高碘酸氧化肝素抑制P-选择素介导的A375细胞粘附

摘要通过选择性化学修饰制备出具有低抗凝血活性的高碘酸氧化肝素, 并系统地分析了其抗肿瘤细胞粘附的活性. 研究结果表明, 具有低抗凝血活性的高碘酸氧化肝素仍然可以显著地抑制P-选择素介导的人恶性黑素瘤A375细胞与血小板间的粘附.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

Modeling the Effect of the Relative Humidity on the Manipulation of Nanoparticles with an Atomic Force Microscope

In the manipulation of nanoparticles, different behaviors are typically observed including sliding, rolling and rotation. Most of investigations in this field have so far focused on describing the interaction forces under vacuum (dry air) environmental condition, while the effect of the relative humidity has been poorly considered. In this work we developed a model for simulating the dynamic nanoparticle motion (rolling and sliding) in an AFM-based manipulation of nanoparticles in a humid environment. In our method, the interaction forces include the adhesion force, mainly consisting of the capillary force and van der Waals force, the normal force and friction forces. We calculated the adhesion force by considering the contributions from the wet and dry portions of the particle. Our stimulations show that nanoparticles smaller than the AFM tip tend to slide before rolling, while in large nanoparticles the rolling occurs first. The particle motion is achieved if the applied force exceeds a critical value and the direction of the rolling movement depends on the applied force angle. Furthermore, small nanoparticles are more easily manipulated by the tip in low-humidity conditions while the manipulations with large nanoparticles need high-humidity conditions. Preliminary results can be used to adjust proper handling force for the accurate and successful assembly of particles.     

Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

Tailoring the trajectory of cell rolling with cytotactic surfaces

Cell separation technology is a key tool for biological studies and medical diagnostics that relies primarily on chemical labeling to identify particular phenotypes. An emergent method of sorting cells based on differential rolling on chemically patterned substrates holds potential benefits over existing technologies, but the underlying mechanisms being exploited are not well characterized. In order to better understand cell rolling on complex surfaces, a microfluidic device with chemically patterned stripes of the cell adhesion molecule P-selectin was designed. The behavior of HL-60 cells rolling under flow was analyzed using a high-resolution visual tracking system. This behavior was then correlated to a number of established predictive models. The combination of computational modeling and widely available fabrication techniques described herein represents a crucial step toward the successful development of continuous, label-free methods of cell separation based on rolling adhesion.     

A simple flow cell system to evaluate in vitro bacterial adhesion on solids

A simple flow cell system was developed for studies of bacterial adhesion on hydrophobic or hydrophilic surfaces in the presence or absence of saliva. The system consists of flow cells made of borosilicate glass tubes (flow cell volume, 0.45 ml) that are connected to syringes mounted on an infusion pump. Hydrophilic or hydrophobic surfaces were obtained by treating the flow cells with acid or dichlorodimethylsilane, respectively. Liquids and bacterial suspensions were withdrawn directly into the flow cells and the flow rate was monitored through the infusion pump. The adhesion of ³H-labeled Streptococcus mutans cells to bare or saliva-coated surfaces was studied in this system by determining the radioactivity retained in the flow cells after rinsing with buffer at different flow rates. The results indicate that factors like the bacterial cell density, the surface properties, the presence of saliva pellicle and the rinsing flow rate significantly affect the adhesion of S. mutans. These results are in agreement with previous findings obtained with other flow cell systems. The present system has the advantages of using small volumes of liquids and allowing defined laminar flow conditions. Moreover, the entire cell, not only selected area spots, are used for the evaluation of bacterial adhesion. No part of the flow cell is re-used, thus avoiding the risk of contamination with other components. The system may therefore be suitable for assays testing the effect of different compounds on bacterial adhesion.     

