Ex vivo expansion of umbilical cord blood hematopoietic progenitor cells by combinations of cytokines

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-γ). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34⁺CD38^? and CD34⁺CD38⁺ subpopulations, respectively. Although absolute CD34⁺CD38^? cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-γ were significantly effective in maintaining CD34⁺CD38^? cells than the other combinations (P < 0.05). After 4 weeks of culture, CD34⁺CD38^? cells disappeared in all combinations of cytokines. Absolute CD34⁺CD38⁺ cell numbers increased in the presence of cytokines. Maximal expansion of CD34⁺CD38⁺ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 ± 3.3 -fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 ± 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34⁺CD38⁺ cells (15.8 ± 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-γ, maximal expansion was inferior to the others but CD34⁺CD38⁺ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.     

Effects of cytokines on hematopoietic progenitor cells in cord blood,in bone marrow,and in peripheral blood mobilized by chemotherapy and G-CSF

We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL] ?3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1–3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.     

Quantification of Nucleated Cells,CD34-Positive Cells and CFU-GM Colonies in Single Bone Marrow Samples and Bone Marrow Harvests Derived from Healthy Children

Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10⁶ cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10⁵ cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10⁶ /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10⁵ cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.     

The in vitro effects of stem cell factor and PIXY321 on myeloid progenitor formation (CFU-GM) from immunomagnetic separated CD34+ cord blood.

Two novel cytokines, stem cell factor (SCF) and PIXY321 (a fusion protein, granulocyte macrophage colony-stimulating factor+IL-3), have recently been demonstrated to enhance in vitro adult myelopoiesis. In this study, we compared the success of separating very early hematopoietic progenitor cells (CD34+) from both cord blood (CB) and adult bone marrow (ABM) and their differential response to SCF, PIXY321, and other later-acting colony-stimulating factors (CSF). Briefly, CD34+ cells were isolated from CB and ABM with an anti-CD34 MAb, HPCA-1, and incubated with various combinations of SCF, PIXY321, and other CSF. The percentage of CD34+ cells was decreased in CB compared to ABM before separation (0.54 versus 1.71%) (p = 0.05). Isolated CD34+ cells from CB and ABM were similar in lineage with respect to CD38, HLA-DR, CD33, and CD5, but decreased in CB with respect to B-lineage expression (CD19, CD10, and CD22) (p = 0.05). SCF increased colony forming unit-granulocyte-macrophage (CFU-GM) formation from CB CD34+ cells compared to unconditioned media and had a significant additive increase with IL-3 (p = 0.006) and granulocyte colony-stimulating factor (p = 0.03). SCF also had an additive increase in CB CFU-GM formation with PIXY321 (p = 0.007). PIXY321 had a similar increase in CFU-GM formation from both CB and ABM CD34+ cells compared to the combination granulocyte macrophage colony-stimulating factor + IL-3. When SCF was added to IL-3, PIXY321, or PIXY321 + IL-6, there was an increase in CFU-GM from CB versus ABM CD34+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Robust Multi-Parameter Single-Platform Quantification of Myeloid and B-Lymphoid CD34 Progenitor Cells in All Clinical CD34 Cell Sources and in Thawed PBSC

As B-lymphoid progenitor cells do not give rise to in vitro colony formation and are unlikely to support myeloid engraftment, we validated a five-color extension of the single platform Stem Cell Enumeration (SCE) kit, to routinely quantify myeloid and B-lymphoid progenitor cells. Fresh samples (n > 20 each) of granulocyte colony stimulating factor mobilized blood (peripheral blood (PB)), cord blood (CB), bone marrow (BM), and apheresis products (APs) were stained in TruCOUNT? tubes and the results were compared with those from the two-color CD45/CD34 reagent combination and the three-color SCE kit. To address repeatability, 10 samples from one AP were prepared by four technicians. Aliquots (n = 15) of four frozen AP were analyzed after thawing. Excellent correlations were observed between the three kits (R² > 0.99), for the quantification of white blood cells and total CD34. The extended kit showed considerable amounts of B-lymphoid progenitors in all CD34 sources (0–20% of all CD34 in PB, AP, and CB; 3–90% in BM). Very similar results were obtained when the same sample was prepared by different technicians. After thawing of frozen AP, the recovery of viable cells varied depending on the freezing medium employed, but the results from the different quantification methods were identical. Most non-viable cells were clearly identified with 7 Aminoactinomycin D (7AAD) but an additional gate in the forward scatter/side scatter was necessary to address dead cells negative for 7AAD. The extended SCE kit allows rapid and exact quantification of viable B-lymphoid and myeloid CD34⁺ cells in all cell sources and in thawed stem cell harvests, and may thus improve the correlation between CD34 number and engraftment kinetics.     

