a single injection. However, the homing efficiency of BMC is low, because the emptying of available niches might be a continuous process after stem-cell death after irradiation. In this article, the death character of CD34 stem cells was directly detected, and a novel method of fractionated (Fr) BMT that aimed to better use each niche that was continuously emptied was developed. METHODS: Apoptosis and necrosis were detected using trivariate fluorescence-activated cell sorting after labeling cells with phycoerythrin-CD34-fluorescein isothiocyanate-Annix V-7-aminoactinomycin D. Fr-BMT was conducted in a model of allogeneic BMT, where the total dosage of BMC was divided into four doses, and each dose was administrated for 4 consecutive days at 24-hr intervals after lethal irradiation. RESULTS: CD34 cells underwent a continuous death process after lethal irradiation and presented three death peaks within a week. Fr-BMT decreased the BMC transplanted but increased the accumulatively homed BMC and enhanced the survival rates of recipients. CONCLUSIONS: Fr-BMT is able to enhance hematopoietic cell homing and engraftment, and this method might prove to be a solution to the previous clinical problem that a single unit of umbilical cord blood is not sufficient for engraftment in adults.