Differential Effects of Ruminant and Industrial 18-Carbon trans-Monounsaturated Fatty Acids (trans Vaccenic and Elaidic) on the Inflammatory Responses of an Endothelial Cell Line

Endothelial dysfunction and inflammation are recognised factors in the development of atherosclerosis. Evidence suggests that intake of industrial trans fatty acids (TFAs) promotes endothelial dysfunction, while ruminant TFAs may have the opposite effect. The aim of this study was to compare the effects of elaidic acid (EA (18:1n-9t); an industrially produced TFA) and trans vaccenic acid (TVA (18:1n-7t); a natural TFA found in ruminant milk and meat) on inflammatory responses of endothelial cells (ECs). ECs (EA.hy926 cells) were cultured under standard conditions and exposed to TFAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments. TFAs were incorporated into ECs in a dose-dependent manner. Preincubation with EA (50 µM) increased production of MCP-1, RANTES, and IL-8 in response to TNF-α, while preincubation with TVA (1 µM) decreased production of ICAM-1 and RANTES in response to TNF-α. Preincubation with EA (50 µM) upregulated toll-like receptor 4 and cyclooxygenase 2 gene expression in response to TNF-α. In contrast, preincubation with TVA (1 µM) downregulated TNF-α induced nuclear factor kappa B subunit 1 gene expression. Preincubation of ECs with EA (50 µM) increased THP-1 monocyte adhesion. In contrast, preincubation of ECs with TVA (1 µM) reduced THP-1 monocyte adhesion, while preincubation of ECs with TVA (50 µM) decreased the level of surface expression of ICAM-1 seen following TNF-α stimulation. The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. These findings suggest differential effects induced by the TFAs tested, fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions in an inflammatory context.     

Antioxidative,Antiapoptotic, and Anti-Inflammatory Effects of Apamin in a Murine Model of Lipopolysaccharide-Induced Acute Kidney Injury

Sepsis is the major cause of acute kidney injury (AKI) in severely ill patients, but only limited therapeutic options are available. During sepsis, lipopolysaccharide (LPS), an endotoxin derived from bacteria, activates signaling cascades involved in inflammatory responses and tissue injury. Apamin is a component of bee venom and has been shown to exert antioxidative, antiapoptotic, and anti-inflammatory activities. However, the effect of apamin on LPS-induced AKI has not been elucidated. Here, we show that apamin treatment significantly ameliorated renal dysfunction and histological injury, especially tubular injury, in LPS-injected mice. Apamin also suppressed LPS-induced oxidative stress through modulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and heme oxygenase-1. Moreover, tubular cell apoptosis with caspase-3 activation in LPS-injected mice was significantly attenuated by apamin. Apamin also inhibited cytokine production and immune cell accumulation, suppressed toll-like receptor 4 pathway, and downregulated vascular adhesion molecules. Taken together, these results suggest that apamin ameliorates LPS-induced renal injury through inhibiting oxidative stress, apoptosis of tubular epithelial cells, and inflammation. Apamin might be a potential therapeutic option for septic AKI.     

Interfacial behavior of immortalized hypothalamic mouse neurons detected by acoustic wave propagation

The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 μM), colchicine (1 μM), cytochalasin B (10 μM) and cytochalasin D (2 μM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.     

Sulfated polysaccharides from red microalgae have antiinflammatory properties in vitro and in vivo

The primary goal of the present research was to determine whether sulfated polysaccharides derived from red microalgae possess anti inflammatory properties when directed against specific parameters of human skin inflammation. These unique biopolymers were studied in both in vitro and in vivo models of skin inflammation. Human subjects were recruited to participatein a study in which the polysaccharide material was applied topically and shown to inhibit cutaneousery thema induced by a known irritant. Leukocyte migration from capillary blood intosites of inflammation is an essential component of the inflammatory process and occurs in a series of steps, two of which are adhesion and chemotaxis. In vitro, the polysaccharide material primarily inhibited the migration of polymorphonuclear leukocytes (PMNs) toward a standard chemoattractant molecule and also partially blocked adhesion of PMNs to endothelial cells. The data obtained strongly suggest that sulfated polysaccharides derived from red microalgae have significant beneficial potential for use in topical products. In addition, the data suggested that the anti inflammatory mechanism for the polysaccharide was, at least in part, due to inhibition of circulating immune cell recruitment toward inflammatory stimuli.     

