Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Spike patterning by Ca2+-dependent regulation of a muscarinic cation current in entorhinal cortex layer II neurons

In entorhinal cortex layer II neurons, muscarinic receptor activation promotes depolarization via activation of a nonspecific cation current (I(NCM)). Under muscarinic influence, these neurons also develop changes in excitability that result in activity-dependent induction of delayed firing and bursting activity. To identify the membrane processes underlying these phenomena, we examined whether I(NCM) may undergo activity-dependent regulation. Our voltage-clamp experiments revealed that appropriate depolarizing protocols increased the basal level of inward current activated during muscarinic stimulation and suggested that this effect was due to I(NCM) upregulation. In the presence of low buffering for intracellular Ca(2+), this upregulation was transient, and its decay could be followed by a phase of I(NCM) downregulation. Both up- and downregulation were elicited by depolarizing stimuli able to activate voltage-gated Ca(2+) channels (VGCC); both were sensitive to increasing concentrations of intracellular Ca(2+)-chelating agents with downregulation being abolished at lower Ca(2+)-buffering capacities; both were reduced or suppressed by VGCC block or in the absence of extracellular Ca(2+). These data indicate that relatively small increases in [Ca(2+)](i) driven by firing activity can induce upregulation of a basal muscarinic depolarizing-current level, whereas more pronounced [Ca(2+)](i) elevations can result in I(NCM) downregulation. We propose that the interaction of activity-dependent positive and negative feedback mechanisms on I(NCM) allows entorhinal cortex layer II neurons to exhibit emergent properties, such as delayed firing and enhanced or suppressed responses to repeated stimuli, that may be of importance in the memory functions of the temporal lobe and in the pathophysiology of epilepsy.     

Regulation of Fungal Infection by a Combination of Amphotericin B and Peptide 2, a Lactoferrin Peptide That Activates Neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 × 10⁸ cells) or Aspergillus fumigatus (1 × 10⁸ cells) was prolonged 3 to 5 days by the injection of 10 μg of peptide 2 (a lactoferrin peptide) and 10 μg of α-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 μg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 μg of amphotericin B. When mice received a combined injection of peptide 2 (10 μg/day) with amphotericin B (0.5 μg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as α-defensin 1, β-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O₂⁻) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O₂⁻-generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47^phox as well as p67^phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

巨细胞病毒感染对动脉粥样硬化的影响

目的：研究巨细胞病毒(CMV)感染和动脉粥样硬化的关系，并探讨其可能机制。方法：通过一个大样本从血清流行病学(ELISA检测患者血清中抗CMV的IgG抗体)和分子生物学(PCR检测动脉粥样硬化病变中CMV特异性基因)方面探讨巨细胞病毒感染和动脉粥样硬化的关系，并检测CMV感染对内皮细胞趋化因子表达的影响。结果：动脉粥样硬化组血清中CMV的阳性率显著高于非动脉粥样硬化组(分别为82．2％和61．0％，P=0．02)；而且动脉粥样硬化斑块中HCMV基因出现率显著高于正常血管组织(13／15和2／7，P=0．01)；CMV感染还可上调内皮细胞：ECV-304表达MCP-1。结论：CMV感染参与动脉粥样硬化的形成和发生，这可能与CMV上调内皮细胞趋化因子表达有关。     

Smad7过度表达抑制3T3细胞增殖和TGF-β1基因表达

研究Smad7过度表达对TGF β1基因表达的调控和对 3T3细胞增殖的影响。实验将Smad7质粒 ,通过脂质体介导转染NIH3T3细胞 ,应用RT PCR方法鉴定转染结果和检测TGF β1mRNA的表达 ,以及用免疫细胞化学检测TGF β1蛋白的表达情况 ,并观察基因转染对细胞增殖的影响。结果显示转染Smad7后 ,NIH3T3细胞中TGF β1mRNA的表达显著减少 (P <0 0 5 ) ,TGF β1蛋白的表达下降 ,细胞增殖明显减缓 (P <0 0 5 )。提示Smad7过度表达能够抑制 3T3细胞的增殖和TGF β1基因的表达 ,可以通过阻断TGF β细胞内信号传导来调控TGF β1的生物学行为。     

