应用多重连接依赖性探针扩增和变性高效液相色谱技术筛查Peutz-Jeghers综合征家系致病基因突变

目的 联合应用多重连接依赖性探针扩增(MLPA)和变性高效液相色谱(DHPLC)技术快速筛查Peutz-Jeghers综合征家系致病基因的突变.方法 收集家系成员的外周血,采用MLPA、PCR-DNA测序等方法分别检测了LKB基因大片段缺失、碱基突变、碱基插入和缺失.同时收集250名正常人外周血,PCR-DHPLC筛查验证突变位点在正常人群中的分布.生物信息学分析突变位点对编码蛋白质结构和功能的影响.结果 2名家系受累成员均携带LKB基因924G＞C点的突变,这一突变位点在家系正常人和正常人群中都不存在.突变导致位于功能结构域的第308位编码氨基酸由色氨酸变为半胱氨酸,变异蛋白质结构和功能发生改变.结论 c.924G＞C位点的突变是一种病理性胚系突变,编码氨基酸的改变(Trp308Cys)是此家系的致病性因素.     

支架介入治疗颈内动脉狭窄和动脉瘤对动脉瘤壁面压力的影响

目的研究当狭窄与动脉瘤毗邻时,使用支架介入治疗后对动脉瘤壁面压力产生的影响。方法使用计算流体动力学分析的方法对动脉瘤模型及狭窄和动脉瘤毗邻的模型进行对比研究。构建3个模型(M1、M2、M3)对压力变化进行分析比较。M1是颈内动脉瘤模型(无狭窄、无支架),在M1中的动脉瘤前构造一段狭窄动脉形成M2,在M2的动脉瘤部位植入支架后形成M3。结果 M2、M1两个模型相比较,轻度狭窄(50%)引起的动脉瘤部位的压力增加约为1.369 9 kPa(10.3 mmHg)(收缩期的峰值时刻),一个心动周期内动脉瘤部位的平均压力增加约为0.572 kPa(4.3 mmHg)。M3、M2两个模型相比较,动脉瘤部位的压力增加大约为1.037 kPa(7.8 mmHg)(收缩期的峰值时刻),一个心动周期内动脉瘤部位的平均压力增加大约为0.399 kPa(3 mmHg)。结论当使用支架治疗狭窄与弯曲颅内动脉瘤毗邻的患者时,轻度狭窄不会导致显著压力增加。载瘤动脉的形状、动脉瘤或动脉狭窄疾病确实对动脉瘤部位压力变化有影响。     

动脉粥样硬化斑块对药物洗脱支架药物扩散影响的数值分析

目的为更真实地反映药物在血管壁内的分布情况,研究动脉粥样硬化斑块药物扩散系数对血管壁组织中药物扩散的影响。方法采用计算流体动力学方法,考虑5种不同药物扩散系数的斑块,研究血管壁组织和斑块中药物浓度的分布。结果随着斑块药物扩散系数的增加,血管壁中药物含量也逐渐增加,但增加的幅度逐渐趋于平缓。结论当斑块扩散系数小于组织时,斑块对血管壁中药物扩散起抑制作用,反之起促进作用。特别是当斑块中的药物扩散系数远大于血管壁时,其不再影响药物在血管壁中的扩散。在今后的研究中考虑斑块的影响是有必要的,这有利于对药物洗脱支架的优化设计。     

The quantitative effect of an accessory ostium on ventilation of the maxillary sinus

