Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin-4(rhIL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN: Open experiment. SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University. MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University) during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA), type 550 ELISA Reader (Bio-Rad, USA). Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhIL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co.Ltd), rat anti-human CD1a monoclonal antibody and FITC-labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co.Ltd). METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhIL-4. ② Different concentrations of dendritic cells[ dendritic cells: neuroblastoma cells=20∶1,50∶1,100∶1（2×108 L-1，5×108 L-1，1×109 L-1）], 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the experimental group. 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the control group. ③ Main surface marker CD1a molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a+ cells. ④ Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect(%)=（1-A experimental well-A effector cell well/A target cell well）×100%.⑤The experimental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES: ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a+ cellular expression rate. ③Lethal effect of dendritic cells on neuroblastoma cells. RESULTS: ①Morphological characters of dendritic cells in the process of induction and differentiation: On the 15th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhIL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a+ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells: Lethal effect of dendritic cells stimulated T cells in each experimental group ( dendritic cells: neuroblastoma cells=100∶1,50∶1,20∶1 respectively) on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41）%，（30.92±5.27）%，(33.57±5.35)%,(26.23±5.20)%, t=3.51，2.98，4.24, P < 0.01）; But the lethal effect of dendritic cells on neuroblastoma was significantly lower when their ratio was 100∶1 and 50∶1 in comparison with 20:1 （t=2.01，2.36, P < 0.05）, and no significant difference in lethal effect existed between the ratio at 100∶1 and 50∶1（t=0.06,P > 0.05）. CONCLUSION: Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.     

The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats.

Glial glutamate transporter-1 (GLT-1) plays an essential role in removing glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP), the present study was undertaken to observe in vivo changes in the expression of GLT-1 and glial fibrillary acidic protein (GFAP) in the CA1 hippocampus during the induction of BIT, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of BIT in rats. Immunohistochemistry for GFAP showed that the processes of astrocytes were prolonged after a CIP 2 days before the lethal ischemic insult, which could protect pyramidal neurons in the CA1 hippocampus against delayed neuronal death induced normally by lethal ischemic insult. The prolonged processes extended into the area between the pyramidal neurons and tightly surrounded them. These changes made the pyramidal layer look like a 'shape grid'. Simultaneously, the prolonged and extended processes showed a great deal of GLT-1. Western blotting analysis showed significant upregulation of GLT-1 expression after the CIP, especially when it was administered 2 days before the subsequent lethal ischemic insult. Neuropathological evaluation by thionin staining showed that DHK dose-dependently blocked the protective role of CIP against delayed neuronal death induced normally by lethal brain ischemia. It might be concluded that the surrounding of pyramidal neurons by astrocytes and upregulation of GLT-1 induced by CIP played an important role in the acquisition of the BIT induced by CIP.     

Epidural hematoma following use of a three-point skull clamp.

We report an unusual case of epidural hematoma secondary to the use of a three-point skull-fixation device. Initially, a posterior-fossa brain tumor with hydrocephalus was diagnosed in a 15-year-old boy. Midline suboccipital craniectomy was performed with the patient placed in the prone position. A Mayfield (Ohio Medical Instrument Co., Cincinnati, OH, USA) skull clamp was used for fixation of the head during the surgical procedure, following which, a huge epidural hematoma developed within 6 hours of surgery, due to skull penetration and fracture at one of the clamp's pin sites located proximate to the coronal suture.     

The CLDN5 locus may be involved in the vulnerability to schizophrenia.

The present study was designed to detect three single nucleotide polymorphisms (SNPs) located on 22q11 that was thought as being of particularly importance for genetic research into schizophrenia. We recruited a total of 176 Chinese family trios of Han descent, consisting of mothers, fathers and affected offspring with schizophrenia for the genetic analysis. The transmission disequilibrium test (TDT) showed that of three SNPs, rs10314 in the 3'-untranslated region of the CLDN5 locus was associated with schizophrenia (chi(2) = 4.75, P = 0.029). The other two SNPs, rs1548359 present in the CDC45L locus centromeric of rs10314 and rs739371 in the 5'-flanking region of the CLDN5 locus, did not show such an association. The global chi-square (chi(2)) test showed that the 3-SNP haplotype system was not associated with schizophrenia although the 1-df test for individual haplotypes showed that the rs1548359(C)-rs10314(G)-rs739371(C) haplotype was excessively non-transmitted (chi(2) = 5.32, P = 0.02). Because the claudin proteins are a major component for barrier-forming tight junctions that could play a crucial role in response to changing natural, physiological and pathological conditions, the CLDN5 association with schizophrenia may be an important clue leading to look into a meeting point of genetic and environmental factors.     

