Neonatal platelets from cord blood and peripheral blood

The haemostatic system in neonates is different from that of adults, possibly contributing to an increased incidence of bleeding disorders, such as intracranial hemorrhage. In this study, we analyzed platelets from cord blood and peripheral blood, collected at three time points after delivery from 20 term and 37 preterm neonates as well as blood from 20 healthy adults. Platelet membrane glycoproteins (GP) were quantified and P-selectin expression and PAC-1 binding ability before and after stimulation with TRAP were analyzed by whole blood flow cytometry. We found no significant differences in neonatal platelets from cord blood and peripheral blood within the first 24?h of life. Platelets from infants less than 30 weeks of gestation expressed lower levels of GP (33271?±?9381 vs. 44085?±?17287 for GPIIIa, P?<?0.05) and were less reactive than platelets from term newborns (4.3?±?3.3 vs. 20.1?±?11.8% PAC-1 positive platelets after stimulation with TRAP, P?<?0.05). A significantly lower level of GPIIb/IIIa expression on platelets from peripheral blood was seen in term newborns as well as preterm infants, compared to adults. There was only a partial enhancement in the degranulation ability (α-granules) (13.4?±?12.3 vs. 50.3?±?16.1% P-selectin positive platelets, P?<?0.05) and no significant increase for PAC-1 binding (13.6?±?10.9 vs. 15.3?±?5.9% PAC-1 positive platelets, P?=?0.8) during the first 12 days of life. In conclusion, we could demonstrate that neonatal platelet reactivity increases with gestational age.     

Differential maturation of megakaryocyte progenitor cells from cord blood and mobilized peripheral blood

OBJECTIVE: In comparison with stem cell transplantation using bone marrow or cytokine-mobilized peripheral blood, cord blood transplantation is characterized by delayed engraftment, in particular platelet recovery. The differences in the kinetics of engraftment may be related to quantitative differences in the numbers of stem cells and megakaryocyte progenitor cells and/or to qualitative differences between megakaryocyte progenitor cells in these grafts. We compared the hematopoietic composition of these grafts and determined the distribution of mature and immature megakaryocyte progenitor cells in cord blood and mobilized peripheral blood and their in vitro kinetic behavior. METHODS: Megakaryocyte progenitor cell subpopulations from cord blood (CB) and mobilized peripheral blood (PBSC) were expanded in vitro in the presence of mpl-ligand. The developmental differences during expansion of megakaryocyte progenitors were analyzed by flow cytometry and progenitor assays. RESULTS: We found that the immature (CD34(+)/CD41(-)) subpopulation from CB contains more than 98% of all megakaryocyte progenitor cells, responsible for 99% of all megakaryocytic cells cultured during 2 weeks. The CB CD34(+)/CD41(+) subpopulation shows no contribution to megakaryocytic cell formation. In contrast, in PBSC the mature (CD34(+)/CD41(+)) subpopulation contains 7% of all megakaryocyte progenitor cells. Moreover, CD34(+) cells from CB and PBSC also showed distinct phenotypic differences during maturation in vitro. PBSC megakaryocyte progenitor cells transiently express both CD34 and CD41 during maturation in vitro, whereas CB progenitor cells transiently lack expression of both markers before differention into (CD34(-)/CD41(+)) megakaryocytic cells. CONCLUSION: The in vitro data indicate the presence of different developmental stages of megakaryocyte progenitor cells in CB as compared to PBSC. These differences in composition and maturation between CB and PBSC may be related to the different kinetics of engraftment following transplantation of these stem cell sources.     

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.     

Antitumor activities of human dendritic cells derived from peripheral and cord blood