The airflow and gas exchange behaviors of the human maxillary sinus were quantified to better understand the effect of an accessory ostium (AO). An anatomically correct numerical domain was constructed using CT data from a male patient with mild nasal obstruction. For the purpose of comparison, a numerical model without an AO was also generated by artificially removing the AO from the original model using CAD software. A steady-flow field through the nasal cavity was simulated using ANSYS-FLUENT v13.0 with a target flow rate of 250 ml/s. The Volume of Fluid (VOF) method was used to investigate the concentration field of nitric oxide (NO) initially filled in the maxillary sinus. The simulation results showed that a transit flow through the maxillary sinus developed in the presence of an AO. As the flow entered the sinus through either a natural or accessory ostium from the middle meatus, the velocity was significantly reduced to a local maximum of approximately 0.034 m/s inside the sinus. This by-pass flow rate through the sinus of 2.186×10(-1) to 3.591×10(-1) ml/s was a small fraction of the total flow rate inhaled from the nostril, but it effectively changed the local flow topology and led to a larger reduction in NO concentration in the maxillary sinus. This more rapid reduction in NO concentration was due to enhanced ventilation activity afforded by convective transport of the transit stream through the flow path connecting the natural ostium and the AO. The inspiration and expiration phases were qualitatively similar in flow pattern except for the flow direction in the maxillary sinus, suggesting that the AO plays a similar physiological role during both inspiration and expiration in terms of ventilation.     

Tumor type and single-cell/mesothelial-like cell pattern of breast carcinoma metastases in pleural and peritoneal effusions

Invasive ductal and lobular breast carcinomas often have different preferred metastasis sites and distinct histomorphologic characteristics. Their metastatic cytomorphologic cell features in body cavity fluids are generally readily recognized, but the single-cell/mesothelial-like pattern and its relationship to the primary tumor type have not been well studied, nor whether metastases have a propensity for certain body cavity sites on the basis of the primary tumor type. To further assess the tumor type and single-cell pattern of breast carcinoma metastases in pleural and peritoneal effusions, we retrospectively studied 853 pleural and peritoneal effusions and correlated the findings with the primary tumor type. When necessary, the single- cell/mesothelial-like pattern was documented immunohistochemically. Metastatic breast carcinomas represented 249 (50.8%) of 490 pleural and 51 (14.0%) of 363 peritoneal effusions. Most metastases in pleural and peritoneal effusions were ductal carcinomas (92.4% and 62.7%, respectively). Lobular carcinoma accounted for only 2 (0.8%) of 249 pleural and 11 (21.6%) of 51 peritoneal effusions. The single-cell/mesothelial-like cell pattern was found in all lobular carcinomas but also in 11 (6.0%) of 184 reviewed ductal carcinomas (nine pleural and two peritoneal). Awareness of these findings and the use of immunohistochemical analyses are necessary for accurately diagnosing metastatic breast carcinoma, especially lobular type.     

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.     

Importance of the cutoff ratio for detecting antibodies against hepatitis A virus in oral fluids by enzyme immunoassay

Multiple studies have examined the use of oral fluids in modified serum-based assays aiming to replace serum in antibody detection for hepatitis A. However, the reliable detection of HAV immunity in oral fluid requires an extremely sensitive assay; most immunoassays designed for serum antibody determination lack sufficient sensitivity for this purpose. Consequently, an “in-house” competitive enzyme immunoassay (EIA) designed specifically for use with oral samples collected using a ChemBio^® device was developed to detect total anti-HAV antibodies (IgG and IgM). This system was compared to an in-house competitive EIA and a commercial EIA considered to be the “gold standard” using corresponding serum samples (n = 225) to determine the accuracy of the assay and to evaluate the importance of the cutoff ratio for the detection of anti-HAV antibodies in oral fluids. When the median serum cutoff and the optimal oral fluid cutoff (ROC analysis) obtained from the in-house competitive EIA were compared, the oral fluid cutoff was found to be 28.8% higher than the serum cutoff. When different oral fluid cutoff values were compared, a reduction of about 17% was shown to be essential to increase test accuracy. At an oral fluid cutoff value of 0.351, sensitivity and specificity were higher, reaching 91.7% and 86.2% (p < 0.001, AUROC = 0.915), respectively. The convenience, accuracy and non-invasive nature of the developed method make it a useful alternative to serum-based assays for discriminating between HAV-immune and non-immune individuals.     

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology.A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory.A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10 PFU/ml (Arm53b and WE54) and 1 PFU/ml (Traub and E350).Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses.This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.