Ubirajara Agero

Front Instabilities and Invasiveness of Simulated Avascular Tumors

We study the interface morphology of a 2D simulation of an avascular tumor composed of identical cells growing in an homogeneous healthy tissue matrix (TM), in order to understand the origin of the morphological changes often observed during real tumor growth. We use the Glazier–Graner–Hogeweg model, which treats tumor cells as extended, deformable objects, to study the effects of two parameters: a dimensionless diffusion-limitation parameter defined as the ratio of the tumor consumption rate to the substrate transport rate, and the tumor-TM surface tension. We model TM as a nondiffusing field, neglecting the TM pressure and haptotactic repulsion acting on a real growing tumor; thus, our model is appropriate for studying tumors with highly motile cells, e.g., gliomas. We show that the diffusion-limitation parameter determines whether the growing tumor develops a smooth (noninvasive) or fingered (invasive) interface, and that the sensitivity of tumor morphology to tumor-TM surface tension increases with the size of the dimensionless diffusion-limitation parameter. For large diffusion-limitation parameters, we find a transition (missed in previous work) between dendritic structures, produced when tumor-TM surface tension is high, and seaweed-like structures, produced when tumor-TM surface tension is low. This observation leads to a direct analogy between the mathematics and dynamics of tumors and those observed in nonbiological directional solidification. Our results are also consistent with the biological observation that hypoxia promotes invasive growth of tumor cells by inducing higher levels of receptors for scatter factors that weaken cell-cell adhesion and increase cell motility. These findings suggest that tumor morphology may have value in predicting the efficiency of antiangiogenic therapy in individual patients.

Defocusing microscopy : An approach for red blood cell optics

Thin transparent objects (phase objects) can become visible in a bright-field light microscope, if the microscope is slightly defocused. Thick transparent objects, like red blood cells (RBC), are seen because some of their parts are always out of focus. By applying our recently developed defocusing microscopy technique to RBC, we are able to interpret RBC bright-field light microscopy images, an old standing problem. From the average image contrast we obtain RBC shape, size, and refractive index. From contrast fluctuations caused by the flicker phenomenon, we obtain RBC bending modulus and cytoplasm viscosity.

Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

Background Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.

Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells

We introduce Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total three-dimensional (3D) imaging of transparent objects. By total 3D imaging, we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic, and hypertonic solutions. For each situation, the shapes of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of red blood cell (RBC) surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine the volume, surface area, sphericity index, and RBC refractive index for each osmotic condition.

Front Instabilities and Invasiveness of Simulated 3D Avascular Tumors

We use the Glazier-Graner-Hogeweg model to simulate three-dimensional (3D), single-phenotype, avascular tumors growing in an homogeneous tissue matrix (TM) supplying a single limiting nutrient. We study the effects of two parameters on tumor morphology: a diffusion-limitation parameter defined as the ratio of the tumor-substrate consumption rate to the substrate-transport rate, and the tumor-TM surface tension. This initial model omits necrosis and oxidative/hypoxic metabolism effects, which can further influence tumor morphology, but our simplified model still shows significant parameter dependencies. The diffusion-limitation parameter determines whether the growing solid tumor develops a smooth (noninvasive) or fingered (invasive) interface, as in our earlier two-dimensional (2D) simulations. The sensitivity of 3D tumor morphology to tumor-TM surface tension increases with the size of the diffusion-limitation parameter, as in 2D. The 3D results are unexpectedly close to those in 2D. Our results therefore may justify using simpler 2D simulations of tumor growth, instead of more realistic but more computationally expensive 3D simulations. While geometrical artifacts mean that 2D sections of connected 3D tumors may be disconnected, the morphologies of 3D simulated tumors nevertheless correlate with the morphologies of their 2D sections, especially for low-surface-tension tumors, allowing the use of 2D sections to partially reconstruct medically-important 3D-tumor structures.

Bulk elastic properties of chicken embryos during somitogenesis

We present measurements of the bulk Youngs moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ~0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryos morphology. By correlating the measured tissue displacements to the displacements calculated from the in-plane force and the model, we obtained the approximate short time linear-elastic Youngs moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca.

The thrombolytic action of a proteolytic fraction (P1G10) from Carica candamarcensis

A group of cysteine-proteolytic enzymes from C. candamarcensis latex, designated as P1G10 displays pharmacological properties in animal models following various types of lesions. This enzyme fraction expresses in vitro fibrinolytic effect without need for plasminogen activation. Based on this evidence, we assessed by intravital microscopy the effect of P1G10 on recanalization of microvessels after thrombus induction in the ear of hairless mice. Video playback of intravital microscopic images allowed measurement of blood flow velocity (mm/s) during the experimental procedure. Groups treated with 5 or 7.5mg/Kg P1G10 showed thrombolysis between 7-15min, without vessel obstruction. Ex vivo experiments demonstrated that platelet activation by ADP is impaired in a dose dependent manner following treatment with P1G10. The P1G10 action on plasma coagulation also showed that prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (aPTT, μg/uL) are increased in a dose dependent manner. In addition, P1G10 displayed fibrinogenolytic and fibrinolytic activities, both in a dose dependent manner. Each of these effects was suppressed by inhibition of the proteolytic activity of the fraction. The antithrombotic action of P1G10 can be explained by proteolytic cleavage of fibrinogen and fibrin, both key factors during formation of a stable thrombus. These results combined with prior evidence suggest that P1G10 has potential as thrombolytic agent.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

BackgroundPlasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated.MethodsConcentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group.ResultsAnaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance.ConclusionsThese findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

Profiling of individual human red blood cells under osmotic stress using defocusing microscopy.

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities. We monitor the RBCs’ radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed. Finally, membrane fluctuation measurements give us information about the cells deformability.