Cytotoxicity assessment of beta-lapachone in endothelial cells

The selective activity of an antineoplastic drug is related to its ability to promote cytotoxic action on tumor cells and preserve the integrity of non-neoplastic cells. Beta-lapachone is extracted from the sawdust of Ipe wood, a thick bark tree from the Ipe wood found in the Brazilian Cerrado biome. This study aimed to evaluate the cytotoxic action of beta-lapachone in an endothelial cell line. The EA.hy926 cells were seeded in two groups, G1 and G2, cultured and exposed to beta-lapachone at concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1 and 3 & 956;M for 24 hours. G1 remained under normal cultivation conditions and G2 was subjected to oxidative stress through an ischemia and reperfusion assay, in a deoxygenated sealed chamber. The cytotoxicity assay was performed using the tetrazolium reduction method. In G1, the cytotoxicity ranged from 0.0 to 10.0%; and in G2 between 0.0 and 6.3%. No statistically significant difference was observed between the obtained values. Moreover, we found no cytotoxic action of beta-lapachone on endothelial cells, and the results point out that the drug might have preserved the cell’s integrity against oxidative stress under the conditions of this experiment. This promising result suggests the possibility of beta-lapachone as a chemotherapy drug with selective activity.

Assessment of Air Pollutant PM2.5 Pulmonary Exposure Using a 3D Lung-on-Chip Model.

Airborne particulate matters have posed significant risk to human health worldwide. Fine particulate matters (PM2.5, aerodynamic diameter <2.5 µm) are associated with increased morbidity and mortality attributed to pulmonary diseases. An advanced in vitro model would benefit the assessment of PM2.5 induced pulmonary injuries and drug development. In this work, we present a PM2.5 exposure model to evaluate the pulmonary risk of fine particulate matter exposure in an organotypic manner with the help of 3D human lung-on-a-chip. By compartmentalized co-culturing of human endothelial cells, epithelial cells, and extra cellular matrix, our lung-on-a-chip recapitulated the structural features of the alveolar-blood barrier, which is pivotal for exogenous hazard toxicity evaluation. PM2.5 was applied to the channel lined with lung epithelial cells to model the pulmonary exposure of fine particulate matter. The results indicated acute high dose PM2.5 exposure would lead to various malfunctions of the alveolar-capillary barrier, including adheren junction disruption, increased ROS generation, apoptosis, inflammatory biofactor expression in epithelial cells and endothelial cells, elevated permeability, and monocyte attachments. Collectively, our lung-on-a-chip model provides a simple platform to investigate the complex responses after PM2.5 exposure in a physiologically relevant level, which could be of great potential in environmental risk assessment and therapeutic treatment development.

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

BACKGROUND: To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. METHODS: Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. RESULTS: Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. CONCLUSIONS: Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor.

Micro-autoradiographic assessment of cell types contributing to 2-deoxy-2-[(18)F]fluoro-D-glucose uptake during ventilator-induced and endotoxemic lung injury.

PURPOSE: The aim of the study was to use micro-autoradiography to investigate the lung cell types responsible for 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) uptake in murine models of acute lung injury (ALI). PROCEDURES: C57/BL6 mice were studied in three groups: controls, ventilator-induced lung injury (VILI), and endotoxin. VILI was produced by high tidal volumes and zero end-expiratory pressure and endotoxin ALI, by intranasal administration. Following FDG injection, the lungs were processed and exposed to autoradiographic emulsion. Grain density over cells was used to quantify FDG uptake. RESULTS: Neutrophils, macrophages, and type 2 epithelial cells presented higher grain densities during VILI and endotoxin ALI than controls. Remarkably, cell grain density in specific cell types was dependent on the injury mechanism. Whereas macrophages showed high grain densities during endotoxin ALI, similar to those exhibited by neutrophils, type 2 epithelial cells demonstrated the second highest grain density (with neutrophils as the highest) during VILI. CONCLUSIONS: In murine models of VILI and endotoxin ALI, FDG uptake occurs not only in neutrophils but also in macrophages and type 2 epithelial cells. FDG uptake by individual cell types depends on the mechanism underlying ALI.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

A novel method for detection of apoptosis.

There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes.

Infection with Plasmodium falciparum during pregnancy leads to chondroitin 4-sulfate-mediated adhesion of the infected red blood cells (IRBCs) in the placenta, causing severe health complications to fetus and the mother. The IRBCs are also frequently found in low density in the umbilical cord of infected placentas. In this study, the CSPGs of umbilical vein and arteries were purified, characterized, and their localization and IRBC-binding abilities were studied. While a versican type CSPG was found both in the vein and arteries, a serglycin type CSPG was present exclusively in the vein. The CSPGs were present at significant level on the endothelial surface of the umbilical vein but not on that of arteries. Although the purified versican and serglycin type CSPGs could bind IRBCs, their binding abilities were significantly less compared to the low sulfated CSPGs of the placenta because of the predominance of 6-sulfated disaccharide moieties in the CS chains. Therefore, IRBCs were unable to bind efficiently onto the umbilical cord endothelial surface. Unexpectedly, however, the IRBCs adhered densely in the blood vessels of fetal villi in the placental tissue sections and sparingly in the blood spaces of the umbilical cord vein, presumably because the CSPG that can efficiently bind IRBCs is present at high levels in the fetal blood vessels and at very low levels in the umbilical cord blood vessels. Since the C4S-adherent IRBCs that enter the fetal blood vessels cannot adhere to the cord endothelial surface and parasites cannot efficiently grow due to fetal hemoglobin toxicity and protection by maternal antibodies, transplacental infection may be quickly cleared without clinical episodes.