High-density lipoprotein subclasses and esterification rate of cholesterol in children: effect of gender and age

Since the development of coronary heart disease (CAD) is affected by a specific pattern of plasma high density lipoprotein (HDL) effects it may be useful to know whether this occurs already in childhood. In this study we evaluated particle size distribution of HDL by gradient gel electrophoresis and the determination of cholesterol esterification rate (FER_HDL) in plasma depleted of apo B lipoproteins in 221 children (108 boys and 113 girls) aged 4 months to 20 years. Total plasma- (TC), low-density lipoprotein- (LDL-C) and HDL- (HDL-C) cholesterol, HDL unesterified cholesterol (HDL-UC) and plasma triglycerides (TG) were also measured. There were no significant gender and age differences with respect to the plasma TC, LDL-TC and TG but concentration of HDL-TC increased with age. Post-pubertal girls had significantly higher relative concentrations of HDL_2b compared to boys (30.4% vs 17.2%), while HDL_3b,c was lower in post-pubertal girls (8.7% vs. 16.5 %). FER_HDL correlated inversely with HDL_2b and positively with HDL_3b,c particles and was significantly higher in boys of the post-pubertal group compared to girls (16.9%/h vs 12.5%/h). While in girls there was a positive correlation between age and HDL-C, HDL-UC and the relative concentration of HDL_2b no significant correlation were observed in boys. In girls the increase in TC showed a significant correlation with a simultaneous increase in HDL-C, HDL-UC and HDL_2b. In boys TC correlated significantly with changes in TG only. When HDL_2b and HDL_3b,c cholesterol levels are calculated from HDL-C concentration and per cent distribution the differences between males and females are further emphasized. These data indicate that HDL particle size distribution is age- and gender-dependent.     

Hypoglycémie hyperinsulinémique persistante du nouveau-né et du nourrisson

Persistent hyperinsulinemic hypoglycaemia of infancy (PHHI) is the most frequent cause of hypoglycaemia in infancy. Clinical presentation is heterogeneous, with variable onset of hypoglycaemia and response to diazoxide, and presence of sporadic or familial forms. Underlying histopathological lesions can be focal or diffuse. Focal lesions are characterised by focal hyperplasia of pancreatic islet-like cells, whereas diffuse lesions implicate the whole pancreas. The distinction between the two forms is important because surgical treatment and genetic counselling are radically different. Focal lesions correspond to somatic defects which are totally cured by limited pancreatic resection, whereas diffuse lesions require a subtotal pancreatectomy exposing to high risk of diabetes mellitus. Diffuse lesions are due to functional abnormalities involving several genes and different transmission forms. Recessively inherited PHHI have been attributed to homozygote mutations for the beta-cell sulfonylurea receptor (SUR1) or the inward-rectifying potassium-channel (Kir6.2) genes. Dominantly inherited PHHI can implicate the glucokinase gene, particularly when PHHI is associated with diabetes, the glutamate dehydrogenase gene when hyperammonaemia is associated, or another locus.     

Interpreting the improved outcome of patients with central nervous system metastases managed in clinical trials compared with standard hospital practice*

The aims were to determine the median survival and prognostic factors of patients with central nervous system (CNS) metastases managed with whole‐brain radiation therapy (WBRT), and to explore selection criteria in recently published clinical trials using aggressive interventions in CNS metastases. A retrospective audit was performed on patients managed with WBRT for CNS metastases. Potential prognostic factors were recorded and analysed for their association with survival duration. The proportion of patients with these factors was also compared with those of patients managed under three recently reported studies investigating aggressive interventions, such as radiosurgery and chemotherapy for CNS metastases. Seventy‐three patients were treated with WBRT for cerebral metastases over a 12‐month period. The median survival of the population was 3.4 months (95% confidence interval: 2.7–4.1), with 6‐ and 12‐month survival rates of 30 and 18%, respectively. Significant prognostic factors for prolonged median survival were Eastern Cooperative Oncology Group status 0–2 (P = 0.015), Medical Research Council neurological functional status 0–1 (P = 0.006), and Recursive Partitioning Analysis Class 2 versus Class 3 (P = 0.020). On multivariate analysis, younger patient age (P = 0.02) and better performance status (P < 0.01) were associated with improved outcome. When comparing these characteristics with selected published studies, our study cohort demonstrated a higher proportion of patients with poor performance status, a greater number of metastases per patient and a higher incidence of extracranial disease. This reflects the selected nature of patients in these published studies. Central nervous system metastases confer a poor prognosis and, for the majority of patients, aggressive interventions are unlikely to improve survival. The use of potentially toxic and expensive treatments should be reserved for those few in whom these studies have shown a potential benefit.     

