Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly

The phospholipid inflammatory mediator, platelet-activating factor (PAF), can stimulate polymorphonuclear leukocyte (PMN) chemotaxis. Conversion of cytoplasmic actin from monomers to filaments is associated with PMN motile functions. Using the fluorescent actin filament stain nitrobenzodiaxole phallicidin, we have investigated PAF's effects on human PMN actin polymerization. Concentrations of PAF between 1 x 10(-11) to 1 x 10(-6) mol/L induced actin filament (F- actin) assembly. An optimal concentration of PAF (1-5 x 10(-8) mol/L) induced a significantly lower rise in relative F-actin content (1.72 +/- 0.07 SEM) than an optimal concentration (5 x 10(-7) mol/L) of the chemotactic peptide FMLP (2.21 +/- 0.06). Unlike FMLP (F-actin content: 1.25 +/- 0.04 at five seconds), PAF stimulation was associated with a delay of more than five seconds (1.04 +/- 0.01 at five seconds) before an increase in F-actin could be detected. F-actin concentration reached maximum levels by 30 to 60 seconds. Prolonged stimulation (20 minutes) with PAF was associated with two phases of polymerization and depolymerization. Like FMLP, the initiation of actin filament assembly by PAF required receptor occupancy, this reaction being totally blocked by the PAF receptor inhibitor, SKI 63-441. As evidenced by the lack of inhibition by nordihydroguaiaretic acid (5 to 20 mumol/L), the production of leukotriene B4 was not required for the PAF-induced changes in F-actin. Like FMLP, PAF's ability to stimulate PMN actin polymerization was inhibited by pertussis toxin (.05 to 2.5 micrograms/mL) but not impaired by the addition of EGTA and/or the calcium ionophore A23187. Preincubation with 1 x 10(-11) to 1 x 10(-8) mol/L PAF for 2 to 60 minutes enhanced the rise in F-actin content induced by low concentrations of FMLP (5 x 10(-12) to 1 x 10(-10) mol/L) indicating that this phospholipid was capable of "priming" the PMN actin polymerization response.     

血管内皮细胞生长因子和碱性成纤维细胞生长因子体外联合诱导外周血单个核细胞定向分化为血管内皮细胞

目的:在体外培养的条件下,应用血管内皮细胞生长因子和碱性成纤维细胞生长因子联合诱导人外周血单个核细胞向血管内皮细胞分化,解决组织工程血管化的种子细胞来源问题。方法:实验于2005-11/2006-05在安徽省立医院中心实验室完成。①血管内皮细胞生长因子(Pepro Tech公司,批号090310);碱性成纤维细胞生长因子(Pepro Tech公司,批号0704CY081);人淋巴细胞分离液Ficoll-paque(天津灏洋生物制品科技有限公司);PE标记的CD31,鼠抗人vWF单克隆抗体(BD Biosciences公司);FITC标记的兔抗鼠IgG(北京中杉金桥公司)。②无菌条件下取健康人外周血20mL,肝素抗凝,Hanks液双倍稀释,按1∶2置于淋巴细胞分离液上层,离心后提取分液层与上层交界部位呈混浊的灰白色层,即为单个核细胞层。③取离心后的细胞,向DMEM-F12培养基中分别加入含体积分数为0.2的胎牛血清、10μg/L血管内皮细胞生长因子、10μg/L碱性成纤维细胞生长因子。以不含血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导剂的DMEM-F12培养基作为空白对照。吹打均匀并计数,按1×1010L-1接种于25cm2培养瓶中,于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,第3天更换培养基,去除未贴壁的细胞,以后每2d换液1次。④倒置相差显微镜下观察细胞单层排列是否呈"铺路石"样结构。运用流式细胞术检测诱导后的细胞表达CD31和vWF情况。透射电镜观察细胞的超微结构。结果:①诱导分化后细胞形态学变化:经血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导后的细胞形态上呈典型的"铺路石"样外观,经历从小圆→梭形→扁平细胞的过程,符合内皮细胞的演变过程。②诱导分化后细胞的表面标志鉴定:诱导分化20d,贴壁细胞中62.5%表达CD31,58.2%表达vWF,54.3%表达CD31/vWF。③培养细胞的超微结构观察:透射电镜下细胞胞浆内可见特征性W-P小体。结论:外周血单个核细胞在血管内皮细胞生长因子和碱性成纤维细胞生长因子体外联合诱导下,可分化为血管内皮细胞。     

