Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

PHA激活的脐血T细胞条件培养液对人骨髓CFU-c的抑制

本文研究了PHA刺激18小时收获的脐血T细胞条件培养液(PHA-TCM)对正常人骨髓CFU-c的影响。结果显示PHA-TCM能够显著抑制CFU-c的生长,这种抑制与PHA-TCM浓度有关。并发现经PHA-TCM作用后M型集落比例明显降低。PHA-TCM中未检出IFN和IL-2活性。进一步研究证实,PHA-TCM中CFU-c抑制活性是一种对酸碱敏感对热相对不敏感的蛋白质,其分子量大于10,000道尔顿。     

利用IL－3／GM－CSF融合蛋白（PIXY－321）扩增脐血造血细胞方法的初步建立

利用单克隆抗体免疫磁珠吸附方法分离脐血ＣＤ３４＋细胞,并观察了ＩＬ３／ＧＭＣＳＦ融合蛋白（ＰＩＸＹ３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ３和ＧＭＣＳＦ单独及联合应用。在液体培养条件下,每毫升２０ｎｇＰＩＸＹ３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血祖细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。     

LY2228820 Dimesylate,a Selective Inhibitor of p38 Mitogen-activated Protein Kinase,Reduces Angiogenic Endothelial Cord Formation in Vitro and in Vivo

LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen-activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor- and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation, with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6, and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK-specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A-stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.     

44例不同孕周脐带脱垂患者的临床资料分析

目的:探讨不同孕周脐带脱垂患者相关因素的差异。方法:回顾性比较分析2012年01月至2017年12月我院收治的44例脐带脱垂患者的临床资料。将患者按照脐带脱垂发生的孕周分为足月组、早产组及流产组,使用SPSS18.0统计软件处理数据。结果:我院近六年脐带脱垂总的发病率为1.829/1000。44例患者中,足月组7人,占15.91%;早产组22人,占50%;流产组15人,占34.09%。三组患者的年龄、产次及孕次均无显著统计学差异(P0.05)。足月组新生儿apgar评分最高,与其它两组相比均有统计学差异(P0.05),早产组apgar评分显著高于流产组(P0.05);足月组剖宫产率为100%,早产组为63.64%,流产组则为13.13%,三组患者剖宫产率比较存在统计学差异(P=0.000),足月组剖宫产率与早产组比较无统计学差异(P=0.075),足月组剖宫产率与流产组比较有统计学差异(P=0.000),早产组剖宫产率与流产组比较有统计学差异(P=0.003)。足月组异常胎方位的发生率显著低于早产组(P=0.038)。早产组胎儿数(单胎、双胎)与足月组及流产组相比均有统计学差异(P0.05),而足月组与流产组胎儿数则无统计学差异(P0.05)。早产组双胎妊娠占比例更高。三组患者发生脐带脱垂的地点比较无统计学差异(P=0.256)。结论:不同孕周是否发生脐带脱垂与患者的年龄、产次、孕次及地点无关。脐带脱垂较多发生于早产者,且早产患者中双胎、异常胎方位发生率更高。一旦发生脐带脱垂,尤其是有机会存活的胎儿,应以最快的方式娩出胎儿,提高新生儿存活几率。     

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age (P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth (P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.     

Down-regulation of human<Emphasis Type="Italic">NDR</Emphasis> gene in megakaryocytic differentiation of erythroleukemia K562 cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues.NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.     

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume,hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34(+) cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34(+) cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34(+) cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 degrees C. Values for the hydraulic conductivity were 3.2 x 10(-8) and 2.8 x 10(-7)cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2 x 10(-7) and 7.4 x 10(-6)cm/s, respectively. Clonogenic assays indicated that the ability of CD34(+) cells to grow and differentiate was significantly impaired outside the limits 0.6-4x isotonic. Based on the Boyle van't Hoff plot, the tolerable limits for cell volume excursion were therefore 45-140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 degrees C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34(+) cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.