结核分枝杆菌Ag85B与IL-12基因真核共表达质粒的构建及表达

目的 :构建结核分枝杆菌Ag85B和鼠IL 12基因的共表达载体pBud85B IL12。方法 :将结核分枝杆菌Ag85B基因和鼠IL 12基因同时克隆入含多启动子的共表达载体pBudCE4 .1中 ,构建真核共表达质粒pBud85B IL12。以pBud85B IL12转染COS 7细胞 ,通过RT PCR及ELISA方法检测目的基因的表达。结果 :在COS 7细胞中同时可检测到Ag85B和IL12的表达。结论 :pBud85B IL12共表达质粒的成功构建 ,为对其免疫原性、免疫反应性及免疫保护作用的进一步研究奠定了基础     

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.     

Efficiently stimulated adult microglia cross‐prime naive CD8+ T cells injected in the brain

Microglia are the major myeloid‐immune cells of the brain parenchyma. In a steady state, microglia monitor their environment for pathogens or damaged cells. In response to neural injury or inflammation, microglia become competent APCs able to prime CD4⁺ and CD8⁺ T lymphocytes. We previously demonstrated that neonatal and adult microglia cross‐present exogenous soluble Ags in vitro. However, whether microglia are able to cross‐present Ag to naive CD8⁺ T cells in vivo, within the brain microenvironment, remains undetermined. Here, we have designed an original protocol in order to exclude the involvement in cross‐presentation activity of peripheral migrating APCs and of CNS‐associated APCs. In C57Bl/6 mice, in which the body but not the head has been properly irradiated, we analyzed the ability of resident microglia to stimulate intracerebrally injected CD8⁺ T cells in vivo. This study demonstrates for the first time that adult microglia cross‐present Ag to naive CD8⁺ T cells in vivo and that full microglia activation is required to overcome the inhibitory constrains of the brain and to render microglia able to cross‐prime naive CD8⁺ T cells injected in the brain. These observations offer new insights in brain‐tumor immunotherapy based on the induction of cytotoxic antitumoral T cells.     

纸基-表面增强拉曼光谱法快速鉴别染色南五味子药材

目的 采用纸基-表面增强拉曼光谱法(SERS)对染色南五味子进行快速鉴别。方法 选用浸泡法制备的银胶纸作为SERS基底,擦拭经乙醇-水溶液润湿的南五味子,银胶纸立即进行SERS检测;先后对银溶胶的浓缩倍数、银胶纸的SERS增强效果及稳定性等因素进行考察。结果 成功鉴别低浓度酸性红、赤藓红染色的南五味子。结论 纸基-SERS法可实现非法染色南五味子的快速、准确、无损的鉴别,有望应用于快检领域。     

Total effective compliance of the vascular bed in essential hypertension

Total effective vascular compliance, hemodynamic parameters, cardiopulmonary (CPBV) and total blood volumes (TBV) were determined in 31 men, including nine normotensive controls and 22 permanent essential hypertensive patients. The effective compliance was calculated from the changes in central venous pressure recorded simultaneously with the changes in blood volume obtained after a rapid dextran infusion. In hypertensives, compliance was significantly reduced (1.55 +/- 0.6 vs 2.25 +/- 0.11 ml./mm. Hg/Kg. in controls) (P less than 0.001) and negatively correlated with blood pressure (P less than 0.01), cardiac index (P less than 0.01), and the CPBV/TBV ratio (P less than 0.01). These results suggest that venous compliance contributes to the control of cardiac output in essential hypertension.     

冠心病介入术后氧自由基的生成及其对脂质过氧化和纤溶的影响

目的:探讨冠心病介入治疗术后氧自由基的生成及其对患者体内脂质过氧化和纤溶状况的影响。方法:48例接受经皮冠脉介入治疗(PCI)术的冠心病患者分别于术前,术后即刻、30 min、1 h、24 h和一周采用ELISA法测定血浆中丙二醛、D-二聚体、血管假性血友病因子相关抗原(vWF Ag)浓度。结果:PCI术后短时间内(0～24 h)血浆中丙二醛、D-二聚体,vWFAg水平均明显升高(P<0．05)。结论:PCI术后短时间内即有大量的氧自由基生成,凝血系统的激活和继发性纤溶功能亢进。     

Detection of melamine based on the suppressed anodic response of uric acid by a Au–Ag nanoparticles modified glassy carbon electrode

