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The incidence of wound infections that do not adequately respond to standard-of-care antimicrobial treatment has been increasing. To address this challenge, a novel antimicrobial magnetic thermotherapy platform has been developed in which a high-amplitude, high-frequency, alternating magnetic field (AMF) is used to rapidly heat magnetic nanoparticles that are bound to Staphylococcus aureus (S. aureus). The antimicrobial efficacy of this platform was evaluated in the treatment of both an in vitro culture model of S. aureus biofilm and a mouse model of cutaneous S. aureus infection. We demonstrated that an antibody-targeted magnetic nanoparticle bound to S. aureus was effective at thermally inactivating S. aureus and achieving accelerated wound healing without causing tissue injury.

The identification of novel, synthetic targeting ligands to endothelial receptors has led to the rapid development of targeted nanoparticles for drug, gene and imaging probe delivery. Central to development and optimization are effective models for assessing particle binding in vitro. Here, we developed a simple and cost effective method to quantitatively assess nanoparticle accumulation under physiologically-relevant laminar flow. We designed reversibly vacuum-sealed PDMS microfluidic chambers compatible with 35 mm petri dishes, which deliver uniform or gradient shear stress. These chambers have sufficient surface area for facile cell collection for particle accumulation quantitation through FACS. We tested this model by synthesizing and flowing liposomes coated with APN (K (D) ~ 300 μM) and VCAM-1-targeting (K (D) ~ 30 μM) peptides over HUVEC. Particle binding significantly increased with ligand concentration (up to 6 mol%) and decreased with excess PEG. While the accumulation of particles with the lower affinity ligand decreased with shear, accumulation of those with the higher affinity ligand was highest in a low shear environment (2.4 dyne/cm(2)), as compared with greater shear or the absence of shear. We describe here a robust flow chamber model that is applied to optimize the properties of 100 nm liposomes targeted to inflamed endothelium.

Application of mechanical force to bonds between selectins and their ligands is a requirement for these adhesion receptors to optimally perform functions that include leukocyte tethering and activation of stable adhesion. Although all three selectins are reported to signal from the outside-in subsequent to ligand binding, E-selectin is unique in its capacity to bind multiple sialyl Lewis x presenting ligands and mediate slow rolling on the order of a micron per second. A diverse set of ligands are recognized by E-selectin in the mouse, including ESL-1, CD44 (HCELL), and PSGL-1 which are critical in transition from slow rolling to arrest and for efficient transendothelial migration. The molecular recognition process is different in humans as L-selectin is a major ligand, which along with glycolipids constitute more than half of the E-selectin receptors on human polymorphonuclear neutrophils (PMN). In addition, E-selectin is most efficient at raising the affinity and avidity of CD18 integrins that supports PMN deceleration and trafficking to sites of acute inflammation. The mechanism is only partially understood but known to involve a rise in cytosolic calcium and tyrosine phosphorylation that activates p38 MAP kinase and Syk kinase, both of which transduce signals from clustered E-selectin ligands. In this review we highlight the molecular recognition and mechanical requirements of this process to reveal how E-selectin confers selectivity and efficiency of signaling for extravasation at sites of inflammation and the mechanism of action of a new glycomimetic antagonist targeted to the lectin domain that has shown efficacy in blocking neutrophil activation and adhesion on inflamed endothelium.

Adhesion to and subsequent extravasation through the endothelial lining of blood vessels is critical for tumor cells to establish metastases. Recent studies have indicated that polymorphonuclear neutrophils (PMNs) may enhance melanoma adhesion to the endothelium (EC) and subsequent extravasation under dynamic flow conditions. However, little is known about hydrodynamics involved in the tumor microenvironment within the microcirculation. In this study, effects of hydrodynamic flow on regulating melanoma cell adhesion to the EC have been investigated. Results indicate that under flow conditions, interactions between melanoma cells and the EC are distinctly different from PMN-EC interactions. Without expressions of surface integrins or sialylated molecules, most melanoma cells that express a high-level of intercellular adhesion molecule (ICAM-1) are not able to effectively adhere to the inflamed EC by themselves. Binding of melanoma cells and PMNs through ICAM-1 on melanoma cells and beta(2) integrins on PMNs has been shown to enhance melanoma cell arrest on the EC. Although PMN tethering on the EC is regulated by both the shear rate and shear stress, melanoma cell adhesion to the EC and subsequent extravasation via tethering PMN on the EC is predominantly regulated by shear rate, which partly is due to the shear-rate-dependent PMN-melanoma aggregation in shear flow. These findings provide a rationale and mechanistic basis for understanding of leukocyte-tumor cell interactions under flow conditions during tumor cell extravasation and metastasis.

A novel biointerface probe was implemented to study the deformability of the neutrophil membrane and cortical cytoskeleton. Piconewton scale forces are applied to the cell using an ultrasensitive and tunable force transducer comprised of an avidin-coated microsphere attached to a biotinylated and swollen red blood cell. Deformations of freshly isolated human neutrophils were observed on the stage of an inverted phase contrast microscope. Force versus probe indentation curves over a cycle of contact, indentation, and retraction revealed three distinct material responses. Small probe deformations (approximately 500 nm) tested over a range of rates (e.g. 100-500 nm/s) revealed predominantly an elastic response. An initial low-slope region in the force-indentation curves (approximately 0.005 pN/nm), typically extending 0.5-1.0 microm from the cell surface was interpreted as probe contact with microvilli extensions. Further deformation yielded a slope of 0.054+/-0.006 pN/nm, indicative of a stiffer cortical membrane. Disrupting cytoskeletal actin organization by pretreatment with cytochalasin D, reduced the slope by 40% to 0.033+/-0.007 pN/nm and introduced hysteresis in the recovery phase. Modeling the neutrophil as a liquid drop with constant surface tension yielded values of cortical tension of 0.035 pN/nm for resting and 0.02 pN/nm for cytochalasin-treated neutrophils. These data demonstrate the utility of the biointerface probe for measuring local surface compliance and microstructure of living cells.