腺病毒载体的研究进展

腺病毒(adenovirus,Ad)作为基因表达载体的研制起始于20世纪60年代初,当时病毒学家观察到腺病毒基因组可与猴类病毒40(SV40)基因组杂交,说明腺病毒基因组可承载异源性基因.此后腺病毒逐步发展成一种重要的载体系统[1-9],并成功地用于基因治疗的体内外实验和临床试验[10-21].目前运用最广的腺病毒载体(adenovirus vector,Adv)是血清5型腺病毒.     

全髋关节置换术假体周围感染的处理（一）

全髋关节置换术（THA）是骨科最常见的手术之一。尽管初次THA术后深部感染率相对低,但其带来的经济负担、残疾、甚至死亡具有潜在的破坏性。由于有越来越多的老年患者需要行关节成形术,感染的预防和应该最优化治疗,以减少患者与医疗系统直接和间接的代价。     

Clinical,biochemical and molecular characterization of peroxisomal diseases in Arabs

Shaheen R, Al‐Dirbashi OY, Al‐Hassnan ZN, Al‐Owain M, Makhsheed N, Basheeri F, Seidahmed MZ, Salih MAM, Faqih E, Zaidan H, Al‐Sayed M, Rahbeeni Z, Al‐Sheddi T, Hashem M, Kurdi W, Shimozawa N, Alkuraya FS. Clinical, biochemical and molecular characterization of peroxisomal diseases in Arabs. Peroxisomes are single membrane‐bound cellular organelles that carry out critical metabolic reactions perturbation of which leads to an array of clinical phenotypes known as peroxisomal disorders (PD). In this study, the largest of its kind in the Middle East, we sought to comprehensively characterize these rare disorders at the clinical, biochemical and molecular levels. Over a 2‐year period, we have enrolled 17 patients representing 16 Arab families. Zellweger‐spectrum phenotype was observed in 12 patients and the remaining 5 had the rhizomelic chondrodysplasia punctata phenotype. We show that homozygosity mapping is a cost‐effective strategy that enabled the identification of the underlying genetic defect in 100% of the cases. The pathogenic nature of the mutations identified was confirmed by immunofluorescence and complementation assays. We confirm the genetic heterogeneity of PD in our population, expand the pool of pathogenic alleles and draw some phenotype/genotype correlations.     

Identifying the translational gap in the evaluation of drug-induced QTc interval prolongation

Aims

Given the similarities in QT_c response between dogs and humans, dogs are used in pre-clinical cardiovascular safety studies. The objective of our investigation was to characterize the PKPD relationships and identify translational gaps across species following the administration of three compounds known to cause QT_c interval prolongation, namely cisapride, d, l-sotalol and moxifloxacin.

Methods

Pharmacokinetic and pharmacodynamic data from experiments in conscious dogs and clinical trials were included in this analysis. First, pharmacokinetic modelling and deconvolution methods were applied to derive drug concentrations at the time of each QT measurement. A Bayesian PKPD model was then used to describe QT prolongation, allowing discrimination of drug-specific effects from other physiological factors known to alter QT interval duration. A threshold of ≥10 ms was used to explore the probability of prolongation after drug administration.

Results

A linear relationship was found to best describe the pro-arrhythmic effects of cisapride, d,l-sotalol and moxifloxacin both in dogs and in humans. The drug-specific parameter (slope) in dogs was statistically significantly different from humans. Despite such differences, our results show that the probability of QT_c prolongation ≥10 ms in dogs nears 100% for all three compounds at the therapeutic exposure range in humans.

Conclusions

Our findings indicate that the slope of PKPD relationship in conscious dogs may be used as the basis for the prediction of drug-induced QT_c prolongation in humans. Furthermore, the risk of QT_c prolongation can be expressed in terms of the probability associated with an increase ≥10 ms, allowing direct inferences about the clinical relevance of the pro-arrhythmic potential of a molecule.     