Effects of recombinant thrombopoietin on the growth of murine primitive and committed hematopoietic progenitors in serum-free culture

BACKGROUND: The aim of the present study was to determine the effect of thrombopoietin (Tpo) in combination with other cytokines on the growth of murine megakaryocytic, granulocyte-macrophage, erythroid and primitive hematopoietic progenitor cells, excluding possibilities of synergistic effects by serum factor(s). METHODS: Serum-free clonogenic assay systems were used for assay of colony forming units in megakaryocytes (CFU-Mk), colony forming units in granulocytes-macrophages (CFU-GM), burst-forming units in erythrocytes (BFU-E) in marrow of normal mice and of high proliferative potential colony forming cells in 5-fluorouracil-treated marrow. RESULTS: Serum-free culture enabled megakaryocyte colony growth by recombinant murine (rm) Tpo and this was synergistically supported with rm interleukin (IL)-3, rm stem cell factor (SCF) or recombinant human (rh) erythropoietin (Epo). Recombinant human IL-6, rhIL-11 and rm granulocyte-macrophage colony stimulating factor (GM-CSF) showed synergistic effects with rmTpo only in the presence of serum. Recombinant murine IL-3 or rmSCF increased the large colonies and mixed-type colonies containing other populations, suggesting that these cytokines promoted the proliferation of immature populations of CFU-Mk. The rmTpo enhanced the growth of granulocyte-macrophage (GM) colonies stimulated by rmGM-CSF or rmIL-3 and erythroid bursts by rhEpo, with or without rmIL-3. The rmTpo also significantly increased HPP colonies in synergy with rmIL-3 only in the presence of serum or rmSCF. CONCLUSION: Serum-free culture is valuable for evaluating synergistic effects of cytokines and Tpo acts not only on megakaryocytic progenitors but also on granulocyte-macrophage, erythroid and primitive progenitor cells in combination with other cytokines, such as rmIL-3 and rmSCF. However, serum or SCF was required for enhancement of the colony growth of primitive progenitors by Tpo.     

An infantile case of cytomegalovirus induced idiopathic thrombocytopenic purpura with predominant proliferation of CD10 positive lymphoblast in bone marrow

An infant with cytomegalovirus infection (CMV) developed idiopathic thrombocytopenic purpura (ITP) at 4 months of age. A bone marrow (BM) aspiration showed a remarkable increase of immature megakaryocytes and prominent proliferation of lymphoblasts. Flow cytometric analysis of the bone marrow cells showed that the predominant cells in the lymphocyte cluster were of B-lineage (CD19) with CD10 (common acute lymphoblastic leukemia antigen) positive. Virus study showed a high titer of CMV antibody. Cytomegalovirus DNA was detected by the polymerase chain reaction (PCR) method in urine, peripheral cells and marrow cells. Low-grade fever, diarrhea and petechiae were accompanied by mild liver dysfunction. Complete remission was made with intravenous high-dose immunoglobulin (IVIg) without progression to overt acute leukemia. The percentage of CD10⁺/CD19⁺ lymphocytes in bone marrow also diminished. We postulated that the proliferation of immature lymphocytes and megakaryocytes in bone marrow was caused by maturation arrest that might result from CMV infection.     

Factors associated with bone marrow stem cell yield for pediatric allogeneic stem cell transplantation: The impact of donor characteristics

The aim of this study was to investigate the effects of donor characteristics on CD34⁺ cell yield in BM harvest. Between April 2010 and November 2013, consecutive donors who underwent BM harvesting in our BM transplantation unit were retrospectively investigated. Donors were classified into two groups: those who donated BM without mobilization (steady‐state BM donors) and those who received G‐CSF for stem cell mobilization (G‐CSF‐primed BM donors). Donor characteristics (age, gender, race, body weight, BMI, and laboratory factors including donor's leukocyte, platelet, and monocyte) and their relationship with total nuclear cell and CD34⁺ cell numbers has been evaluated. A total of 64 healthy related donors (29 males/35 females, median age 11.2 years; 49 [76.6%] younger than 18 and 36 [56.3%] younger than 12 years) were included in the study. The median CD34⁺ cell yield in the harvest was 0.12×10⁶/L (0.02‐0.21) in SS‐BM donors and 0.18×10⁶/L (0.09‐0.67) in GP‐BM donors (P=.03). Median of CD34⁺ cell count given to recipients was 2.6×10⁶/recipient body weight (1.3‐19.3) in SS‐BM yields and 3.8×10⁶/recipient body weight (1.1‐10.2) in GP‐BM yields, respectively. Multiple regression analysis showed that donor height and pre‐G‐CSF platelet were the most important parameters to obtain a sufficient BM harvest. Our data suggest that the shorter donors and the donors with higher thrombocyte counts may offer more hematopoietic stem cell. The height and thrombocyte count of the donors should be taken into consideration before planning the targeted CD34⁺ cell count especially for pediatric donors.     