A cooperative, sequential process of molecular recognition governs leukocyte capture, rolling, and arrest on inflamed endothelium. Flowing neutrophils are captured via heterotypic adhesive interactions mediated by endothelial E-selectin, whereas homotypic interactions between neutrophils are mediated by L-selectin. To elucidate how each selectin facilitates the transition to CD18-mediated stable adhesion, E-selectin and L-selectin were expressed at defined site density in a murine pre-B-cell line. Direct observation of two-body collisions revealed that 30% of neutrophil interactions with E-selectin transfectants formed doublets at low shear rate G = 14 s(-1) whereas a threshold shear rate 14 s(-1)or = Gor = 10 s(-1) was necessary for L-selectin adhesion. Adhesion via L-selectin resisted rupture at high shear stress, while E-selectin tethered doublets remained intact longer once formed. Moreover, higher expression of L-selectin (1100 sites/microm2) than that of E-selectin (220 sites/microm2) was required for comparable heterotypic adhesion efficiency. With a threefold rise in active CD18 upregulated on chemotactically stimulated neutrophils, homotypic adhesion efficiency increased 10-fold compared to less than 5-fold for heterotypic adhesion to selectin transfectants. Co-expression of E-selectin and ICAM-1 boosted adhesion efficiency threefold more than either receptor alone over the range of active CD18 expression. These data are the first to quantify adhesion efficiency mediated by selectin tethering and conformational activation of beta2-integrin in neutrophils in shear flow.

Of the white blood cells that traverse the circulation, the polymorphonuclear leukocytes commonly called neutrophils, are the most numerous, numbering ∼5000 per microliter of blood. In a sense, these cells are the emergency response unit within the body's circulatory highway since they are the first to be recruited at sites of tissue trauma, infection, or inflammation. The chief function of neutrophils is the capture, phagocytosis, and degradation of foreign invaders. To carry out this critical function, neutrophils sense infection via chemotactic receptors that trigger cellular activation upon ligation of as few as 10–100 bacterial or chemokine peptides. Despite this acute sensitivity to stimuli, neutrophils circulate in healthy individuals largely in a passive state, with a very low efficiency of capture and arrest on quiescent endothelium. Here, we define the efficiency as the fraction of all cells passing by a given length of vessel wall that is captured and achieves firm adhesion. Within seconds of chemotactic signaling, adhesion molecules expressed on the plasma membrane of neutrophils are activated and a rapid boost in the efficiency of stable adhesion is detected. This occurs for both homotypic neutrophil–neutrophil adhesion and neutrophil capture and adhesion on inflamed endothelium. With such large numbers of circulating neutrophils, and with their inherent capability to rapidly adhere to postcapillary venular endothelium, the body has evolved a diverse set of mechanisms to regulate the inflammatory cascade that begins with intracellular signaling and leads to cell arrest and extravasation at sites of tissue insult. In this article we will focus on the interplay between particle–fluid dynamics that involve the shear and normal forces that transport cells transversely and axially within the vessel, and the activation and interaction of adhesion molecules that involve receptor–ligand bond formation that enables the neutrophils to resist wall shear stress and adhere to specific sites of inflammation. © 2002 Biomedical Engineering Society.

Affinity regulation of integrin function plays an important role during both leukocyte-endothelial and leukocyte-leukocyte interactions. We compared the roles of Mn(2+) (Manganese) and Gd(3+) (Gadolinium) in regulating leukocyte CD18-integrin function. We observed that: (i) Both cations prolonged neutrophil homotypic aggregation following chemoattractant IL-8 stimulation, with Gd(3+) being effective at doses two orders of magnitude (10 microM range) lower that Mn(2+). (ii) While both Gd(3+) and Mn(2+) mediate homotypic cell aggregation via L: -selectin and CD18 integrins, their effects on the integrin subunits, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), was different. Gd(3+) altered both LFA-1 and Mac-1 function, while the dominant effect of Mn(2+) was on Mac-1. This effect of Gd(3+) on LFA-1 function was confirmed in cell-free studies that measured the binding of recombinant ICAM-1 to LFA-1 immobilized on beads. (iii) Both ions augmented the binding of 327C, an antibody that recognizes active CD18 on human neutrophils, both in the presence and absence of exogenous IL-8. The effects of Mn(2+) was more pronounced since it caused 3-4-fold increase in mAb 327C binding to neutrophils compared to Gd(3+) which increased antibody binding by only approximately 80%. 327C binding was partially reduced by Ca(2+). Further, 327C binding induced by Mn(2+) did not correlate tightly with cell adhesion function. (iv) In studies that monitored intracellular Ca(2+) ([Ca(2+)](i)), the addition of Mn(2+) but not Gd(3+) to neutrophils altered [Ca(2+)](i) levels. Overall, while both Gd(3+) and Mn(2+) stabilize high affinity CD18 mediated cell adhesion, Gd(3+) affects integrin conformation while Mn(2+) may also trigger other effects.

Address correspondence to Scott I. Simon, Department of Biomedical Engineering, University of California at Davis, 451 E. Health Sciences Dr., Davis, CA 95616, USA. Electronic mail: sisimon@ ucdavis.edu The online version of the original article can be found at doi: 10.1007/s10439-012-0698-x. Annals of Biomedical Engineering, Vol. 41, No. 3, March 2013 ( 2012) pp. 610 DOI: 10.1007/s10439-012-0709-y

Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity beta(2) integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner.