时间分辨荧光免疫技术在乙肝病毒标志物检测中的应用

目的以微粒子酶免疫分析（MEIA）为参照，探讨时间分辨荧光免疫技术（TRFIA）定量测定乙肝病毒标志物的临床应用价值。方法采用TRFIA与酶免疫吸附试验技术（ELISA）对623份随机标本做5项乙肝病毒标志物测定，其中95份标本用MEIA复测。结果TRFIA法检测乙肝五项指标与MEIA法比较，差异无显著性（P〉0．05）。结论TRFIA法测定乙肝五项指标的相对特异性、灵敏度及符合率较ELISA法高，为临床疗效观察及乙肝疫苗的接种提供科学依据。     

One-step quantitative thyrotropin assay for the detection of hypothyroidism in point-of-care conditions

OBJECTIVES: Different screening strategies for early diagnosis of hypothyroidism have been discussed increasingly. We demonstrate the applicability of a miniaturized microparticle assay format for rapid and quantitative determination of increased thyrotropin (TSH) concentrations in serum. DESIGN AND METHODS: Porous microparticles were used as solid phase for a noncompetitive, one-step, kinetic immunoassay with varying incubation times and time-resolved fluorescence detection. RESULTS: The analytical (mean of zero + 3 SD) and functional (CV <15%) detection limits were 1.5 and 6.0 mIU/L for 2-min, 0.5 and 1.5 mIU/L for 7-min, and 0.2 and 0.5 mIU/L for 15-min assays, respectively. A good correlation was found with the Chiron Diagnostics ACS:180 assay (slopes 0.885-1.051, y-intercepts < +/- 0.20 mIU/L, S(y logical or, bar below x) 0.98, n = 20). CONCLUSION: The kinetic TSH assay provides reproducible and quantitative information on thyroid status within minutes and is applicable for the detection of hypothyroidism in point-of-care (POC) conditions.     

Modifying theophylline microparticle surfaces via the sequential deposition of poly(vinyl alcohol-co-vinyl acetate) copolymers

The aim of this study was to investigate the manner in which amphiphilic poly(vinyl alcohol-co-vinyl acetate) copolymers (PVA-Ac) assembled on drug surfaces and use this information to generate a novel bi-layer polymer coating for a theophylline microparticle. Three grades of PVA-Ac, differing in hydrolysis degree and monomer distribution, were synthesised, characterised by nuclear magnetic resonance and shown to interact with theophylline when suspended in water. PVA-Ac deposition at the solid/liquid interface was driven by polymer hydrogen bond formation in a process that induced consequential structural changes in the macromolecule architecture. The most hydrophobic grades of the copolymer appeared to adsorb in a multistage process that passed through a series of equilibrium points. The PVA-Ac surface allowed two grades of the copolymer to be sequentially adsorbed and this resulted in the fabrication of a microparticle with desirable characteristics for pharmaceutical formulation production.     

Trophoblast deportation part II: a review of the maternal consequences of trophoblast deportation

The deportation of trophoblast debris from the placenta was first documented over 100 years ago, and today we know that the deported material ranges from multinucleated syncytial knots/sprouts to trophoblast-derived nanoparticles. However little is known about the effect of trophoblast debris on maternal physiology since it is difficult to investigate these effects in vivo in women. Animal models have been reported but they have provided relatively little information. Most of our current knowledge regarding the effects of trophoblast debris on maternal systems is provided by studies using trophoblast debris obtained from in vitro models of the human placenta. Herein we review the animal models and the in vitro studies, which, between them, suggest that deported trophoblast material may play a role in tolerising the maternal immune system during normal pregnancy, and conversely that in pathological pregnancies aberrant maternal immune and/or endothelial/vascular responses may result from a change in either the quantity or quality of deported trophoblast debris.     

Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma

Introduction

In an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1 - 6 °C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasma's hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage.

Materials and Methods

FFP units (n = 30) were thawed at 37 °C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography).

Results

In FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408 × 10³/L). FFP-5 showed a decline in MP count by 50% (p < 0.0001), and procoagulant activity by 29% (p < 0.0001). FFP-5 exhibited only 54% (p < 0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p < 0.0001) and a longer delay in reaching maximum clot (p < 0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it.