HCMV感染患儿HLA-DR、CD4+CD25+调节性T细胞及IL-17、IL-27表达水平与肝损害的相关性研究

目的 探讨人巨细胞病毒（HCMV）感染患儿HLA-DR、CD4⁺CD25⁺调节性T细胞（CD4⁺CD25⁺Treg）以及IL-17、IL-27的表达水平与肝功能损害的相关性。方法 以21例HCMV感染肝功能损害患儿和21例HCMV感染非肝功能损害患儿为研究对象。流式细胞术检测两组外周血CD4⁺CD25⁺Treg、HLA-DR细胞的表达;ELISA检测血浆IL-17及IL-27表达。结果 与非肝损组比较,肝损组患儿CD4⁺CD25⁺Treg比率较低,IL-17、IL-27表达水平较高,差异均有统计学意义（P < 0.05）。IL-17及IL-27水平与CD4⁺CD25⁺Treg细胞比率呈负相关（P < 0.01）。结论 CD4⁺CD25⁺Treg细胞介导的免疫耐受失衡及IL-17、IL-27等炎症因子过度激活可能参与HCMV感染肝功能损害发生。     

The impact of donor age and sex on the nucleated cell count and CD34 count in healthy bone marrow donors

BM remains an important source of stem cells. The BM characteristics change with age but the estimation of CD34 calculation of one CD34+ cell per 100 nucleated cells is used for all donors including pediatric donors in the operating room before getting the actual CD34 count. In order to see whether this formula is applicable for pediatric donors, we designed a retrospective study to see the affect of the age and sex on the BM NCC, CD34 count, and CD34/NCC ratios. Ninety‐eight BM collections from 91 related donors were evaluated retrospectively (median age: nine yr [1.5–54 yr]; M/F: 41/50). A significant negative correlation was found between the donor age and NCC (r = −0.229, p < 0.05), CD34 count (r = −0.563, p < 0.01), and CD34/NCC (r = −0.664, p < 0.01). The negative correlation for CD34 count and CD34/NCC persisted in female and male donor groups. When donors younger than 16 yr of age were compared with the older donor group, the median NCC, median CD34 count, and CD34/NCC were significantly lower in the older group (p < 0.01). Age and sex have to be taken into consideration to avoid unnecessary high‐volume collections and increased operating room time in the younger donors.     

Prognostic impact of CD200 and CD56 expression in pediatric B-cell acute lymphoblastic leukemia patients

This study aimed to determine the prognostic impact of CD200 and CD56 expression in pediatric B cell acute lymphoblastic leukemia (B-ALL) patients, both of which have been implicated in immune tolerance and previously suggested as independent risk factors. CD200 has a central role in immune tolerance that protects stem cells and other critical tissues from immune damage. The expression of CD200/CD56 in leukemic blasts were assessed in leukemic blasts before chemotherapy in 43 bone marrow (BM) and/or peripheral blood (PB) samples by flow cytometry. Twenty eight of 43 B-ALL cases (65%) showed CD200 positive expression, 5 of 43 cases (11.6%) showed CD56 expression, and only 2 patients (4.7%) expressed both CD200 and CD56. Patients with CD200⁺ and CD56⁺ were significantly associated with lower platelet count; less tendency for induction of remission response as compared to negative ones (p = .01 for both). The overall survival (OS) and DFS were significantly shorter in CD200⁺ and CD56⁺ cases as compared to those with CD200^? and CD56^? expression. In conclusion, CD200 and/or CD56 positive expression in B-ALL at diagnosis suggest a poor prognosis and may be associated with biological aggressiveness.     

