保元汤加减对实验大鼠肺孢子虫作用的电镜观察

目的　探索中医药治疗卡氏肺孢子虫肺炎的途径。　方法　应用中药保元汤加减煎剂给实验大鼠灌胃 ,通过透射电镜观察其对实验大鼠肺内肺孢子虫的作用。　结果　实验组较对照组大鼠肺内肺孢子虫有明显改变。其中肺孢子虫胞浆内大量空泡形成 ;包囊壁明显破坏 ;虫体有溶解现象。　结论　保元汤加减煎剂 ,对实验大鼠体内的卡氏肺孢子虫具有抑制和杀灭作用。     

Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

Hyperplastic Lymphoid Tissue in HIV/AIDS: An Electron Microscopic Study

The purpose of this study was to characterize the ultrastructure of lymphoid tissue from HIV/AIDS patients and to evaluate it as a reservoir and source of HIV. HIV has been demonstrated in lymph nodes and tonsils and adenoids, by immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM), to be associated with germinal center (GC) follicular dendritic cells (FDC). The presence of HIV in the larger gastrointestinal tract-associated lymphoid tissue (GALT) has been much less studied. Whether FDC themselves are productively infected by HIV in any of the lymphoid sites is controversial. Lymph nodes, tonsils, and gastrointestinal biopsies were fixed in neutral buffered glutaraldehyde and prepared for TEM. Mature HIV particles were abundant in GC of hyperplastic lymph nodes, tonsils, and the GALT. They were enmeshed within an electron-dense matrix associated with an all-encompassing branching FDC network of processes. HIV particles were seen budding from both FDC and lymphocytes. The greatest numbers of particles were seen in hyperplastic lymphoid tissue from untreated individuals and in lymph nodes co-infected with opportunistic organisms, such as Mycobacterium aviumcomplex. In addition to HIV, unidentifiable “particles” of varying sizes, possibly including other viruses, were regularly seen in association with FDC. Ultrastructural study graphically demonstrated the abundance of HIV particles associated with the complex FDC network of hyperplastic lymph nodes, tonsils, and GALT. HIV was shown to productively infect FDC, as well as lymphocytes.     

Protective effects of melatonin against spinal cord injury induced oxidative damage in rat kidney: A morphological and biochemical study

Spinal cord injury (SCI) induced oxidative stress affects multiple organ systems including the kidney. We studied the possible protective effects of melatonin on SCI-induced oxidative damage in renal tissues of rats. Wistar albino rats (n = 24) were exposed to SCI and divided into vehicle- or melatonin-treated SCI groups. Melatonin was administred intraperitoneally at a dose of 10 mg/kg for seven days. Renal tissues were investigated by light and electron microscopy. Furthermore, tissue malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) and superoxide dismutase (SOD) activities were also determined. In the vehicle-treated SCI group, the renal histology was disturbed compared to controls, whereas the melatonin-treated SCI group showed significantly reduced degeneration of renal tissue as seen by both light and electron microscopy. MDA levels, MPO and SOD activities were increased and GSH levels were decreased in the vehicle-treated SCI group compared to controls. On the other hand, decreased MDA levels and MPO activities and increased GSH levels were observed in the melatonin-treated SCI group compared to vehicle-treated SCI group. These results showed that experimentally induced SCI caused oxidative stress in the rat kidney, whereas melatonin treatment reduced oxidative stress, suggesting that it may be used as a complementary therapy of renal problems occurring following SCI.     

Cytotoxic and genotoxic potential of respirable fraction of composite dust on human bronchial cells

ObjectiveTo determine the cytotoxic and genotoxic potential of the respirable fraction of composite dust (<4 μm) on human bronchial epithelial cells.MethodsComposite sticks of three commercial dental composites (Filtek Supreme XTE, Grandio, Transbond XT) were ground in an enclosed plexiglass chamber with a rough dental bur (grain-size 100 μm) and the generated airborne respirable dust was collected in a personal cyclone on a teflon filter (pore size 5 μm). Immediately after particle collection, the dust was quantified gravimetrically and the particles were suspended in cell culturing medium. Next, human bronchial epithelial cells (16HBE14o-) were exposed to the suspensions (3 μg/ml–400 μg/ml). After 24 h, cell viability (WST-1 assay) and membrane integrity (LDH assay) were evaluated. Furthermore, the genotoxic effect of a sub-cytotoxic concentration (50 μg/ml) of composite dust was evaluated by the comet assay after 3 h exposure and cell cycle disturbances were analyzed by flow cytometry. Cellular uptake of particles was evaluated by transmission electronic microscope (TEM).ResultsFor all three tested composite materials, a decrease in metabolic activity of 10–35% was observed when the cells were exposed to the highest concentrations (100 μg/ml–400 μg/ml). Toxicity was partially linked to membrane disruption especially after 72 h exposure. All tested composites provoked a mild genotoxic effect after short-term exposure compared to the control groups. TEM revealed that respirable particles of all tested composites were taken up by the cells.SignificanceThe respirable fraction of composite dust only showed cytotoxic effects at the highest concentrations, whereas mild genotoxicity was observed after exposure to a sub-cytotoxic concentration.     

