微粒体在缺血性卒中的作用

在血管内皮功能障碍、急慢性血管炎症和高凝状态下均可发现由血小板、白细胞和内皮 细胞释放的微粒体。不同种类的微粒体在缺血性卒中病理过程中的作用有所不同：缺血性卒中患者 外周血白细胞微粒升高，高水平的白细胞微粒体能加重血管内皮细胞损伤，血浆中长期及高水平的 血小板微粒体可诱导微血管血栓形成。另外，不同种类的微粒体均能反映血管内皮损伤的程度，诱 导内皮细胞功能失调，加重内皮功能障碍，同时微粒体中携带的信使RNA和微小核糖核酸等能促进内 皮细胞增殖，促进动脉粥样硬化斑块内新生血管生成，使斑块更易破裂出血。     

Evaluation of particulate acellular vaccines against Brucella ovis infection in rams

The attenuated Brucella melitensis Rev 1 vaccine, used against brucellosis infection, interferes with serological diagnosis tests, may induce abortions in pregnant animals, and may infect humans. In order to overcome these drawbacks, we developed acellular vaccines based on a Brucella ovis antigenic complex (HS) containing outer membrane proteins and R-LPS entrapped in poly(anhydride) conventional and mannosylated nanoparticles (NP-HS and MAN-NP-HS) or in poly(?-caprolactone) microparticles (HS-PEC) as antigen delivery systems and immunoadjuvants. Brucellosis free rams were vaccinated subcutaneously with a single dose of particles containing 3 mg of HS, and challenged 6 months thereafter. Protection was evaluated by clinical, bacteriological and serological examinations, in comparison with non-vaccinated control rams. HS-PEC vaccine induced protection (7 out of 13 animals were infected) equivalent to that induced by the reference Rev 1 vaccine (8/14). In contrast, animals immunized with NP-HS were not protected, showing similar results to that obtained in the control unvaccinated rams. Furthermore HS-PEC vaccine did not interfere against B. melitensis serodiagnostic tests. In summary, HS-PEC microparticles could be used as a safe and effective vaccine against brucellosis in rams.     

Localization and quantification of biodegradable particles in an intestinal cell model: The influence of particle size

The physicochemical parameters influencing particle crossing through the epithelial barrier have been largely studied and size has been shown as a crucial factor. Most part of this work has been done with polystyrene (PS) particles; however, few studies were conducted with well characterized biodegradable polymeric nanoparticles, more suitable for drug delivery. The aim of the present work was to study the influence of the size of well characterized biodegradable polymeric particles on cellular uptake by Caco-2 cells. Poly (d,l-lactide-co-glycolide acid) (PLGA) particles loaded with a fluorescent dye, 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiO), were prepared by the emulsion evaporation process. Five batches of particles with narrow size distribution (100, 300, 600, 1000, and 2000 nm) were produced using selective centrifugation. Surface properties (zeta potential, hydrophobicity and residual surfactant rate) were similar among all batches. Preliminary study showed that after growth and differentiation, cell counting differed from plate to plate. Therefore, a quantitative method using fluorescence spectroscopy was developed to estimate the number of particles interacting with a single cell. The results were compared to the interaction obtained with polystyrene particles, commonly used as particle model. The interaction was clearly dependant on particle size and concentration. Particles in the range of 100 nm presented a higher interaction when compared to larger particles. More than 6000 (d,l-lactide-co-glycolide acid) particles and 200,000 polystyrene particles were quantified per cell. Cellular localisation of particles by confocal microscopy showed the association of the poly (d,l-lactide-co-glycolide acid) particles with the cells. Small particles were observed intracellularly, whereas particles larger than 300 nm were associated with the apical membranes. Interestingly, some of the 100 nm PLGA particles were localized in the nuclei of the cells.     

Proteomics of microparticles after deep venous thrombosis

Background

Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells.

Methods

To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05.

Results

Two proteins (Galectin-3 Binding Protein, [Gal3BP], R = 1.76 and Alpha-2 macroglobulin [A2M] R = 1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R = 0.65).

Conclusions

These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.     

Uptake of microparticles into the epithelium of human nasopharyngeal lymphoid tissue

The M cells of nasopharyngeal lymphoid tissue (NALT) have been considered to play an important role for vaccine delivery systems in humans. A number of investigations have reported particle uptake data in NALT of rodents. However, there have been no reports indicating any involvement of the nasopharyngeal lymphoid tissue in human vaccination. In the present study, we investigated whether the epithelium of human adenoid tissues might incorporate fluorescent microparticles using electron and fluorescent microscopy. The dissected adenoid tissues were incubated with various sizes and concentrations of fluorescent microparticles for 120 min at 37°C. Furthermore, the effect of surface coatings of microparticles with cations on the uptake into the epithelium of adenoid tissues was investigated. Transmission electron microscopy revealed that microparticles were taken up by the M cells of human nasopharyngeal lymphoid tissues. The NALT-M cells showed greater uptake of the smallest particles, 0.2 μm in diameter, than those of 0.5, 1.0, or 2.0 μm diameter. It was also revealed that surface coatings with poly-l-lysin or chitosan resulted in efficient uptake into the NALT. These results indicate that nasal administration of antigenic microparticles, which were coated with cationic materials, probably leads to a useful method of transnasal vaccination against respiratory and intestinal infections in humans.     

