Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland

The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here ∼65000 μm²) was fulfilled by examining tissue areas along the edges of ultrathin sections. (2) The size of the counting frame was determined by measuring the lengths of the section margins (minus a guard area) by means of a Morphomat. This value was multiplied by the width of the investigated tissue zone, corresponding to the diameter of the electron microscope viewing screen. (3) Disector counting was carried out simultaneously on both sections (bidirectional disector) to improve efficiency. In the present study tiny synaptic bodies (SBs) were quantitated by disector in a rat pineal gland, yielding ∼30 SBs/1000 μm³. By contrast, single section profile counts of SBs amounted to 90 SBs/20000 μm². Since the presently described adaptation of the disector is time-consuming, it is proposed to determine a proportion factor allowing to estimate number of structures per volume based on single section profile counts. This would decrease the evaluation time by more than 50%.     

Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.     

Spray-dried fucoidan microparticles for pulmonary delivery of antitubercular drugs

Pulmonary tuberculosis accounts for 80% of cases and the delivery of antitubercular drugs into the lungs allows targeting the infected organ and, possibly, reducing systemic drug toxicity. This work aimed at using fucoidan as matrix of inhalable microparticles that associate two first-line antitubercular drugs, for an application in pulmonary tuberculosis therapy. Fucoidan is composed of fucose and sulphated sugar residues, moieties described as being recognised by surface receptors of alveolar macrophages, which host mycobacteria. Inhalable fucoidan microparticles loaded with antitubercular drugs were successfully produced with high association efficiencies of either isoniazid (95%) or rifabutin (81%). The microparticles evidenced no cytotoxicity on lung epithelial cells (A549). However, rifabutin-loaded microparticles showed a certain degree of toxicity on macrophage-like cells (THP-1) at the highest tested concentration (1?mg/mL). Furthermore, microparticles showed favourable aerodynamic properties for deep lung delivery (MMAD 2.0–3.8?µm) and, thus, show potential for an application as inhalable tuberculosis therapy.     

In vivo biocompatibility of the PLGA microparticles in parotid gland

Poly(lactic-co-glycolic acid) (PLGA) microparticles are used in various disorders for the controlled or sustained release of drugs, with the management of salivary gland pathologies possible using this technology. There is no record of the response to such microparticles in the glandular parenchyma. The purpose of this study was to assess the morphological changes in the parotid gland when injected with a single dose of PLGA microparticles. We used 12 adult female Sprague Dawley rats (Rattus norvegicus) that were injected into their right parotid gland with sterile vehicle solution (G1, n=4), 0.5 mg PLGA microparticles (G2, n=4), and 0.75 mg PLGA microparticles (G3, n=4); the microparticles were dissolved in a sterile vehicle solution. The intercalar and striated ducts lumen, the thickness of the acini and the histology aspect in terms of the parenchyma organization, cell morphology of acini and duct system, the presence of polymeric residues, and inflammatory response were determined at 14 days post-injection. The administration of the compound in a single dose modified some of the morphometric parameters of parenchyma (intercalar duct lumen and thickness of the glandular acini) but did not induce tissue inflammatory response, despite the visible presence of polymer waste. This suggests that PLGA microparticles are biocompatible with the parotid tissue, making it possible to use intraglandular controlled drug administration.     

Solid lipid microparticles containing loratadine prepared using a Micromixer

Solid lipid microparticles were investigated as a taste-masking approach for a lipophilic weak base in a suspension. The idea was that the drug concentration in the aqueous phase of a suspension might be reduced by its partitioning into the solid lipid particles. Loratadine, as a model drug, was used to prepare Precirol® ATO 5 microparticles by a Micromixer. The effects of three process variables: drug loading, PVA concentration and water/lipid ratio on the microparticle size, encapsulation efficiency, surface appearance, in-vitro release and drug partitioning in a suspension were studied. Loratadine release was slow in simulated saliva and very fast at the pH of stomach. In suspension of loratadine lipid microparticles, drug was released into the aqueous phase to the same concentration as in a drug suspension. Therefore, the usefulness of these microparticles for taste-masking in liquids is limited. However, they might be useful for taste-masking in solid dosage forms.     

Imbalance between the circulating endothelium-derived apoptotic microparticles and the endothelial colony-forming units of progenitor cells in patients undergoing diagnostic coronary angiography

PurposeThe involvement of the circulating endothelium-derived microparticles (EMPs) and the endothelial progenitor cells (EPCs) has been shown in the pathogenesis of coronary artery disease (CAD). The current study aimed to explore whether the Friesinger index is associated with the levels of the apoptotic CD144+/CD31+/annexin V+ ​EMPs and the number of endothelial colony-forming units of progenitor cells in patients undergoing coronary angiography.Patients and methodsFifty-seven patients with a median age of 62 years (range: 48–84 years) were enrolled. Quantification of the apoptotic CD144⁺/CD31⁺/annexin V⁺ EMPs was performed by flow cytometry. The number of endothelial colony-forming units defined by CFU-Hill was assessed by cell culture.ResultsThere was a positive correlation between the Friesinger index and the circulating levels of the apoptotic CD144⁺/CD31⁺/annexin V⁺ EMPs (rho=0.817, p<0.001), whereas a negative correlation was found with the number of CFU-Hill (rho ​= ​− 0.649, p<0.001). Multivariable logistic analysis showed that the risk of having moderate/severe CAD was five times greater among male patients (OR:5.32; 95% CI: 1.19 - 16.33; p=0.038) and almost one and a half times higher among those with a higher level of apoptotic CD144⁺/CD31⁺/annexin V⁺ EMPs (OR:1.74; 95% CI: 1.23 - 2.28; p=0.001). Finally, the circulating levels of apoptotic EMPs labelled for CD144⁺/CD31⁺/annexin V⁺ presented a good discrimination of moderate/severe CAD, with an AUC of 0.85 (95% CI ​= ​0.74 - 0.96; p< 0.001).ConclusionsModerate or severe CAD is associated with increased levels of apoptotic EMPs and reduced EPC colony-forming capacity, increasing the occurrence of endothelial injuries.     

