Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study, we report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd(2)O(3)) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd(2)O(3) nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd(2)O(3) nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r(1) and r(2) values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd(2)O(3). Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is, an extremely high MR signal at the cellular and molecular level.

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H 2 O 2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H 2 O 2 and did not undergo apoptosis. Exogenously added H 2 O 2 , together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H 2 O 2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H 2 O 2 production, whereas H 2 O 2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H 2 O 2 to commit suicide.

Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A 4 (LXA 4 ) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA 4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/ CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA 4 . Furthermore, we found that LXA 4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA 4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA 4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.Periodontitis is one of the most prevalent inflammatory diseases in humans, the key etiologic agent being the Gramnegative anaerobic rod Porphyromonas gingivalis (54). This bacterium not only is involved in tooth loss but also may cause recurrent bacteremias and contribute to systemic disorders, such as cardiovascular disease (10,22,23,39,46,65). P. gingivalis expresses a broad range of virulence factors, such as cysteine proteinases (gingipains), fimbriae, lipopolysaccharide (LPS), and capsular polysaccharide. Infection with the bacterium may lead to chronic inflammation in which hyperresponsive neutrophils contribute to host-mediated tissue destruction. P. gingivalis has been found in human atherosclerotic plaques (15,27) and has been shown to promote the phenotypic switch of murine monocytes into foam cells, e.g., by inducing reactive oxygen species (ROS) generation and the oxidation of lowdensity lipoprotein (LDL) (31,38,57).We have recently reported that the exposure of human blood to P. gingivalis causes the formation of atherogenic LDL through a gingipain-mediated cleavage of apoB-100...