Myelotoxic effects of the bifunctional alkylating agent bizelesin on human,canine and murine myeloid progenitor cells

 Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies have found bizelesin to be more toxic to beagle dogs than to rodents and that myelosuppression was the dose-limiting toxicity. This toxicity was dose- and time-dependent in all species. Due to the significant difference in the in vivo myelotoxicity between species, it was important to determine which one most closely resembles humans on a pharmacodynamic basis. Therefore, hematopoietic clonal assays were utilized to evaluate the effects of bizelesin on granulocyte-macrophage (CFU-gm) colony formation. Marrow cells were exposed in vitro to bizelesin (0.001–1000 nM) for 1 or 8 h and then assayed for colony formation. There was a 3-log difference in drug concentration at which 100% colony inhibition occurred (1 or 8 h) for murine CFU-gm versus human or canine CFU-gm. The IC₇₀ value after an 8-h bizelesin exposure for human CFU-gm (0.006±0.002 nM) was 2220-times lower than for murine CFU-gm (13.32±8.31 nM). At any given concentration, an 8 h drug exposure resulted in greater colony inhibition than a 1 h exposure for all species (P <0.05). Increasing exposure time from 1 to 8 h increased toxicity to human and canine CFU-gm much more than to murine CFU-gm. The clinically formulated drug solution was a more potent inhibitor of human colony formation than drug dissolved in DMSO. The IC₇₀ value after a 1-h exposure was 1.7 times lower for human CFU-gm with formulated bizelesin (0.106±0.105 nM) than bulk drug in DMSO (0.184±0.044 nM). The results of these in vitro clonal assays were qualitatively consistent with those seen in whole animal studies, suggesting that bizelesin will be a potent myelosuppressive agent in the clinic. Since the dose-limiting toxicity in preclinical models is myelosuppression and the in vitro sensitivity of human and canine CFU-gm is similar, the canine maximum tolerated dose (MTD) is better than the murine MTD to determine a safe starting dose for phase I clinical trials. Received: 27 July 1995/Accepted: 1 February 1996     

Main-chain flexibility and hydrophobicity of ionenes strongly impact their antimicrobial activity: an extended study on drug resistance strains and Mycobacterium

The spread of antibiotic-resistant pathogens and the resurgence of tuberculosis disease are major motivations to search for novel antimicrobial agents. Some promising candidates in this respect are cationic polymers, also known as synthetic mimics of antimicrobial peptides (SMAMPs), which act through the membrane-lytic mechanism. Development of resistance toward SMAMPs is less likely than toward currently employed antibiotics; however, further studies are needed to better understand their structure–activity relationship. The main objective of this work is to understand the cross-influence of hydrophobicity, main-chain flexibility, and the topology of ionenes (polycations containing a cationic moiety within the main-chain) on activity. To fulfill this goal, a library of ionenes was developed and compared with previously investigated molecules. The obtained compounds display promising activity against the model microorganisms and drug-resistance clinical isolates, including Mycobacterium tuberculosis. The killing efficiency was also investigated, and results confirm a strong effect of hydrophobicity, revealing higher activity for molecules possessing the flexible linker within the polymer main-chain.

A high significance of the main chain flexibility and an unexpected effect of hydrophobicity on the biological activity in series of ionenes was observed. The most potent among the tested polycations showed high activity toward clinical bacterial isolates.     

异一枝蒿酮酸的结构

从新疆一枝蒿(Artemisia rupestris L.)中分得一个新成分,命名为异一枝蒿酮酸(isorupestonic acid),根据光谱(IR,UV,MS.NMR),X-ray晶体衍射及CD谱分析,确定其结构及绝对构型。并经X-ray晶体衍射及CD谱分析修正了一枝蒿酮酸的绝对构型。     