Study of the effect of atorvastatin on the interaction between ICAM-1 and CD11b by live-cell single-molecule force spectroscopy

The interaction between the cell adhesion molecule CD11b and its ligand ICAM-1 plays an important role in inflammatory responses in the disease of atherosclerosis. Atorvastatin is a commonly prescribed statin drug which has been considered as one of the most potent therapeutic agents for atherosclerosis due to its lipid-lowering effect. Recently, there is a growing body of evidence that atorvastatin has anti-inflammatory effect. We have applied the advanced method of live-cell single-molecule force spectros...     

Photoprotective effects of bucillamine against UV-induced damage in an SKH-1 hairless mouse model

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.     

Adhesion of Pseudomonas fluorescens on magnetic surfaces

The adhesion of Pseudomonas fluorescens (ATCC 700830) to perpendicularly polarized magnetic surfaces was recently discovered. The findings have found that the magnetic free surfaces from different magnetic polarities have different profound effects on the P. fluorescens bacterial adhesion to its surfaces. These phenomena can be explained by the surface magnetic effect, which was found to affect the surface free energy. An in situ experiment, by contrast microscopy and under static conditions, was conducted to determine the influence of magnetic surfaces, that are polarized under different external magnetizing field strengths, on bacterial adhesion. The effect of different magnetic polarities on the surface free energy has also been investigated.     

Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength

The S-layer present at the outermost cell surface of some lactobacillus species is known to convey hydrophobicity to the lactobacillus cell surface. Yet, it is commonly found that adhesion of lactobacilli to solid substrata does not proceed according to expectations based on cell surface hydrophobicity. In this paper, the role of cell surface hydrophobicity of two lactobacillus strains with and without a surface layer protein (SLP) layer has been investigated with regard to their adhesion to hydrophobically or hydrophilically functionalized glass surfaces under well-defined flow conditions and in low and high ionic strength suspensions. Similarly, the interaction of the lactobacilli with similarly functionalized atomic force microscope (AFM) tips was measured. In a low ionic strength suspension, both lactobacillus strains show higher initial deposition rates to hydrophobic glass than to hydrophilic glass, whereas in a high ionic strength suspension no clear influence of cell surface hydrophobicity on adhesion is observed. Independent of ionic strength, however, AFM detects stronger interaction forces when both bacteria and tip are hydrophobic or hydrophilic than when bacteria and tip have opposite hydrophobicities. This suggest that the interaction develops in a different way when a bacterium is forced into contact with the tip surface, like in AFM, as compared with contacts developing between a cell surface and a macroscopic substratum under flow. In addition, the distance dependence of the total Gibbs energy of interaction could only be qualitatively correlated with bacterial deposition and desorption in the parallel plate flow chamber.     

Blockade of airway inflammation and hyper-responsiveness by an angiopoietin-1 variant, COMP-Ang1

Inflammation of the asthmatic airway is usually accompanied by increased vascular permeability and plasma exudation. Angiopoietin-1 (Ang1) has potential therapeutic applications in preventing vascular leakage. Recently, we developed a soluble, stable, and potent Ang1 variant, COMP-Ang1. COMP-Ang1 is more potent than native Ang1 in phosphorylating the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 receptor in lung endothelial cells. We have used a mouse model for allergic airway disease to determine effects of COMP-Ang1 on allergen-induced bronchial inflammation and airway hyper-responsiveness. These mice develop the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyper-responsiveness, increased levels of Th2 cell cytokines (IL-4, IL-5, and IL-13), adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), and chemokines (eotaxin and RANTES), and increased vascular permeability. Intravenous administration of COMP-Ang1 reduced bronchial inflammation and airway hyper-responsiveness. In addition, the increased plasma extravasation in allergic airway disease was significantly reduced by the administration of COMP-Ang1. These results suggest that COMP-Ang1 attenuates airway inflammation and hyper-responsiveness, prevents vascular leakage, and may be used as a therapeutic agent in allergic airway disease.     