The phenotypical and functional characteristics of cord blood monocytes and CD14(-/low)/CD16(+) dendritic cells can be relevant to the development of cellular immune responses after transplantation

Umbilical cord blood (UCB) has been used as an alternative source of haematopoietic progenitors for transplantation presenting advantages over bone marrow (BM) that are related with known shortages of newborns' immune system at adaptive and innate levels. Using flow cytometry, we studied the expression of Toll-like receptors (TLRs) and chemokine receptors (CKRs) and the production of pro-inflammatory cytokines by monocytes and CD14(-/low)/CD16(+)DCs from peripheral blood (PB; n=10), and umbilical cord blood (UCB; n=10). CKRs and cytokines were studied before and after stimulation of cells with LPS plus IFN-gamma. We also identified the two populations in normal bone marrow samples (BM; n=5). BM presented lower frequencies of both studied populations when compared to UCB and PB. CD14(-/low)/CD16(+)DCs presented a pattern of TLR expression different from mature monocytes reflecting distinct functions for these two populations. UCB cells presented reduced expression of TLR-4 and lower capability to produce cytokines prior stimulation. The populations studied presented different patterns of CKR expression reflecting distinct migratory pathways. Moreover, UCB cells presented higher expressions of CXCR4 and CCR7 that may be involved in immune system maturation and stem cell homing. Monocytes and CD14(-/low)/CD16(+)DCs present functional and phenotypical characteristics that may contribute to the lower incidence and severity of GVHD.     

The effect of overweight and obesity on psoriasis patients in Chinese Han population: a hospital‐based study

Background Accumulating evidence indicates that psoriasis is associated with increased risk of overweight and obesity. However, few studies have investigated this relationship in Chinese Han population. Objective The aim of this study was to explore the relationship between overweight/obesity and psoriasis and to evaluate the overweight/obesity effect on the clinical features of psoriasis in Chinese Han population. Methods A hospital‐based study was conducted, which involved in 4452 patients and 1166 controls of Chinese Han through epidemiological investigation. Controls used in the study were individuals without psoriasis from health examination centre, and other skin disease patients from outpatient department. Results Compared with the control group, a significantly greater prevalence of overweight and obesity was observed in psoriasis patients. The estimated ORs were 1.301 (95% CI, 1.105–1.531) and 1.680 (95% CI, 1.134–2.491) respectively. The disease severity of psoriasis measured by psoriasis area and severity index (PASI) was statistically correlated with body mass index (BMI) (r = 0.184, P < 0.01). Moreover, a high proportion of overweight patients had affected hands or/and feet, buttocks, trunk, legs, arms and arthritis (P < 0.01). Conclusions Our study suggested that psoriatic patients have a higher prevalence of overweight and obesity compared with non‐psoriatic patients in Chinese Han population. Overweight and obesity has different risk effect on severity and manifestations of psoriasis and might be useful for better evaluating psoriasis clinically.     

Effects of autonomic balance and fluid and electrolyte changes on cardiac function in infarcted rats: A serial study of sexual dimorphism

Premenopausal women are known to show lower incidence of cardiovascular disease than men. During myocardial infarction (MI), homeostatic responses are activated, including the sympathetic autonomic nervous system and the rennin–angiotensin–aldosterone system, which is related to the fluid and electrolyte balance, both aiming to maintain cardiac output. This study sought to perform a serial evaluation of sexual dimorphism in cardiac autonomic control and fluid and electrolyte balance during the development of MI‐induced heart failure in rats. Experimental MI was induced in male (M) and female (F) adult (7–9 weeks of age) Wistar rats. The animals were placed in metabolic cages to assess fluid intake and urine volume 1 and 4 weeks after inducing MI (male myocardial infarction (MMI) and female myocardial infarction (FMI) groups). They subsequently underwent echocardiographic evaluation and spectral analysis of heart rate variability. After completing each protocol, the animals were killed for postmortem evaluation and histology. The MMI group showed earlier and more intense cardiac morphological and functional changes than the FMI group, although the extent of MI did not differ between groups (P > 0.05). The MMI group showed higher sympathetic modulation and sodium and water retention than the FMI group (P < 0.05), which may partly explain both the echocardiographic and pathological findings. Females subjected to infarction seem to show attenuation of sympathetic modulation, more favourable fluid and electrolyte balances, and better preserved cardiac function compared to males subjected to the same infarction model.