We demonstrated a sensitive electrochemical method for the determination of nonelectroactive melamine (Mel) using a modified glassy carbon electrode (GCE), with uric acid (UA) as the signal reporter. To increase the anodic response of UA, GCE was coated with Au–Ag nanoparticles and a Nafion thin film (Au–Ag/Nafion/GCE). The sensing mechanism was based on the competitive adsorption behavior of Mel on the Au–Ag/Nafion/GCE, which reduces the electroactive surface area of nanoparticles and thus hinders anodic response of UA. Under optimal conditions and the use of an analytical method of differential pulse voltammetry, this modified electrode detected Mel concentrations ranging from 2.5 to 70 nM, with a detection limit of 1.8 nM. The Au–Ag/Nafion/GCE demonstrated satisfactory reproducibility and stability, with relative standard deviations (RSDs) of 9.3% and 7.1%, respectively. The proposed electrochemical method was then successfully used to determine the Mel content in spiked milk powder and cat food samples, with RSDs of 1.7%–9.3% and recoveries of 92.4%–103.7%.     

Liposuction in Dercum's disease: impact on haemostatic factors associated with cardiovascular disease and insulin sensitivity

Berntorp E, Berntorp K, Brorson H, Frick K (University of Lund, Malmö, Sweden). Liposuction in Dercum's disease: impact on haemostatic factors associated with cardiovascular disease and insulin sensitivity. J Intern Med 1998; 243 : 197–201.

Objective

To study the impact of adipose tissue removal by liposuction on factors associated with increased risk of cardiovascular atherosclerotic disease within the coagulation and fibrinolytic system and glucose metabolism.

Design, setting and subjects

Liposuction was performed in 53 patients with Dercum's disease. The levels of fibrinogen, von Willebrand factor antigen (VWF:Ag) and plasminogen activator inhibitor type 1 activity (PAI-1) were measured preoperatively, and 2 weeks, 4 weeks and 3 months postoperatively. In a subsample of 10 patients, insulin sensitivity was determined before and 2–4 weeks after surgery using the 2-h euglycaemic hyperinsulinaemic clamp technique. The study was performed as a single-centre study.

Main outcome measure

Fibrinogen, PAI-1 and VWF:Ag levels, and glucose uptake before and after removal of adipose tissue.

Results

Weight reduction was sustained throughout the follow-up period with a mean decrease from 90.7 to 86.6 kg (P < 0.0001). There was a slight increase in levels of coagulation factors 2 and 4 weeks postoperatively, probably in reaction to the surgical trauma. After 3 months the values had returned to preoperative levels except for PAI-1, which still showed a slight increase (P < 0.05). In the subsample of 10 patients, glucose uptake was improved (P < 0.05) from a short-term perspective after surgery.

Conclusion

Surgical removal of adipose tissue, without change in lifestyle, does not seem to improve the levels of coagulation and fibrinolytic factors associated with cardiovascular atherosclerotic disease, whereas glucose takeup may be facilitated and insulin sensitivity increases from a short-term perspective.

     

Evaluation and performance characteristics of the Q Hemostasis Analyzer, an automated coagulation analyzer

The Q Hemostasis Analyzer (Grifols, Barcelona, Spain) is a fully-automated random-access multiparameter analyzer, designed to perform coagulation, chromogenic and immunologic assays. It is equipped with a cap-piercing system. The instrument was evaluated in a hemostasis laboratory of a University Hospital with respect to its technical features in the determination of coagulation i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen and single coagulation factors V (FV) and VIII (FVIII), chromogenic [antithrombin (AT) and protein C activity] and immunologic assays [von Willebrand factor antigen (vWF:Ag) concentration], using reagents from the analyzer manufacturer. Total precision (evaluated as the coefficient of variation) was below 6% for most parameters both in normal and in pathological ranges, except for FV, FVIII, AT and vWF:Ag both in the normal and pathological samples. No carryover was detected in alternating aPTT measurement in a pool of normal plasma samples and in the same pool spiked with unfractionated heparin (> 1.5 IU/mL). The effective throughput was 154 PT, 66 PT/aPTT, 42 PT/aPTT/fibrinogen, and 38 PT/aPTT/AT per hour, leading to 154 to 114 tests performed per hour, depending of the tested panel. Test results obtained on the Q Hemostasis Analyzer were well correlated with those obtained on the ACL TOP analyzer (Instrumentation Laboratory), with r between 0.862 and 0.989. In conclusion, routine coagulation testing can be performed on the Q Hemostasis Analyzer with satisfactory precision and the same apply to more specialized and specific tests.