Contrast-enhanced imaging of SPIO-labeled platelets using magnetomotive ultrasound

The ability to image platelets in vivo can provide insight into blood clotting processes and coagulopathies, and aid in identifying sites of vascular endothelial damage related to trauma or cardiovascular disease. Toward this end, we have developed a magnetomotive ultrasound (MMUS) system that provides contrast-enhanced imaging of superparamagnetic iron oxide (SPIO) labeled platelets via magnetically-induced vibration. Platelets are a promising platform for functional imaging contrast because they readily take up SPIOs and are easily harvested from blood. Here we report a novel MMUS system that accommodates an arbitrarily thick sample while maintaining portability. We employed a frequency- and phase-locked motion detection algorithm based on bandpass filtering of the differential RF phase, which allows for the detection of sub-resolution vibration amplitudes on the order of several nanometers. We then demonstrated MMUS in homogenous tissue phantoms at SPIO concentrations as low as 0.09 mg/ml Fe (p < 0.0001, n = 6, t-test). Finally, we showed that our system is capable of 3-dimensional imaging of a 185 μL simulated clot containing SPIO-platelets. This highlights the potential utility for non-invasive imaging of platelet-rich clots, which would constitute a fundamental advance in technology for the study of hemostasis and detection of clinically relevant thrombi

Modulation of macrophage phenotype by cell shape.

Phenotypic polarization of macrophages is regulated by a milieu of cues in the local tissue microenvironment. Although much is known about how soluble factors influence macrophage polarization, relatively little is known about how physical cues present in the extracellular environment might modulate proinflammatory (M1) vs. prohealing (M2) activation. Specifically, the role of cell shape has not been explored, even though it has been observed that macrophages adopt different geometries in vivo. We and others observed that macrophages polarized toward different phenotypes in vitro exhibit dramatic changes in cell shape: M2 cells exhibit an elongated shape compared with M1 cells. Using a micropatterning approach to control macrophage cell shape directly, we demonstrate here that elongation itself, without exogenous cytokines, leads to the expression of M2 phenotype markers and reduces the secretion of inflammatory cytokines. Moreover, elongation enhances the effects of M2-inducing cytokines IL-4 and IL-13 and protects cells from M1-inducing stimuli LPS and IFN-γ. In addition shape- but not cytokine-induced polarization is abrogated when actin and actin/myosin contractility are inhibited by pharmacological agents, suggesting a role for the cytoskeleton in the control of macrophage polarization by cell geometry. Our studies demonstrate that alterations in cell shape associated with changes in ECM architecture may provide integral cues to modulate macrophage phenotype polarization

Real-time imaging of Toxoplasma -infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers

Peripheral blood monocytes are actively infected by Toxoplasma gondii and can function as “Trojan horses” for parasite spread in the bloodstream. Using dynamic live-cell imaging, we visualized the transendothelial migration (TEM) of T. gondii-infected primary human monocytes during the initial minutes following contact with human endothelium. On average, infected and uninfected monocytes required only 9.8 and 4.1 minutes, respectively, to complete TEM. Infection increased monocyte crawling distances and velocities on endothelium, but overall TEM frequencies were comparable between infected and uninfected cells. In the vasculature, monocytes adhere to endothelium under the conditions of shear stress found in rapidly flowing blood. Remarkably, the addition of fluidic shear stress increased the TEM frequency of infected monocytes 4.5-fold compared to static conditions (to 45.2% from 10.3%). Infection led to a modest increase in expression of the high affinity conformation of the monocyte integrin Mac-1, and Mac-1 accumulated near endothelial junctions during TEM. Blocking Mac-1 inhibited the crawling and TEM of infected monocytes to a greater degree than uninfected monocytes, and blocking the Mac-1 ligand, ICAM-1, dramatically reduced crawling and TEM for both populations. These findings contribute to a greater understanding of parasite dissemination from the vasculature into tissues

Transendothelial migration of Toxoplasma‐infected monocytes

Peripheral blood monocytes are actively infected by Toxoplasma gondii and can function as 'Trojan horses' for parasite spread in the bloodstream. Using dynamic live-cell imaging, we visualized the transendothelial migration (TEM) of T. gondii-infected primary human monocytes during the initial minutes following contact with human endothelium. On average, infected and uninfected monocytes required only 9.8 and 4.1 min, respectively, to complete TEM. Infection increased monocyte crawling distances and velocities on endothelium, but overall TEM frequencies were comparable between infected and uninfected cells. In the vasculature, monocytes adhere to endothelium under the conditions of shear stress found in rapidly flowing blood. Remarkably, the addition of fluidic shear stress increased the TEM frequency of infected monocytes 4.5-fold compared to static conditions (to 45.2% from 10.3%). Infection led to a modest increase in expression of the high-affinityconformation of the monocyte integrin Mac-1 (CD11b/CD18), and Mac-1 accumulated near endothelial junctions during TEM. Blocking Mac-1 inhibited the crawling and TEM of infected monocytes to a greater degree than uninfected monocytes, and blocking the Mac-1 ligand, ICAM-1, dramatically reduced crawling and TEM for both populations. These findings contribute to a greater understanding of parasite dissemination from the vasculature into tissues

Physical and mechanical regulation of macrophage phenotype and function

Macrophages are tissue-resident immune cells that play a critical role in maintaining homeostasis and fighting infection. In addition, these cells are involved in the progression of many pathologies including cancer and atheroscloerosis. In response to a variety of microenvironmental stimuli, macrophages can be polarized to achieve a spectrum of functional phenotypes. This review will discuss some emerging evidence in support of macrophage phenotypic regulation by physical and mechanical cues. As alterations in the physical microenvironment often underlie pathophysiological states, an understanding of their effects on macrophage phenotype and function may help provide mechanistic insights into disease pathogenesis