AIM: To observe the biological specialization of humanperipheral blood dendritic cells (DC) and cord blood derivedDC and its effects on effector cells killing humanhepatocarcinoma cell line BEL-7402 in vitro.METHODS: The DC biological characteristics were detectedwith immunohistochemical and MTT assay. Two antitumorexperimental groups are: peripheral blood DC and cordblood DC groups. Peripheral blood DC groups used LAKcells as the effector cells and BEL-7402 as target cells, whilecord blood DC groups used CTL induced by tumor antigentwice pulsed DC as effector cells and BEL-7402 as targetcells, additional peripheral blood DC and cord blood DC areadded to observe its stimulating activities to effector cells.The effector's cytotoxicity to tumor cells were detected withneutral red colorimetric assay at two effector/target ratios of5:1 and 10: 1.RESULTS: Peripheral blood DC and cord blood DC highlyexpressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100protein. The stimulating activities to lymphocyteproliferation were compared between experimental groups(DC added) and control group (no DC added). In sixexperiment subgroups, the DC/lymphocyte ratio wassequentially 0.25: 100, 0.5: 100, 1: 100, 2: 100, 4: 100 and 8:100, A values(x± s) were 0.75396± 0.009, 0.84916± 0.010,0.90894± 0.012, 0.98371 ± 0.007, 1.01299 ± 0.006 and 1.20384± 0.006 in peripheral blood DC groups and 0.77650 ± 0.005,0.83008± 0.007, 0.92725 ± 0.007, 1.05990 ± 0.010, 1.15583 ±0.011, 1.22983 ± 0.011 in cord blood DC groups. A value was0.59517 ± 0.005 in control group. The stimulating activitieswere higher in experimental groups than in control group ( P＜ 0.01 ), which were increased when the DC concentrationwas enlarged ( P ＜ 0.01 ). Two differently derived DCs hadthe same phenotypes and similar stimulating activities ( P ＞0.05). In peripheral blood DC groups, the cytotoxicity (x ±s) of the LD groups (experimental groups) and L groups(control group) was 58.16% ± 2.03% (5: 1), 46.18% ±2.25% (10:l) and 38.13% ± 1.29% (5: 1) and 65.40% ±1.56% (10: 1), respectively; in cord blood DC groups, TDgroups (experimental groups ) and T groups (controlgroups) were 69.71% ± 2.33% (5: 1 ), 77.64% ± 1.94% ( 10:1) a. nd 56.89% ± 1.82% (5:1) and 60.99% ± 1.42% (10:1),respectively. The cytotoxicity activities were enhanced withincreased effector/target ratio ( P ＜ 0.01 ). At the sameeffector/target ratio, the cytotoxicity of experimental groupswas higher than that of control groups ( P ＜ 0.01 ). Thecytotoxicity of cord blood DC groups were higher than ofperipheral blood DC groups ( P ＜ 0.01 ).CONCLUSION: Peripheral blood DC and cord blood DC aremature DCs in morphology and function, both can enhancethe effector cell killing activities to hepetocarcinoma cells.DC pulsed with tumor antigen can induce ligher specific CTLactivity than unpulsed DC.     

Neonatal rotavirus infection and its relation to cord blood antibodies

Among 274 neonates born at the maternity services of an urban hospital in India, 36.1% of the infants shed rotavirus in feces (as detected by ELISA) by 72 h of life. The excretion rate increased to 70.3% among the 120 infants who stayed for 5 days or more at the hospital. Diarrhoeal symptoms of mild and self-limited nature were observed only in 19.2% of the rotavirus excretors, the remaining being asymptomatic. Among the 98 infants who received supplement feeds, 49% acquired rotavirus infection as against 24.7% of the 150 exclusively breast fed infants (p less than 0.001). Viral RNA in the feces of all rota positive infants showed the same electropherotype, indicating infection from a common source. The mean percentage rotavirus inhibitory activity of cord sera in the infected and non-infected infants was 50.2 +/- 21.7 and 56.6 +/- 19.2 respectively (p greater than 0.05), suggesting that cord blood antibodies do not offer significant protection against neonatal rotavirus infection.     

人脐血和外周血树突状细胞诱导抗肝癌活性

目的　观察人脐血和外周血树突状细胞 (dendriticcells ,DC)的生物学特性及其对效应细胞体外杀伤人肝癌细胞株BEL 740 2 (B)的影响。方法　免疫组化和MTT法检测DC的生物学特性。抗肿瘤实验则分两大组 ,人外周血DC组为B +LAK +DC ,人脐血DC组为B +DC CTL ,检测效应细胞的细胞毒活性。结果　①人外周血和脐血DC均高表达HLA ABC、HLA DR、HLA DQ、CD54和S 10 0蛋白 ;刺激淋巴细胞增殖的效应均比对照组明显增高 (P <0 0 1) ,两种来源DC无显著性差异 (P >0 0 5)。②人外周血DC组和脐血DC组的细胞毒活性均为实验组 >对照组 (P <0 0 1) ,人脐血DC组 >人外周血DC组 (P <0 0 1)。结论　人外周血和脐血DC均为形态和功能成熟的DC ,均能明显增强效应细胞杀伤肝癌细胞的活性 ;而经肿瘤抗原致敏DC诱导的特异性CTL杀伤活性更强。提示临床上可根据患者的具体情况选择DC的来源     

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Background

Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes.

Design and Methods

We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated.

Results

Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival.

Conclusions

Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.     