Effects of amifostine on clonogenic mesenchymal progenitors and hematopoietic progenitors exposed to radiation

PURPOSE: To determine the radiation sensitivities of mesenchymal progenitors and hematopoietic progenitors, and to determine the in vitro effects of amifostine on hematopoietic and mesenchymal progenitors exposed to radiation. METHODS: Radiosensitivity of mesenchymal progenitor cells was determined by exposing marrow low-density cells to radiation at doses of 100 to 800 cGy. Mesenchymal cell colonies were established by plating 2.5 x 10(5) marrow low-density cells in long-term marrow culture medium (LTCM). The size, frequency, and cellular composition of the mesenchymal progenitor cells were scored after 14 days of incubation. Mesenchymal progenitor cells were subdivided into progenitors forming fibroblast and adipocyte mixed colonies (CFU-FA), and pure fibroblast colonies (CFU-F). Hematopoietic progenitors were assessed by methylcellulose-based assay. RESULTS: Radiation at 100 cGy caused a mild decrease in CFU-F and CFU-FA derived colonies by 12% and 13%, respectively; 200 cGy decreased CFU-F by 36% and CFU-FA by 52%; 400 cGy decreased CFU-F by 50% and CFU-FA by 86%; and 600 cGy decreased CFU-F by 24%, with total absence of CFU-FA. Pretreatment with amifostine protected 100% of CFU-F at 100 and 200 cGy, 84% at 400 cGy, 46% at 600 cGy, and 14% at 800 cGy. With CFU-FA colonies amifostine pretreatment provided only minimal radioprotection. For hematopoietic progenitors radiation at 100 cGy reduced CFU-GM by 74% but had no significant effect on CFU-GEMM and BFU-E. Radiation at 200 cGy decreased CFU-GEMM by 72%, BFU-E by 54%, and CFU-GM by 84%; 400 cGy further decreased CFU-GEMM by 83%, BFU-E by 81%, and CFU-GM by 93%. Pretreatment with amifostine resulted in twofold stimulation of CFU-GEMM and BFU-E colonies. All BFU-E colonies were protected up to 200 cGy. For CFU-GEMM amifostine pretreatment resulting in 68% at 200 cGy and 31% at 400 cGy. For CFU-GM colonies it was 54% at 100 cGy, 32% at 200 cGy, and 12% at 400 cGy. CONCLUSIONS: Mesenchymal progenitor cell subpopulations are differentially sensitive to radiation. Amifostine protects both mesenchymal and hematopoietic progenitors against radiation injury, though the level of protection appears to be dependent upon the sensitivities of these progenitor cells to radiation. Amifostine is a potent stimulant of BFU-E and CFU-GEMM progenitor colonies.     

CD4+CD25+Foxp3+调节性T细胞与IL-33在儿童哮喘发病机制中的作用

目的 探讨CD4+CD25+Foxp3+调节性T细胞(Treg)与IL-33在儿童哮喘发病中的作用.方法 采用流式细胞仪检测45例哮喘患儿(哮喘组)、50例呼吸道合胞病毒感染喘息患儿(喘息组)及40例健康儿童(对照组)外周血CD4+CD25+Foxp3+Treg细胞百分比,采用ELISA法检测各组外周血血清IFN-γ、IL-4、IL-5及IL-33浓度,进行比较分析.结果 哮喘组患儿体内CD4+CD25+Foxp3+Treg 水平较喘息组及对照组均降低(P<0.05);哮喘组患儿体内IL-33水平较喘息组及对照组均升高(P<0.05),哮喘组患儿体内CD4+CD25+Foxp3+Treg与IL-33呈负相关(r=-0.156,P<0.01).结论 在哮喘患儿发病机制中,CD4+CD25+Foxp3+Treg与IL-33可能存在相互作用.     

手足口病患儿Th17、CD4+CD25+调节性T细胞的变化及意义

目的 研究手足口病患儿外周血Th17、CD4+CD25+调节性T细胞（CD4+CD25+Treg）的变化。方法 以89例手足口病患儿为研究对象,其中普通型55例,重型34例,另选取30例健康儿童为对照组;采用流式细胞术检测各组外周血CD4+CD25+Treg细胞、Th17细胞在CD4+T淋巴细胞中所占的比例;采用ELISA法检测IL-10、TGF-β和IL-17在各组的表达水平。结果 与对照组相比,重型和普通型手足口病患儿的Th17细胞、IL-17水平均明显升高（均P<0.05）,而CD4+CD25+Treg细胞、IL-10、TGF-β水平则显著下降（均P<0.05）,且手足口病患儿外周血Th17、IL-17水平随病情严重而增高,而CD4+CD25+Treg细胞、IL-10、TGF-β水平随病情严重而降低。结论 手足口病患儿外周血Th17细胞应答增强,而CD4+CD25+Treg细胞应答降低,Th17细胞与CD4+CD25+Treg细胞比例失衡可能在手足口病发病机制中具有重要作用。     