Delivery and Biodistribution of Traceable Polymeric Micellar Diclofenac in the Rat

The nonsteroidal anti-inflammatory drugs elevate cardiovascular risk, perhaps, due to their accumulation in the heart and kidneys. We designed nanodelivery systems for cardiotoxic diclofenac to reduce its presence in these organs. Diclofenac ethyl ester (DFEE) was encapsulated in traceable micelles based on poly(ethylene oxide)-b-poly(ε-caprolactone) (DFEE-PCL-TM) or poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (DFEE-PBCL-TM). Diclofenac pharmacokinetics and tissue distribution were studied after intravenous (iv) and intraperitoneal administration of the nanoformulations and compared with those after iv doses of free diclofenac (n = 3-6/group). The average diameters for DFEE-PBCL-TM and DFEE-PCL-TM were 37.2 ± 0.06 and 45.1 ± 0.06 nm, respectively. Drug concentration dropped below the assay sensitivity after free drug administration in 6 h, but persisted for 24 h following DFEE-PBCL-TM (2.3 ± 1.4 μg/mL) and DFEE-PCL-TM (1.9 ± 0.6 μg/mL) iv administration. The diclofenac heart:blood and kidney:blood ratios were 5- to 12-fold lower with the nanoformulations than with free diclofenac. Near-infrared fluorescence measurements in tissues suggested exposure patterns to nanocarriers parallel with those achieved for delivered diclofenac by nanoformulations. Administration of DFEE-PCL-TM by iv or intraperitoneal injection, resulted in comparable pharmacokinetics and 6 h postdose near-infrared fluorescence in the heart, kidneys, liver, and spleen. When compared to each other, DFEE-PBCL-TM showed significantly lower diclofenac levels in the heart compared to DFEE-PCL-TM (0.3 ± 0.03 vs. 0.5 ± 0.1 μg/g). Developed nanoformulations of diclofenac prolonged diclofenac circulation and reduced its presence in the heart and kidneys, strongly suggesting cardiac-safe delivery vehicles for diclofenac.     

Iron-Oxide Nanocluster Labeling of Clostridium novyi-NT Spores for MR Imaging–Monitored Locoregional Delivery to Liver Tumors in Rat and Rabbit Models

PurposeTo label Clostridium novyi-NT spores (C. novyi-NT) with iron oxide nanoclusters and track distribution of bacteria during magnetic resonance (MR) imaging-monitored locoregional delivery to liver tumors using intratumoral injection or intra-arterial transcatheter infusion.Materials and MethodsVegetative state C. novyi-NT were labeled with iron oxide particles followed by induction of sporulation. Labeling was confirmed with fluorescence microscopy and transmission electron microscopy (TEM). T2 and T2* relaxation times for magnetic clusters and magnetic microspheres were determined using 7T and 1.5T MR imaging scanners. In vitro assays compared labeled bacteria viability and oncolytic potential to unlabeled controls. Labeled spores were either directly injected into N1-S1 rodent liver tumors (n = 24) or selectively infused via the hepatic artery in rabbits with VX2 liver tumors (n = 3). Hematoxylin-eosin, Prussian blue, and gram staining were performed. Statistical comparison methods included paired t-test and ANOVA.ResultsBoth fluorescence microscopy and TEM studies confirmed presence of iron oxide labels within the bacterial spores. Phantom studies demonstrated that the synthesized nanoclusters produce R2 relaxivities comparable to clinical agents. Labeling had no significant impact on overall growth or oncolytic properties (P >.05). Tumor signal-to-noise ratio (SNR) decreased significantly following intratumoral injection and intra-arterial infusion of labeled spores (P <.05). Prussian blue and gram staining confirmed spore delivery.ConclusionsC. novyi-NT spores can be internally labeled with iron oxide nanoparticles to visualize distribution with MR imaging during locoregional bacteriolytic therapy involving direct injection or intra-arterial transcatheter infusion.     

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.     

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.