Development of duplex real-time PCR for detection of two DNA respiratory viruses

A method was developed for the detection and quantitation of HAdV (human adenovirus) and HBoV (human bocavirus) based on a duplex real-time PCR, the AB PCR, using a Smartcycler instrument. A control real-time PCR was carried out on albumin DNA to standardise the non-homogenous respiratory samples. No cross-reactivity was observed with viruses or bacteria that could be found in the respiratory tract. The diagnosis rate using the AB PCR on clinical samples was 10.7%: 3.4% for HBoV detection, 6.9% for HAdV detection and 0.3% double detection HBoV–HAdV. The clinical and epidemiological characteristics of the HAdV- and HBoV-infected patients were evaluated. In the HAdV-positive group and the HBoV-positive group the samples were classified according to the severity of the disease. The HAdV viral load did not appear to be linked to the severity of the disease. Conversely, the difference between the two HBoV groups, severe and non-severe, was significant statistically when the comparison was based on the viral load (P = 0.006) or after adjustment of the viral load to the number of cells in the samples (P = 0.02).     

DNA梯度化分析软件系统在细胞凋亡定量检测中的应用

目的:检验我们设计的DNA梯度化分析软件在细胞凋亡定量检测中的应用价值 方法:用Camptothesin(CAM)诱导U937细胞株凋亡,通过该系统计算DNA琼脂糖凝胶电泳后DNA的梯度值,并与凋亡细胞胞浆寡聚核小体的Elisa检测值进行相关性分析.结果:随着CAM浓度的增加,DNA琼脂糖凝胶电泳后梯度化程度越明显,测出的梯度值也越大,而且与胞浆寡聚核小体的Elisa检测值呈正相关(r=0.989,p<0.01)结论:利用DNA梯度化分析系统可对琼脂糖凝胶电泳后的凋亡细胞DNA片段,进行定量分析,提供简便直接的方法.     

Comparison of methodologies for the in vivo assessment of <Superscript>18</Superscript>FLT utilisation in colorectal cancer

Fluorine-18 3-deoxy-3-fluorothymidine (¹⁸FLT) is a tissue proliferation marker which has been suggested as a new tumour-specific imaging tracer in positron emission tomography (PET). The objectives of this study were to investigate the pharmacokinetics of ¹⁸FLT in patients with colorectal cancer, defining methodologies for the quantitative analysis of the in vivo ¹⁸FLT uptake and subsequently assessing the accuracy of semi-quantitative measures. Dynamic acquisitions over a single field of view of interest identified by computed tomography were carried out for up to 60 min following injection of ¹⁸FLT (360±25 MBq). Dynamic arterial blood sampling was carried out in order to provide a blood input function. Simultaneous venous samples were also taken in order to investigate their potential utilisation in deriving a hybrid input function. Arterial and venous blood samples at 5, 15, 30, 60 and 90 min p.i. were used for metabolite analysis. Eleven patients with primary and/or metastatic colorectal cancer were studied on a lesion by lesion basis (n=21). All acquired images were reconstructed using ordered subsets expectation maximisation and segmented attenuation correction. Time-activity curves were derived by image region of interest (ROI) analysis and image-based input functions were obtained using abdominal or thoracic aorta ROIs. Standardised uptake values (SUVs) were calculated to provide semi-quantitative indices of uptake, while non-linear regression (NLR) methodology in association with a three-compartment model and Patlak analysis were carried out to derive the net influx constant K _i . The metabolite analysis revealed two radioactive metabolites, with the parent compound representing ~80% of the total radioactivity in the 30-min plasma sample. In the case of NLR, better fits were obtained with a 3k model (i.e. k ₄=0) for both lesion and bone marrow time-activity curves. For the same lesions, a high correlation was observed between the K _i derived from either Patlak analysis or NLR(3k) and the corresponding SUVs. Our results also suggest that the quantitative behaviour of ¹⁸FLT in vivo (up to 60 min p.i.) may be characterised using a 3k model or Patlak analysis in combination with image-derived input functions. The good correlation found between the SUVs (at 60 min) and K _i values supports the use of semi-quantitative indices to assess the proliferation rate of colorectal cancer lesions in vivo with ¹⁸FLT.The work included in this paper was selected for consideration in the Marie-Curie award during the European Association of Nuclear Medicine 2002 meeting in Vienna.     