Relationship of intracellular magnesium of cord blood platelets to birth weight

Magnesium (Mg(2+)) has an important role in insulin action, and insulin stimulates Mg(2+) uptake in insulin-sensitive tissues. Impaired biologic responses to insulin are referred to as insulin resistance. Diabetic patients and obese subjects are reported to have intracellular magnesium ([Mg(2+)](i)) deficiency. Many epidemiologic studies have disclosed that restricted fetal growth has been associated with increased risk of insulin resistance in adult life. We studied the relationship of [Mg(2+)](i) in cord blood platelets to birth weight. The subjects were 19 infants who were small for gestational age (SGA) and 45 who were appropriate for gestational age (AGA). By using a fluorescent probe, mag-fura-2, we examined the basal and insulin-stimulated [Mg(2+)](i) of platelets in the cord blood. Cord plasma insulin-like growth factor-1 (IGF-1)and leptin levels were determined with the use of enzyme-linked immunosorbent assay (ELISA). Birth weight was correlated with cord plasma IGF-1 (P < .001) and leptin (P < .005). Mean basal [Mg(2+)](i), but not plasma Mg(2+), was lower in the SGA than in the AGA group (291 +/- 149 micromol/L v 468 +/- 132 micromol/L, P < .001). The basal [Mg(2+)](i) was significantly correlated with the birth weight (P < .001) as well as birth length (P < .001). At 60 seconds after stimulation with insulin, there was no significant difference in stimulated [Mg(2+)](i) between the SGA and AGA groups. Although the SGA group had low [Mg(2+)](i), the platelets had good potentiality to compensate for low [Mg(2+)](i). [Mg(2+)](i) reflects the extent of fetal growth. Decreased [Mg(2+)](i) in SGA might underlie the initial pathophysiologic events leading to insulin resistance.     

脐血、外周血内皮祖细胞分化成内皮细胞的实验研究

目的探讨人的脐血、外周血内皮祖细胞（endothelialprogenitorcells,EPCs）体外分离、纯化、诱导扩增和分化为内皮细胞的可行性,并检测其表型和功能。方法新鲜脐血和健康成年人的外周血,使用Ficoll密度梯度离心法得单个核细胞,在M199培养基中体外培养,3d后去除悬浮细胞,继续培养,诱导EPCs增殖和分化。流式细胞仪检测EPCs标志CD34和内皮细胞特异性标志CD31表型,RTPCR检测ecNOS,flk1/KDR基因水平表达,免疫组化验证蛋白水平表达,并进一步通过NO活性的变化检测内皮细胞的功能。结果流式细胞仪检测,外周血单个核细胞（peripheralbloodmononuclearcells,PBMC）刚分离时,CD34阳性表达率为（1.1±0.8）%,培养3d后为（16.9±6.2）%。细胞形态观察发现,刚分离的单个核细胞呈圆形,形态小,3d后有明显集落形成,7d后梭形细胞线样排列,随培养时间增加,细胞形态逐渐变大,呈现出典型铺路石样改变。脐血单个核细胞（umbilicalcordbloodmononuclearcells,CBMC）和PBMC培养10d后,CD31阳性表达率分别为（76±17）%和（82±9）%。RTPCR检测有内皮细胞特异性成分ecNOS,flk1/KDR的表达。免疫组化染色,细胞膜和细胞浆中有弥漫性棕色出现,呈阳性反应,证实了蛋白水平的表达。培养10d的贴壁细胞随着VEGF浓度增加,NO生成增加,具有内皮细胞的功能。结论脐血,外周血EPCs体外分离,纯化,诱导培养后的贴壁细胞表型检测,大部分细胞具有内皮系标志物,并具有产生NO功能。     

Unrelated peripheral blood and cord blood hematopoietic stem cell transplants for thalassemia major

Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment available for thalassemia major. While sibling donors are usually used, unrelated bone marrow transplants have also recently been found to be successful. In this report of two patients, we show that, besides using unrelated bone marrow, peripheral blood stem cell and cord blood transplantations from unrelated donors are viable options. We believe a significant contributory factor in both our cases was the large cell dose used.     

The phenotypic and functional characteristics of umbilical cord blood and peripheral blood natural killer cells

Allogeneic hematopoietic cell transplantation can be curative for patients with high-risk acute leukaemia. Umbilical cord blood (UCB) is an increasingly used source of allogeneic stem cells for patients who are in need of a transplant, but do not have a sibling donor. This review highlights the similarities and differences between the natural killer (NK) cells obtained from adult peripheral blood (PB) and UCB. These two cell sources show similar percentages of NK cells, including the major CD56^dim and CD56^bright subpopulations. UCB also contains an additional CD56⁻CD16⁺ subset, not typically found in PB. In addition, there are a number of progenitor cell populations in UCB that can give rise to NK cells. Some studies showed that UCB NK cells express a relatively higher percentage of inhibitory receptors (CD94/NKG2A and killer-cell immunoglobulin-like receptors) and less adhesion molecules. Resting UCB NK cells also show significantly less cytotoxicity compared to PB NK cells. However, following cytokine stimulation, the cytotoxicity of UCB NK cells can be rapidly increased to levels that are comparable to PB NK cells. Activation and expansion protocols for UCB NK cells are briefly reviewed. Lastly, we outline the early use of UCB NK cells in clinical trials.