妊娠中晚期暴露于可卡因对子代海马发育的影响

目的　建立妊娠中晚期暴露于可卡因的小鼠动物模型 ,研究可卡因对子代海马的发育是否具有影响以及这种影响在可卡因诱发的神经行为异常发病机理中可能所起的作用。方法　运用水迷宫方法观察可卡因对子鼠青春期空间辨别能力的影响 ,分别采用甲苯胺蓝染色技术和免疫组织化学结合图象分析技术观察海马中锥体细胞和神经胶质细胞的发育情况。结果　妊娠中晚期暴露于可卡因的小鼠子代在青春期海马锥体细胞极性紊乱 ,发育不良 ,未迁移到正常位置 ;神经胶质细胞数目减少。水迷宫结果显示可卡因可引起子代寻找平台的潜休期延长。结论　妊娠中晚期暴露于可卡因可引起子代青春期海马发育异常 ,这可能在可卡因引起子代空间辨别能力下降的发病机理中起着重要作用。     

脑胶质瘤中血脑屏障的超微结构特点

８例胶质瘤病例，标本分别取自同一病例的瘤体，瘤周水肿区和邻近脑组织。瘤体和瘤周水肿区血脑屏障超微结构变化无明显差异，内皮肿胀，毛细血管腔狭小，腔面有稀疏的绒毛样突起，内皮为无孔型，胞质内胞饮泡较多，内皮紧密连接增长，细胞连接间隙增宽，基膜完整，基膜完整，胶质膜缺损。     

RAAS系统在继发性高血压疾病中的应用进展

在人体体内分布最广泛的内分泌调节系统是肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosteron system,RAAS),该系统在内分泌调节环节中发挥着至关重要的作用.在内分泌系统中,可以通过RAAS来实现对人体体内血量流动和外周阻力的控制,并且还可以通过RAAS来进一步调节体内血压、水和电解质的平衡,确保人体环境能够保持在一个稳定的状态.组成RAAS的主要部分有肾素、血管紧张素和醛固酮.针对RAAS各个组成部分进行深入研究之后,发现在RAAS系统中的一些部分与嗜铬细胞瘤、副神经节瘤等继发性高血压疾病密切相关.通过检测RAAS的组分可以辅助诊断因RAAS系统失调所导致的多种疾病.本文将从RAAS组分的检测方法及其在继发性高血压中的临床应用进行综述.     

Effect of once-daily fluticasone furoate nasal spray on nasal symptoms in adults and adolescents with perennial allergic rhinitis.

BACKGROUND: Intranasal corticosteroids are recommended as first-line therapy for the treatment of allergic rhinitis. Fluticasone furoate is a novel enhanced-affinity glucocorticoid for the treatment of allergic rhinitis. OBJECTIVE: To compare the efficacy and safety of intranasal fluticasone furoate with those of vehicle placebo nasal spray in adult and adolescent patients with perennial allergic rhinitis (PAR). METHODS: After screening (7-14 days), patients 12 years and older with confirmed PAR were randomized to receive fluticasone furoate, 110 microg once daily, or placebo once daily intranasally for 4 weeks in this double-blind, multicenter study. The primary end point was mean change from baseline during the entire treatment period in daily reflective total nasal symptom score (rTNSS), recorded on diary cards by patients, using a 4-point categorical scale. RESULTS: The mean reduction from baseline during the treatment period in daily rTNSS was significantly greater in fluticasone furoate recipients than in placebo recipients (P = .005). This finding was supported by significantly greater mean reductions in morning rTNSS and evening rTNSS (P = .004 and P = .011, respectively). A significantly greater mean reduction in instantaneous morning predose TNSS with fluticasone furoate compared with placebo (P = .006) confirmed the efficacy of once-daily administration. Fluticasone furoate was also significantly more effective than placebo in overall response to therapy (P = .005). CONCLUSIONS: Fluticasone furoate nasal spray, 110 microg once daily, effectively relieved nasal symptoms of PAR in adults and adolescents 12 years and older.     

甲氨喋呤对胶原诱导性关节炎大鼠滑膜骨桥蛋白和整合素αVβ3表达的影响

目的 探讨甲氨喋呤对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠滑膜骨桥蛋白(osteopontin,OPN)及其受体整合素αvβ3表达的影响及其作用机制.方法 建立CIA大鼠模型,分为模型组和甲氨喋呤(MTX)组,后者用MTX进行干预治疗;采用免疫组织化学染色技术检测滑膜组织OPN和整合素αvβ3的表达,ELISA方法检测血清TNF-α水平.结果 ① CIA模型组和MTX组大鼠的滑膜OPN、整合素αvβ3表达均明显高于正常大鼠对照组(均为P<0.01);与模型组比较,MTX组OPN和整合素αVβ3的表达减少(均为P<0.01). ②正常对照组和MTX组大鼠血清TNF-α水平显著性低于模型组大鼠(均为P<0.01).结论 滑膜OPN和整合素αvβ3异常表达在CIA发病中具有重要意义.MTX通过下调滑膜OPN和整合素αvβ3的表达,降低血清TNF-α水平从而达到治疗CIA的作用.