Placental Microparticles,DNA, and RNA in Preeclampsia

Preeclampsia is a common disorder of the second half of pregnancy that complicates 2% to 7% of all pregnancies worldwide and remains a major cause of maternal and fetal morbidity and mortality. Although the origin of the disease is still elusive, population-based studies have suggested that it might implicate genetic, immunologic, or physiologic factors. On the other hand, there is no doubt that the placenta plays an important role in its development. In preeclampsia, the shedding of placenta debris, such as syncytiotrophoblast microparticles (STBMs) and DNA and messenger RNA molecules, into the maternal peripheral blood is increased. The analysis of this material may give new insight into placentation and the underlying etiology of this disorder, as well as yield new tracks of research for the understanding of the molecular mechanisms, leading to the generation of the clinical symptoms.     

Air pollution‐associated procoagulant changes: the role of circulating microvesicles

Summary. Background: Epidemiological studies suggest an association between exposure to particulate matter (PM) in air pollution and the risk of venous thromboembolism (VTE). Objectives: To investigate the underlying pathophysiological pathways linking PM exposure and VTE. Patients and methods: We assessed potential associations between PM exposure and coagulation and inflammation parameters, including circulating microvesicles, in a group of 233 patients with diabetes. Results: The numbers of circulating blood platelet‐derived and annexin V‐binding microvesicles were inversely associated with the current levels of PM_2.5 or PM₁₀, measured on the day of sampling. Recent past exposure to PM₁₀, up to 1 week prior to blood sampling, estimated at the patients’ residential addresses, was associated with elevated high‐sensitivity C‐reactive protein (CRP), leukocytes and fibrinogen, as well as with tissue factor (TF)‐dependent procoagulant changes in thrombin generation assays. When longer windows of past exposure were considered, up to 1 year preceding blood sampling, procoagulant changes were evident from the strongly increased numbers of red blood cell‐derived circulating microvesicles and annexin V‐binding microvesicles, but they no longer associated with TF. Past PM exposure was never associated with activated partial thromboplastin time (aPTT), prothrombin time (PT), or factor (F) VII, FVIII, FXII or D‐dimers. Residential distance to a major road was only marginally correlated with procoagulant changes in FVIII and thrombin generation. Conclusions: Increases in the number of microvesicles and in their procoagulant properties, rather than increases in coagulation factors per se, seem to contribute to the risk of VTE, developing during prolonged exposure to air pollutants.     

Non-invasive endotracheal delivery of paclitaxel-loaded alginate microparticles

Aerosolized chemotherapeutics leads to higher, localized and continuous concentrations of active agents in lung tissue with lower side effects for other organs. The present study was performed on jugular vein cannulated rats which endothracheally received 4 mg/kg of free paclitaxel powder (Free-PTX), paclitaxel-loaded alginate microparticles (PTX-ALG-MPs) and i.v. paclitaxel (Anzatax^®). Pharmacokinetic parameters for Free-PTX and PTX-ALG-MPs contain higher AUC, mean residence time (MRT),half-life and bioavailability, with lower elimination constant (k_e). Statistical analysis showed that the amount of paclitaxel per gram of lung tissue after 0.5, 6 and 24 h after administration of Free-PTX was lower than PTX-ALG-MPs. Lung tissue AUC for Free-PTX was lower than PTX-ALG-MPs. According to the obvious advantages obtained, such as dose lowering and increasing paclitaxel residence time and half-life. It should be noted that cell cytotoxicity test on normal airway cell lines was not examined in this study but due to previous reports on safety of inhaled paclitaxel, it can be suggested that pulmonary delivery of paclitaxel can be a useful non-invasive route of administration compared with i.v administration.     

明胶海绵微粒TACE治疗Barcelona临床肝癌分期B期肝细胞癌对外周血中髓系来源抑制性细胞的影响

目的探讨应用明胶海绵微粒TACE(GSMs-TACE)治疗Barcelona临床肿瘤(BCLC)分期B期肝细胞癌(HCC)对患者外周血中髓系来源抑制性细胞(MDSCs)的影响。方法对5例临床确诊为BCLC B期的HCC患者(HCC组)行GSMs-TACE治疗。采用流式细胞仪分别检测患者术前、术后10天及术后30天外周血中MDSCs频率(即MDSCs细胞团占HLA-DR~-细胞群的比例)。另收集7名健康志愿者(正常对照组),于HCC组术前同期进行MDSCs频率检测。比较HCC患者术前及术后不同时间MDSCs频率的差异及HCC组术前与正常对照组同期MDSCs频率的差异。结果 HCC组GSMs-TACE术前外周血中MDSCs频率为(30.26±12.12)%,术后10天降至(10.22±3.79%),术后30天降至(7.33±3.38)%,总体差异有统计学意义(P0.001);两两比较显示术后30天(P0.001)及术后10天(P=0.011)均明显低于术前。HCC组术前MDSCs频率明显高于正常对照组同期水平[(30.26±12.12)%vs (3.41±1.89)%,t=5.876,P0.001)。结论 BCLC B期HCC患者经GSMs-TACE治疗后外周血中MDSCs频率显著降低;GSMs-TACE对患者的机体免疫功能具有正向调节作用。