中药中绿原酸的毛细管区带电泳分析

本文提出了一种测定金银花和含金银花的八种中成药中绿原酸标志物的毛细管区带电泳分析方法。所用的背景电解质为含有10mmol.L-1磷酸盐和20mmolL-1硼酸盐的pH7.00的缓冲溶液,并含有5％的乙醇以提高样品的溶解度。测定的线性范围在27.7～665μg.ml-1,相关系数r=0.9996。测定金银花和含金银花的八种中成药中的绿原酸可以在匕分钟内完成。本分析方法简单、快速、适用面广。     

肺癌微血管定量分析与预后的关系

应用免疫组化LSAB法技术以FⅧ因子相关抗原标记血管内皮细胞对199例原发性肺癌组织内微血管进行了定量观察，结果表明：淋巴结有转移组的肺癌微血管密度为53．6±24．2个／200×，无转移组的为35．7±19．8个／200×；淋巴结有转移组的微血管腔面积为5181．2±2522．4，无转移组为3491．1±2083．7，两者在两组间差异均具显著性（P＜0．01，P＜0．02）。微血管密度在5年以上生存组为32．7±20．8个／200×，半年内死亡组为49．1±30．5个／200×，两者差异具有显著性意义（P＜0．01），微血管腔面积在三组不同生存期的患者间差异亦具显著性（P＜0．01），腔面积不同，术后生存时间不同，提示微血管定量分析可以作为临床判断肺癌预后的指标之一。     

高血压微循环与体循环动力学临床研究系列——Ⅰ.高血压病人球结膜微循环定量分析的临床研究

采用显微电视和微机图像处理系统对60例高血压病人、30例血压正常者,进行球结膜微循环定量分析。结果,高血压病人组:微动脉口径(A_3、A_4)缩小;单根和区域血管灌流(V_3、Q_s、Q_L ASR)减少;毛细血管扩散距离(DD)增大。硝苯吡啶治疗后,微动脉口径、区域灌流和毛细血管交换等指标均有较好改善。还发现,微动脉(A3、A_4)口径与舒张压之间呈良好负相关(r=-0.86、-0.62,P<0.01);微动脉面积密度与舒张压之间呈负相关趋向;而微静脉面积密度与舒张压之间则呈正相关趋向。     

基因扩增检测四种感染人的疟原虫种类、数量的方法

目的建立对四种感染人的疟原虫种类、数量进行基因检测的方法。方法根据恶性疟、三日疟、卵形疟、间日疟的18SrRNA基因序列,设计属、种特异性引物和TaqMan探针,用荧光定量扩增反应确定标本中的基因拷贝数,用电泳区别各种疟原虫,并对2份疟疾血样进行检测。结果建立的检测方法对疟疾血样进行检测,荧光定量PCR具有良好的反应性,通过电泳能区分检测标本中的疟原虫株。结论建立的方法能够特异而灵敏地检测出标本中疟原虫的种类、数量,适合疟疾防治检测的需要。     

Implantation of neuronal suspensions into contusive injury sites in the adult rat spinal cord

Summary Implants of various types of neuronal and nonneuronal tissue have shown promise for the amelioration of certain disorders of the adult mammalian brain. Implants may also have therapeutic potential for some lesions of the spinal cord. To examine the feasibility of implantation for clinically relevant spinal cord injuries, we have implanted cells into injury sites produced by a well-characterized and standardized rat model of contusive injury. To reduce the possibility of the implantation procedure itself causing damage to the spinal cord, the tissue was dissociated and a suspension of cells introduced into the cord via a small bore needle. To test the implantation procedure, dissociated adult rat dorsal root ganglia were used because of the ease with which these neurons could be distinguished after implantation. The extent to which functional deficits were produced or exacerbated by the implantation procedure was assessed by behavioral tests of groups of rats that had been implanted (implant controls), contused (injury only) or contused and implanted (injury-implant). Survival of the implanted neurons was assessed by quantitative morphological analysis of histological sections taken through the injury/implant sites at different times following injury. In addition, the histopathology of the contusive injury sites was compared for rats that had or had not received immediate or delayed implants. Results indicated that cell suspensions could be implanted into the spinal cord without causing a functional deficit in an otherwise uninjured animal or exacerbating a standardized incomplete contusive injury. Implanted neurons survived for at least 4 weeks in all contusion sites whether implantation was performed immediately following injury or after a delay or 1 week. There was no significant difference in neuron survival among the groups at 2 h, 18 h or 1 week after implantation. The average number of surviving neurons expressed as a percentage of those counted immediately after implantation was about 90% at 2 h, 50% at 18 h and 30% at 1 week. However, at 4 weeks after implantation, significantly more neurons survived in the delayed group as compared to the immediate group. The results demonstrate the feasibility of implanting dissociated nervous tissue into the sites of clinically relevant experimental contusive injuries and lay the groundwork for investigating possible beneficial effects of implants of different types of neural or glial cell suspensions in the treatment of contusive spinal cord injuries.Suported by the Stroke and Trauma Program, NIH NINCDS, NO1-NS-2-2310 and NO1-NS-7-2301