Angiopoietin-1 variant, COMP-Ang1 attenuates hydrogen peroxide-induced acute lung injury

Reactive oxygen species (ROS) play a crucial role in acute lung injury. Tissue inflammation, the increased vascular permeability, and plasma exudation are cardinal features of acute lung injury. Angiopoietin-1 (Ang1) has potential therapeutic applications in preventing vascular leakage and also has beneficial effects in several inflammatory disorders. Recently developed COMP-Ang1 is more potent than native Ang1 in phosphorylating tyrosine kinase with immunoglobulin and EGF homology domain 2 receptor in endothelial cells. However, there are no data on effects and related molecular mechanisms of COMP- Ang1 on ROS-induced acute lung injury. We used hydrogen peroxide (H2O2)-inhaled mice to evaluate the effect of COMP-Ang1 on pulmonary inflammation, bronchial hyper-responsiveness, and vascular leakage in acute lung injury. The results have revealed that VEGF expression, the levels of IL-4, TNF-alpha, IL-1beta, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in lungs, the levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and NF-kappaB in nuclear protein extracts, phosphorylation of Akt, and vascular permeability were increased after inhalation of H2O2 and that the administration of COMP-Ang1 markedly reduced airway hyper-responsiveness, pulmonary inflammation, plasma extravasation, and the increases of cytokines, adhesion molecules, and VEGF in lungs treated with H2O2. We have also found that the activation of HIF-1a and NF-kB and the increase of phosphoinositide 3-kinase activity in lung tissues after H2O2 inhalation were decreased by the administration of COMP-Ang1. These results suggest that COMP-Ang1 ameliorates ROS-induced acute lung injury through attenuating vascular leakage and modulating inflammatory mediators.     

Dynamic adhesion behavior of micrometer-scale particles flowing over patchy surfaces with nanoscale electrostatic heterogeneity

The dynamic adhesion behavior of micrometer-scale silica particles is investigated numerically for a low Reynolds number shear flow over a planar collecting wall with randomly distributed electrostatic heterogeneity at the 10-nanometer scale. The hydrodynamic forces and torques on a particle are coupled to spatially varying colloidal interactions between the particle and wall. Contact and frictional forces are included in the force and torque balances to capture particle skipping, rolling, and arrest. These dynamic adhesion signatures are consistent with experimental results and are reminiscent of motion signatures observed in cell adhesion under flowing conditions, although for the synthetic system the particle–wall interactions are controlled by colloidal forces rather than physical bonds between cells and a functionalized surface. As the fraction of the surface (Θ) covered by the cationic patches is increased from zero, particle behavior sequentially transitions from no contact with the surface to skipping, rolling, and arrest, with the threshold patch density for adhesion (Θ_crit) always greater than zero and in quantitative agreement with experimental results. The ionic strength of the flowing solution determines the extent of the electrostatic interactions and can be used to tune selectively the dynamic adhesion behavior by modulating two competing effects. The extent of electrostatic interactions in the plane of the wall, or electrostatic zone of influence, governs the importance of spatial fluctuations in the cationic patch density and thus determines if flowing particles contact the wall. The distance these interactions extend into solution normal to the wall determines the strength of the particle–wall attraction, which governs the transition from skipping and rolling to arrest. The influence of Θ, particle size, Debye length, and shear rate is quantified through the construction of adhesion regime diagrams, which delineate the regions in parameter space that give rise to different dynamic adhesion signatures and illustrate selective adhesion based on particle size or curvature. The results of this study are suggestive of novel ways to control particle–wall interactions using randomly distributed surface heterogeneity.     

An Acoustically Driven Microliter Flow Chamber on a Chip (μFCC) for Cell–Cell and Cell–Surface Interaction Studies

A novel method for pumping very small volumes of liquid by using surface acoustic waves is employed to create a microfluidic flow chamber on a chip. It holds a volume of only a few μl and its planar design provides complete architectural freedom. This allows for the reconstruction of even complex flow scenarios (e.g. curvatures, bifurcations and stenosis). Addition of polymer walls to the planar fluidic track enables cell culturing on the chip surface and the investigation of cell–cell adhesion dynamics under flow. We demonstrate the flexibility of the system for application in many areas of microfluidic investigations including blood clotting phenomena under various flow conditions and the investigation of different stages of cell adhesion.     

Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.