阵发性睡眠性血红蛋白尿症患儿骨髓CD34+CD59+细胞的体外扩增及克隆变化趋势的研究

目的：研究阵发性睡眠性血红蛋白尿症（PNH）患儿骨髓CD34+CD59+细胞的分离、纯化及体外扩增的条件,并对扩增后细胞的长期造血能力进行评估,为探索PNH患儿新的治疗途径提供实验依据。方法：采用磁珠-流式细胞仪二步分选法,从PNH患儿骨髓中分选出CD34+CD59+细胞,在不同细胞因子组合条件下进行体外扩增,并培养集落形成细胞（CFCs）及长期培养始动细胞（LTC-IC）。结果：体外扩增最适宜的细胞因子组合为干细胞因子+白细胞介素（IL）-3 +IL-6+FLT3配基+巨核细胞生成素+红细胞生成素,最适宜的扩增时机为第7天,此时CD34+CD59+细胞的扩增倍数为30.4±6.7倍。CD34+CD59+细胞在扩增以后,仍为CD59+细胞,能保持较好的形成CFCs的能力,仍能形成 LTC-IC,与扩增前相比差异无统计学意义;但PNH患儿CD34+CD59+细胞的扩增能力低于正常对照的CD34+细胞（P<0.01）。结论：PNH患儿的CD34+CD59+细胞能够进行体外扩增,扩增后的细胞仍然具备长期造血重建的能力,并且无向PNH克隆转化的趋势,说明对PNH患儿进行自体移植在临床上有可行性。     

草分枝杆菌对哮喘小鼠CD4+CD25+调节性T细胞和Th17细胞平衡的影响

目的：评估早期应用草分枝杆菌F.U.36注射液干预治疗对哮喘小鼠CD4+CD25+调节性T细胞（Treg）和Th17细胞平衡的影响,探讨草分枝杆菌的免疫调节作用。方法：将30只雌性BALB/c小鼠随机分为对照组、哮喘组和草分枝杆菌治疗组（治疗组）,每组10只。通过注射和雾化吸入鸡卵蛋白（OVA）制备哮喘模型;治疗组于第1次致敏前2周腹腔注射草分枝杆菌F.U.36注射液0.57?μg,隔日1次,共3次;对照组以生理盐水代替致敏液。所有小鼠于末次激发后24 h处死,取小鼠左肺组织作病理切片观察炎症改变;同时利用流式细胞仪检测各组小鼠脾单个核细胞CD4+CD25+ Treg细胞、Th17细胞占CD4+T细胞的百分比;酶联免疫吸附试验（ELISA）检测各组小鼠血清和支气管肺泡灌洗液中细胞因子IL-10、IL-17的表达水平。结果：哮喘小鼠脾单个核细胞CD4+CD25+ Treg细胞百分比及IL-10的表达水平较对照组明显降低（P<0.05）,Th17细胞的百分比及IL-17表达水平较对照组明显增高（P<0.05）;治疗组小鼠CD4+CD25+ Treg细胞百分比及IL-10表达水平较哮喘组明显升高（P<0.05）,而Th17细胞百分比及IL-17表达水平较哮喘组明显降低（P<0.05）。结论：早期应用草分枝杆菌F.U.36干预性治疗哮喘小鼠,可增加CD4+CD25+ Treg细胞的数目并促进IL-10的产生,同时抑制Th17细胞的表达及IL-17的产生。     

The effect of short-course high-dose methylprednisolone on peripheral blood CD34+ progenitor cells of children with acute leukemia during remission induction therapy

This study was undertaken to determine the effect of short-course high-dose methylprednisolone (HDMP) treatment on peripheral blood (PB) CD34+ progenitor cells during remission induction treatment in 11 children with newly diagnosed acute leukemia (7 with ALL, 4 with AML) whose bone marrow (BM) cells expressed fewer than 5% CD34 at the time of diagnosis. All children who had no infection were given HDMP as a single daily oral dose of 30 mg/kg for the first four days of induction therapy. The number of CD34+ progenitor cells were determined by flow cytometry before and after four days of HDMP treatment. While the number of PB blast cells significantly decreased after only a four-day course of HDMP treatment, the number of PB CD34+ progenitor cells increased in all patients. In addition, after four days of HDMP treatment polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC) increased significantly (p < 0.05). We suggest that the potential beneficial effects of HDMP in the induction treatment of acute leukemia may occur partly by the stimulation of PB CD34+ hematopoietic progenitor cells in a short period of time.     