角质形成细胞的培养及其表皮生长因子受体表达

目的:在成功分离人皮肤角质形成细胞的基础上,观察表皮生长因子受体在人皮肤角质形成细胞中的表达情况。方法:实验于2006-3/10在北京大学深圳医院中心实验室进行。采用dispase Ⅱ-trypsin两步消化法获取表皮基底层细胞,用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液进行培养。小鼠皮肤成纤维母细胞的预处理:向对数生长期的小鼠皮肤成纤维母细胞培养液中加入丝裂霉素C至终浓度为4mg/L,37℃下培养4h,弃去培养液,用D-Hank’s液洗3次,加入浓度为0.25g/L的胰蛋白酶消化,分离出细胞,离心(200g,5min),用黄素腺嘌呤二核苷酸培养液悬浮细胞,计数,以5.0×104/cm2的密度种于培养皿内,37℃、体积分数0.05的CO2培养箱下培养。角质形成细胞的培养:将分离的角质形成细胞悬浮在黄素腺嘌呤二核苷酸培养液中,以2.0×104/cm2的密度接种在前1天经丝裂霉素C处理的小鼠皮肤成纤维母细胞滋养层上,37℃、体积分数0.05的CO2培养箱下培养。24h换液,以后每3d换1次液。采用免疫细胞化学的方法检测表皮生长因子受体的表达,采用复合逆转录聚合酶链反应检测角质形成细胞中表皮生长因子受体mRNA的表达。结果:采用dispaseⅡ消化法分离了真皮和表皮,获得较多的角质形成细胞,可以避免真皮成纤维细胞的污染。人皮肤角质形成细胞在黄素腺嘌呤二核苷酸培养液中培养5d可见明显的集落,约10d可长满单层。免疫细胞化学显示表皮生长因子受体在细胞表面有明显的表达,复合逆转录聚合酶链反应显示表皮生长因子受体mRNA有明显的表达。结论:用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液可以较好地培养原代人皮肤角质形成细胞,表皮生长因子受体在细胞表面有明显的表达,这些结果为与表皮生长因子受体相关的皮肤病(如银屑病)的研究奠定了基础。     

富血小板血浆对人骨髓间充质干细胞成骨分化的抑制效应

目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。     

Zinc protoporphyrin in anemia of chronic disorders

Hematofluorometric determination of zinc protoporphyrin (ZPP) is a screening method for the assessment of iron deficiency (ID). Chronic disorders are frequently accompanied by anemias of unclear origin, most probably caused by an impairment of iron metabolism. We investigated the relevance of ZPP for the detection of derangements of iron metabolism in anemias of chronic disorders (ACD). In 19 patients with ACD caused by chronic inflammatory non-neoplastic diseases, ZPP was determined and correlated with ferritin, transferrin saturation, and hemoglobin (Hb). Marrow sideroblast counts and semiquantitative grading of the marrow hemosiderin were performed in all patients to exclude ID and to show the decreased iron bioavailability. In all ACD patients who exhibited the typical laboratory findings of disturbed iron metabolism, such as hypoferremia, decreased transferrin saturation, decreased bone marrow sideroblasts, and increased marrow hemosiderin, strongly elevated ZPP levels were found (131 +/- 23 mumol/mol heme). ZPP returned to normal after successful treatment of the underlying disease. This is shown in three patients with polymyalgia rheumatica. We conclude that the fluorometric determination of ZPP allows detection and quantification of derangements of iron metabolism associated with chronic inflammatory disorders. By recording the derangements quantitatively, ZPP allows monitoring of therapy of chronic inflammatory diseases.     

Urokinase mediates fibrinolysis in the pulmonary microvasculature

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in fibrinolysis remains unsettled. The contribution of uPA may depend on the vascular location, the physical properties of the clot, and its impact on tissue function. To study the contribution of urokinase within the pulmonary microvasculature, a model of pulmonary microembolism in the mouse was developed. Iodine 125 ((125)I)-labeled fibrin microparticles injected intravenously through the tail vein lodged preferentially in the lung, distributing homogeneously throughout the lobes. Clearance of (125)I-microemboli in wild type mice was rapid and essentially complete by 5 hours. In contrast, uPA(-/-) and tissue-type plasminogen activator tPA(-/-) mice, but not uPAR(-/-) mice, showed a marked impairment in pulmonary fibrinolysis throughout the experimental period. The phenotype in the uPA(-/-) mouse was rescued completely by infusion of single chain uPA (scuPA). The increment in clot lysis was 4-fold greater in uPA(-/-) mice infused with the same concentration of scuPA complexed with soluble recombinant uPAR. These data indicate that uPA contributes to endogenous fibrinolysis in the pulmonary vasculature to the same extent as tPA in this model system. Binding of scuPA to its receptor promotes fibrinolytic activity in vivo as well as in vitro. The physical properties of fibrin clots, including size, age, and cellular composition, as well as heterogeneity in endothelial cell function, may modify the participation of uPA in endogenous fibrinolysis. (Blood. 2000;96:1820-1826)