Paracentral Corneal Dellen： A Rare Sign of Graves Ophthalmopathy

Purpose:To report a rare sign, paracentral corneal dellen that developed in a middleaged female patient with Graves ophthalmopathy.Methods:A paracentral corneal dellen developed in the left eye in a 42-year-old woman who was diagnosed as Graves ophthalmopathy. The patient had remarkable upper eyelid retraction, upper eyelid lag and upward motility restriction. The Graves ophthalmopathy was classified as Grade Ⅳ according to NOSPECS classification.Results:Local artificial tear film and 0.3％ Tobramycin eye drops were administered to both eyes.Twenty-four hours later, the left corneal dellen disappeared.Conclusions:Graves ophthalmopathy can lead to paracentral corneal dellen because of severe upper eyelid retraction and upward motility restriction of the eye in spite of the lack of lagophthalmos. Artificial tear drop and antibiotic eye drop therapy helped even though the patient did not have corneal exposure. Eye Science 2003;19:174-175.     

特发性震颤的临床特点分析

目的探讨特发性震颤（essential tremor,ET）的临床特点。方法回顾性分析98例ET患者的临床资料。结果98例ET中男56例,女42例。发病年龄6～72岁,平均43．08±18．18岁。病程1～48年,平均14．04±11．39年。48例（48．98％）患者有阳性家族史。临床主要表现为单症状的姿势性震颤,累及部位依次为手98例（100％）、头38例（38．78％）、下肢28例（28．57％）、咽喉部16例（16．33％）、下颏10例（10．20％）等。64例患者做了饮酒试验,其中58例（90．63％）有酒精反应。84．62％的患者服用盐酸阿罗洛尔治疗有效。结论特发性震颤的临床表现以单症状姿势性震颤为主,手及头部受累明显,多数患者对酒精有反应,盐酸阿罗洛尔治疗有效。     

双侧内囊前肢毁损术治疗难治性强迫症疗效及随访研究

目的　对难治性强迫症进行双侧内囊前肢毁损术治疗 ,评定手术疗效并进行 2年随访 ,以进一步探索脑外科手术对难治性强迫症的疗效 ,并探讨手术治疗的良好适应症。方法　对 2 8例难治性强迫症患者进行双侧内囊前肢毁损术治疗 ,并分别在手术前、手术后二周、手术后三月、手术后六月、手术后一年、手术后二年进行Y BOCS、HAMA、HAMD量表评定及术后疗效评定。结果　 (1)强迫症患者手术后各期Y BOCS评分、HAMA评分与手术前比较均下降 ,有极显著差异 (P <0 .0 0 1) ;(2 )手术后各期Y BOCS的强迫思维评分均有明显下降 (P <0 .0 0 1) ,强迫行为在手术后 1年和 2年 ,与手术前比较无明显改变 (P >0 .0 5 ) ;(3)手术后 2年的总有效率为 5 3.5 % ,明显低于手术后 3月的总有效率 (P <0 .0 1)。结论　采用双侧内囊毁损术有相当的治疗效果 ,对于难治性强迫症患者可作为一种补充治疗手段 ;手术治疗对强迫行为的长期疗效较差 ,以严重的强迫思维为主的难治性强迫症患者为手术更好的适应症。     

NADPH-Diaphorase-Positive Neurons in Primate Cerebral Cortex Colocalize with GABA and Calcium-Binding Proteins

Neurons in the monkey cerebral cortex containing nicotinamideadenine dinucleotide phosphate-diaphorase (NADPH-d) can he dividedinto two distinct types, both nonpyramidal. Type I neurons havea large soma (diameter 20–50 µm), a dense NADPH-dhistochemical reaction. and are distributed throughout the cortex,but mainly in the subcortical white matter, and are mostly aspiny.Type II cells have a small soma (< 20 µm) with lightNADPH-d reactivity and are distributed primarily in the supragranularlayers, particularly layers II and upper III. The numericaldensity of type II cells is much greater than that of type I.Type I neurons also stain for GABA and a few intracortical typeI cells contain calbindin. All type II cells found here arecolocalized with both GABA and calbindin. Neither type I nortype II cells are stained for parvalbumin. Together with previous observations that almost all corticalNADPH-d cells in various subprimates are like type I cells,we suggest that type II cells may form a group of NADPH-d-richneurons differentiated in higher mammalian cortex from a subpopulationof calbindin-containing GABAergic interneurons, and these nitricoxide-synthesizing cells may play a role in control of intracorticalneuronal activity characteristic of higher cerebral functionsin advanced mammals.     

对数期继代对南方红豆杉细胞培养动力学的影响

目的 解决南方红豆杉细胞生长缓慢及代谢水平低下的问题。方法 对对数期(20d)和静止期(30d)的红豆杉细胞分别进行继代，在整个生长周期中测定了培养基中的碳源、氮源、磷酸盐的变化并分析了红豆杉细胞生长及紫杉醇的合成情况。结果 对数期继代细胞吸收碳源和硝态氮早于静止期继代的细胞，且前者的比生长速度是后者的1．5倍，紫杉醇含量提高了近4倍。结论 对数期继代有利于生物量的积累及紫杉醇的合成。