干细胞因子联合粒细胞集落刺激因子对单侧输尿管梗阻大鼠骨髓干细胞及内皮祖细胞的动员作用

目的：探讨干细胞因子(SCF)联合粒细胞集落刺激因子（G-CSF）对单侧输尿管梗阻(UUO)大鼠骨髓干细胞及内皮祖细胞的动员作用。方法：56只Wistar大鼠随机分为7组：正常对照组、SCF组、G-CSF组、SCF联合G-CSF组（SCF-G组）、假手术组、UUO组、SCF联合G-CSF用于UUO组（UUO+SCF-G组）。实验第5天采集血标本后：①流式细胞仪检测静脉血单个核细胞中CD34+、CD34+/CD133+细胞;②检测血清谷丙转氨酶、谷草转氨酶、尿素氮、肌酐水平。结果：①对照组大鼠静脉血单个核细胞中CD34+细胞为（0.13±0.01）%,假手术组CD34+细胞为（0.24±0.06）%与对照组比较差异无显著性（P>0.05）;其余各组与对照组比较CD34+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组（3.04±0.42）%及SCF-G组（2.10±0.28）%增高最为明显;②对照组大鼠静脉血单个核细胞中CD34+/CD133+细胞为（0.02±0.01）%,假手术组CD34+/CD133+细胞（0.05±0.02）%与对照组比较差异无显著性（P>0.05）;其余各组与对照组比较CD34+/CD133+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组（0.73±0.17）%增高最为明显;③7组血清尿素氮,肌酐、谷丙转氨酶水平无明显增高,UUO组谷草转氨酶水平较其他6组增高,差异有显著性。结论：SCF和G-CSF对干细胞和内皮祖细胞的动员效果并非完全呈平行关系,联合使用可提高内皮祖细胞和干细胞的动员率,短期内未见肝、肾毒副作用。［中国当代儿科杂志,2007,9（2）：144-148］     

Unrelated CD3/CD19-Depleted Peripheral Stem Cell Transplantation for Hurler Syndrome

For patients with mucopolysaccharidosis type IH (MPS1-H; Hurler syndrome), early allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice. One boy and one girl aged 20.5 and 22 months, respectively, with MPS1-H received a conditioning regimen consisting of thiotepa, fludarabine, treosulfan, and ATG. Grafts were peripheral blood stem cells from unrelated donors (10/12 and 11/11 matched), that were manipulated by CD3/CD19 depletion and contained 20.3 and 28.2 × 10⁶ CD34+ cells/kg body weight, respectively. Both patients achieved stable hematopoietic engraftment and stable donor chimerism. Neither acute or chronic graft-versus-host disease (GVHD) nor other severe transplant-related complications occurred. At a follow-up of 48 and 37 months, both patients are alive and well with normal levels of α-L-iduronidase and have made major neurodevelopmental progress. Treosulfan-based conditioning offers the advantage of reduced toxicity; the use of unrelated CD3/CD19-depleted peripheral stem cell grafts allows transfusion of high CD34+ cell numbers together with a “tailored” number of CD3+ cells as well as engraftment facilitating cells in order to achieve rapid hematopoietic engraftment while reducing the risk of graft rejection and GVHD. This regimen might be an additional option when unrelated donor HSCT is considered for a patient with MPS1-H.     

Prolonged neutropenia due to antihuman neutrophil antigen 2 (CD177) antibody after bone marrow transplantation

We describe a patient who presented with prolonged neutropenia due to anti‐human neutrophil antigen (HNA)‐2 (CD177) antibody after allogeneic bone marrow transplantation. A granulocyte immunofluorescence test showed bimodal expression of antineutrophil antibody that resulted from specific binding of anti‐HNA‐2 to CD177⁺ neutrophils from healthy donors. The patient did not respond to granulocyte colony stimulating factor, which is able to upregulate CD177 expression on neutrophils. The low percentage of CD177⁺ cells in the few remaining neutrophils supports the possibility of elimination of CD177‐upregulated neutrophils. These findings might explain an inferior response to neutrophil growth factors in neutropenia secondary to anti‐HNA‐2 antibody.