Conclusions

Decline in MP with storage contributes to FFP-5's reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.     

血浆D-二聚体测定对急性缺血性结肠炎的早期诊断价值

目的观察血浆D-二聚体水平在急性缺血性结肠炎发病过程中的变化,探讨其在急性缺血性结肠炎早期诊断中的价值。方法采用微粒子酶免分析法检测36例急性缺血性结肠炎(观察组)患者入院第1、7、14天血浆D-二聚体水平,并与34例健康体检者(对照组)比较;分析D-二聚体水平与急性缺血性结肠炎病变范围的关系。结果入院第1天及治疗第7、14天,观察组患者血浆D-二聚体水平均显著高于对照组,差异有统计学意义(P<0.01)。观察组中,全结肠病变患者血浆D-二聚体水平显著高于乙状结肠、降结肠、横结肠、升结肠病变患者,降结肠及乙状结肠病变患者血浆D-二聚体水平亦显著高于乙状结肠、降结肠、横结肠、升结肠病变患者,差异均有统计学意义(P<0.05)。结论急性缺血性结肠炎患者血浆D-二聚体水平较健康者显著升高,故通过检测血浆D-二聚体水平可以早期诊断急性缺血性结肠炎;病变范围广患者的血浆D-二聚体水平显著高于病变范围小患者。     

Anticoagulant effects of an antidiabetic drug on monocytes in vitro

Introduction

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.

Methods

We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).

Results

Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.

Conclusions

Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.     

A novel method for the direct fabrication of growth factor-loaded microspheres within porous nondegradable hydrogels: Controlled release for cartilage tissue engineering

Because of similar mechanical properties to native cartilage, synthetic hydrogels based on poly(vinyl alcohol) (PVA) have been proposed for replacement of damaged articular cartilage, but they suffer from a complete lack of integration with surrounding tissue. In this study, insulin-like growth factor-1 (IGF-1), an important growth factor in cartilage regeneration, was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles embedded in the PVA hydrogels in a single step based on a double emulsion. The release of IGF-1 from these hydrogels was sustained over 6 weeks in vitro. Poly(glycolic acid) (PGA) fiber scaffolds were wrapped around the hydrogels, seeded with chondrocytes, and implanted subcutaneously in athymic mice. The release of IGF-1 enhanced cartilage formation in the layers surrounding the hydrogels, in terms of the content of extracellular matrix components and mechanical properties, and increased integration between the cartilage layers and the hydrogels, according to gross observation of the cross-sections and histology. The compressive modulus of the cartilage-hydrogel constructs without IGF-1 was 0.07 ± 0.02 MPa, compared to 0.17-0.2 MPa for hydrogels that contained IGF-1. The biochemical and mechanical markers of cartilage formation were not different between the low and high concentrations of IGF-1, despite an order of magnitude difference in concentration. This study shows that the sustained release of IGF-1 can enhance tissue formation and points to a possible strategy for effecting integration with surrounding tissue.     

Immune response after oral administration of the encapsulated malaria synthetic peptide SPf66

The synthetic peptide SPf66 adsorbed on alum is one of the few Plasmodium falciparum vaccines which have been tested in field trials. We previously reported that subcutaneous administration of SPf66 loaded PLGA microparticles (MP) enhances the antibody response to this antigen compared to the conventional alum formulation. We now evaluate the suitability of polymeric formulations to obtain systemic immune responses by gastric intubation of Balb/c mice. Formulations composed of 1:1 mixtures of PLGA 50:50 and 75:25 (lactic:glycolic) microparticles were administered by the oral route, and when animals were boosted 3 weeks later significant systemic IgG antibody responses were elicited, comparable to alum triple shot and superior to the aqueous vaccine given by the oral route. The finding of IgG2a isotype for PLGA-vaccinated mice compared to the absent levels of this isotype for the alum-vaccinated group could be interpreted as a sign of Th1-like immune response and cellular immune response activation. Our results confirm that using the appropriate schedule the oral administration of PLGA particles is suitable to obtain systemic immune responses to the carried antigen.