Microfluidic cell counter/sorter utilizing multiple particle tracing technique and optically switching approach

This paper proposes a novel microfluidic system based on a computer controlled digital image processing (DIP) technique and optical tweezers for automatic cell/microparticle recognition, counting and sorting in a continuous flow environment. In the proposed system, the cells/microparticles are focused electrokinetically into a narrow sample stream and are then driven through the region of interest (ROI), where they are recognized and traced in real time using a proprietary DIP system. Synchronized control signals generated by the DIP system are then used to actuate a focused IR laser beam to displace the target cells from the main sample stream into a neighboring sheath flow, which carries them to a downstream collection channel where they are automatically counted. Experimental trials show that the microchip is capable of continuously sorting and counting microparticles with diameters of 5 and 10 μm. In addition, a sample composed of yeast cells and polystyrene (PS) beads is successfully sorted and collected with a 100% of yield ratio and 91.9% of recovery ratio. The proposed system provides a simple, low-cost, high-performance solution for cell manipulation in microfluidic devices. The preliminary results of the current paper had been presented at the 5th IEEE Conference on Sensors (IEEE Sensors 2006), Daegu, Korea, Oct. 22–25, 2006.     

Vaccine protocols for enhanced immunogenicity of exogenous antigens

Vaccination protocols aim at the delivery of exogenous antigen (Ag) to antigen-presenting cells (APCs) concurrent with the activation of APCs by adjuvants. Activated APCs then cross-present the Ag, cross-prime T effector cells, and activate B cells. Classical protocols rely on a mixture of both Ag and the adjuvant. However, a disadvantage of this strategy is that simultaneous “loading” and activation of APCs is not guaranteed. As a consequence, heterogeneous APC populations will be generated, including APCs being either Ag-presenting or only activated, thus rendering the adaptive immune response suboptimal. Therefore, novel strategies are needed that provide both constituents to the same APC in order to generate a homogeneous Ag-presenting and activated cell population. Here we show that these requirements can be fulfilled via two distinct methods, either by covalently linking Ag to the adjuvant or by co-encapsulating Ag and adjuvant into biodegradable microparticles. These novel vaccine protocols allow the generation of robust T-cell and B-cell responses that match immunogenicity of live vectors. Their characteristics with regard to efficacy, flexibility, and clinical applicability are discussed.     

疫苗微粒鼻腔给药系统的研究进展

疫苗微粒鼻腔给药作为一种新型非侵入性疫苗给药系统,具有能延长免疫时间、增强免疫效果、提高病人顺应性等优点。综述疫苗鼻腔免疫诱导机制、常用的微粒系统、免疫效果影响因素及其评价方法及目前该给药系统存在的问题,旨在为研究开发疫苗微粒鼻腔给药系统提供新的思路和方法。     

细胞膜微粒对血管内皮细胞功能的影响

目的 探讨细胞膜微粒(Mp)在激素相关性股骨头缺血坏死(ANFH)发生机制中所起的作用.方法 选择体外培养的第3代人脐静脉内皮细胞(HUVEC)作为研究对象.从健康人、股骨头坏死患者和使用大剂量激素治疗的非股骨头坏死患者血液中分离出的细胞膜微粒作为刺激因子.将不同来源的细胞膜微粒与HUVEC共同培养一段时间后观察细胞形态学变化,并用流式细胞仪检测细胞培养基中膜微粒种类和数量的改变,反转录聚合酶链反应(RT-PCR)检测内皮细胞凋亡基因的表达.结果 倒置相差显微镜观察细胞:发现各组内皮细胞的形状、密度、生长速度和细胞核的形态差异无统计学意义.流式细胞仪分析显示,CD62E+/CD31+代表细胞活跃程度.共培养48 h后,激素治疗的实验组CD62E+/CD31+明显低于对照组(P=0.035),其他各实验组和对照组差异无统计学意义(P＞0.05).RT-PCR分析显示,fas/β-肌动蛋白代表凋亡基因的表达.激素性ANFH的实验组fas/β-肌动蛋白明显高于对照组(0.776±0.230＞0.669±0.148,P=0.006).激素治疗的实验组高于对照组(0.914±0.226＞0.832±0.200,P=0.005).其余各实验组和对照组之间差异无统计学意义(如:酒精性ANFH 0.810±0.252＞0.781±0.194,P＞0.05).结论 使用激素患者的细胞膜微粒在体外有促进血管内皮细胞凋亡的作用.激素性股骨头坏死患者的细胞膜微粒在体外能够增加血管内皮细胞凋亡基因的表达,但是对内皮细胞释放膜微粒无明显影响.健康人和酒精性股骨头坏死患者的细胞膜微粒在体外对血管内皮细胞的凋亡无明显影响.     

化学发光微粒子免疫分析法与酶联免疫法测定乙肝标志物的比较

目的:比较化学发光微粒子免疫分析法(CMIA)与酶联免疫(ELISA)两种方法检测乙肝病毒免疫标志物的优缺点。方法:用上述两种方法的检测试剂及仪器对200例临床血清标本分别进行检测,然后进行结果分析。结果:ELISA法和CMIA法对乙肝表面抗原的检测灵敏度均能达到0.1IU/ml。HBsAg,HBsAb,HBeAg及HBeAb的检测相互符合率均在95%以上,但对核心抗体的检测,两者符合率为87.5%。结论:两种方法的检测性能相差不大,但CMIA法检测血清乙肝标志物的自动化程度比ELISA法高,并能定量检测HBsAg和HBsAb,可动态观察疗效和监测病情。