Imaging macrophages in trehalose with SIMS

Phagocytosis is a major component of the animal immune system where apoptotic cellular material, metabolites, and waste are safely processed. Further, efficient phagocytosis by macrophages is key to maintaining healthy vascular systems and preventing atherosclerosis. Single-cell images of macrophage phagocytosis of red blood cells, RBCs, and polystyrene microspheres have been chemically mapped with TOF-SIMS. We demonstrate here cholesterol and phosphocholine localizations as relative to time and activity.     

Exact determination of phagocytic activity of alveolar macrophages toward polymer microspheres by elimination of those attached to the macrophage membrane

A method for exact determination of phagocytic activity of alveolar macrophage (M) cells toward synthetic microspheres (MS) by optical microscopy was developed. We examined the effectiveness of the treatment of M samples with trypsin, acid or xylene to remove the polystyrene latex microspheres (PSL MS) attached to M cell membranes during their phagocytosis by M cells. We found that centrifugation, which was employed to collect M samples after incubation with MS, affected significantly the efficiency of the various treatments. Of the three treatments, xylene treatment without centrifugation was the most effective to determine the phagocytic activity of M cells, as xylene dissolved the PSL MS on the cell surface almost completely. This treatment was also effective in the case of poly(lactic-co-glycolic acid) MS (PLGA MS), which have been commonly used as an efficient vehicle for drug delivery system.     

Effects of liposomal phophatidylserine on phagocytic uptake of liposomes by macrophage-like HL-60RG cells

The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.     

Surface charge of PLA microparticles in regulation of antigen loading,macrophage phagocytosis and activation,and immune effects in vitro

The use of microparticles（MPs） as adjuvants has attracted increasing interest in vaccine delivery systems.Many physiochemical characteristics of MPs including hydrodynamic size,surface properties,and morphology can regulate the immune response.Surface charge is an important characteristic of MPs,but how it affects their adjuvanticity remains unknown.In this study,we prepared uniform-sized polylactide MPs coated with various polymers of different positive charge,and investigated how the surface charge affected antigen loading and macrophage phagocytosis and activation in vitro.A higher surface charge greatly enhanced antigen loading and antigen internalization into macrophages,promoted the expression of MHC 11 and CD80,and increased the secretion level of TNF-a.Taken together,these results indicated that surface charge was an important parameter for improving the adjuvanticity of MPs.     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.     

Synthesis and evaluation of the bioactivity of simplified analogs of the seco-pseudopterosins; progress toward determining a pharmacophore

The pseudopterosins are marine natural products that display significant anti-inflammatory and wound healing properties. We describe the synthesis of six structural analogs of the seco-pseudopterosin-like core that are devoid of all but one of the stereocenters found in the carbocyclic core of the natural products. Our targets were selected in an attempt to identify the minimal pharmacophore for the pseudopterosins and their seco analogs. A deliberate effort was made to utilize a conservative synthetic approach based upon the use of well-established reactions, which enabled us to develop routes that proved to be efficient, practical, and easy to implement. The results of several bioassays, including an assessment of the ability to inhibit phagocytosis and to competitively bind to the adenosine receptor A_2A, demonstrate that greatly simplified structural analogs of the pseudopterosins and their seco forms are capable of maintaining several of the important bioactivities that characterize the natural products, and do so with comparable efficacy. Those systems bearing two rather than one oxygen atom appended directly to the aromatic ring are the more effective binding agents. This observation may provide a significant clue regarding the key structural features of the minimal pharmacophore.     

Real-time monitoring of cell migration,phagocytosis and cell surface receptor dynamics using a novel,live-cell opto-microfluidic technique

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic “Lab-in-a-Trench” (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell–cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective–diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.     

Role of lipid rafts in innate immunity and phagocytosis of polystyrene latex microspheres

Understanding of the association of phagocytosis of polymers with signaling of innate immunity of macrophages is the major purpose of this study. Polymer conjugates have been utilized for clinical therapy of cancer and infections, such as Mycobacterium tuberculosis, as effective vectors of drug-delivery systems. They are incorporated through phagocytosis into macrophages and activate innate immunity signaling, which plays a crucial role in its therapeutic and side effects. Macrophage phagocytosis of polystyrene latex microspheres was examined and assayed by treatment of macrophages with the cholesterol depletor methyl-β-cyclodextrin (MβCD) or the sphingolipid depletor n-octyl-β-D-glucopyranoside (OGP). Expressions of various mRNAs during phagocytosis were quantified by real-time PCR. Phagocytosis of polystyrene latex microspheres by various macrophages, such as murine monocyte-derived macrophage J774, rat alveolar macrophage NR8383, and murine Kupffer cell KC13-2, was suppressed by treatment with MβCD or OGP in a concentration-dependent manner. The expression of mRNAs of TNFα, IL-1β, IL-6 and CXCL10 genes induced by lipopolysaccharide (LPS) was not suppressed by treatment with MβCD in J774 cells. Moreover, genes that were induced by LPS were up-regulated even in the absence of LPS by the phagocytosis of polymer conjugates, but such up-regulations were not suppressed by the treatment with MβCD. It was shown that lipid rafts play a significant role in incorporation of polymer conjugates through phagocytosis of macrophages, but their association with signal transduction in innate immunity is very limited.     

Biodistribution of doxorubicin and nanostructured ferrocarbon carrier particles in organism during magnetically controlled drug delivery

Biodistribution of doxorubicin and ferrocarbon carrier particles in organism during and after magnetically controlled anti-tumor drug delivery and deposition was studied. Animal tests show high concentration of the cytostatic drug in the target zone, while its concentration is three orders of magnitude lower in bloodstream and other organs. A significant depot of the drug remains on the deposited particles days after the procedure. Macrophages actively phagocytose the ferrocarbon (FeC) particles and remain viable long enough to carry them to the lymph nodes.