In vivo biocompatibility of diamond-like carbon films containing TiO2 nanoparticles for biomedical applications.

Hybrid diamond-like carbon (DLC) with incorporated titanium dioxide (TiO2) nanoparticle coatings have low friction coefficient, high wear resistance, high hardness, biocompatibility, and high chemical stability. They could be employed to modify biomedical alloys surfaces for numerous applications in biomedical engineering. Here we investigate for the first time the in vivo inflammatory process of DLC coatings with incorporated TiO2 nanoparticles. TiO2-DLC films were grown on AISI 316 stainless-steel substrates using plasma-enhanced chemical vapor deposition. The coated substrates were implanted in CF1 mice peritoneum. The in vivo cytotoxicity and biocompatibility of the samples were analyzed from macrophage lavage. Analysis in the first weeks after implantation could be helpful to evaluate the acute cytotoxicity generated after a possible inflammatory process. The in vivo results showed no inflammatory process. A significant increase in nitric oxide production on the uncoated substrates was confirmed through cytometry, and the coated substrates demonstrated biocompatibility. The presence of TiO2 nanoparticles enhanced the wound healing activity, due to their astringent and antimicrobial properties. DLC and TiO2-DLC coatings were considered biocompatible, and the presence of TiO2 nanoparticles reduced the inflammatory reactions, increasing DLC biocompatibility.

In vitro treatment of Toxoplasma gondii with copper(II) complexes induces apoptosis-like and cellular division alterations.

Toxoplasma gondii is the causative agent of toxoplasmosis, which is one of the most common parasitic diseases in the world. This pathogen causes severe damage to immunocompromised hosts, and the most frequently used therapy is the combination of pyrimethamine and sulfadiazine, which has side effects. Thus, there is a need for new therapies that target T. gondii. Herein, we present the anti-Toxoplasma effect of two new copper(II) complexes: [(H2L1) Cu (µ-Cl)2 Cu(H2L1)] Cl2·5H2O (1) and [(H2L2) Cu (µ-Cl)2 Cu(H2L2)] Cl2·6H2O (2). Complexes (1) and (2) irreversibly controlled parasite growth in vitro, with IC50 values of 0.78µM and 3.57µM, respectively, after 48h. These complexes induced part of the tachyzoite population to convert to bradyzoites, which eventually die. The cell death mechanism was unknown, but signs of apoptosis, such as membrane blebs and nuclear fragmentation, and necrosis, such as plasma membrane disruption, intense cytoplasm vesiculation and the release of cellular contents, were seen. In addition, complex (2) interfered with the correct disposition of the inner membrane complex of the parasite, affecting cell division. These results indicate that these copper complexes have potential effects against T. gondii and may be used as drugs in the future or serve as prototypes for the development of new drugs to treat toxoplasmosis.

Reduction of Toxoplasma gondii Development Due to Inhibition of Parasite Antioxidant Enzymes by a Dinuclear Iron(III) Compound.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide range of vertebrate cells. Here, we describe the cytotoxic effects of the dinuclear iron compound [Fe(HPCINOL)(SO4)]2-µ-oxo, in which HPCINOL is the ligand 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells. This compound was not toxic to LLC-MK2 cells at concentrations of up to 200 µM but was very active against the parasite, with a 50% inhibitory concentration (IC50) of 3.6 µM after 48 h of treatment. Cyst formation was observed after treatment, as indicated by the appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and transmission electron microscopy characteristics. Ultrastructural changes were also seen in T. gondii, including membrane blebs and clefts in the cytoplasm, with inclusions similar to amylopectin granules, which are typically found in bradyzoites. An analysis of the cell death pathways in the parasite revealed that the compound caused a combination of apoptosis and autophagy. Fluorescence assays demonstrated that the redox environment in the LLC-MK2 cells becomes oxidant in the presence of the iron compound. Furthermore, a reduction in superoxide dismutase and catalase activities in the treated parasites and the presence of reactive oxygen species within the parasitophorous vacuoles were observed, indicating an impaired protozoan response against these radicals. These findings suggest that this compound disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite death.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays.

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Nitric oxide is not involved in the killing of Trypanosoma cruzi by chicken macrophages.

It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-gamma and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii.

Further studies on the phagocytic capacity of chicken thrombocytes.

Thrombocytes, functional analog to mammalian platelets, have been described as the primary circulating phagocyte in chicken blood when challenged with bacteria (Chang and Hamilton, 1979). In order to determine if the phagocytic capacity could be extended to protozoa, interaction of chicken thrombocytes with tachyzoites of Toxoplasma gondii and bloodstream trypomastigotes of Trypanosoma cruzi was performed. Interaction with Staphylococcus aureus and Escherichia coli was also performed using fluoresceinated and living bacteria, to be examined by fluorescence microscopy (after ethidium bromide staining) and transmission electron microscopy (after ruthenium red fixation). Using these approaches it was possible to distinguish internalized from attached bacteria. T. cruzi was only found attached to the thrombocyte surface while T. gondii could be observed within the cell. To determine if T. gondii invasion was active or by phagocytosis, interaction was performed under conditions where active penetration and phagocytosis were inhibited by previous fixation of the parasites or treatment of thrombocytes with cytochalasin D, respectively. Interactions with fixed T. gondii showed only attached parasites. Cytochalasin D treated thrombocytes could still be found with internalized T. gondii. By fluorescence and transmission electron microscopy it was possible to observe a small number of bacteria internalized by thrombocytes. These findings show that T. gondii invade thrombocytes through an active penetration process and these blood cells cannot be considered as the primary circulating phagocyte in chicken.

Coculture of chicken thrombocytes and monocytes: morphological changes and lectin binding

Chicken leukocytes were separated from blood on a Percoll cushion following adherence on coverslips, resulting in a coculture of thrombocytes and monocytes. This system was characterized by light microscopy, scanning and transmission electron microscopy, by lectin binding and actin localization in thrombocytes. During the first 24 hours nuclear condensation, cytoplasmic shrinkage, detachment and death of thrombocytes were observed. In contrast, monocytes gradually increased their spreading capacity, specially after thrombocyte detachment from the coverslips. During culture, a large number of fucose and beta-galactose residues were expressed on the surface of thrombocytes, revealed by the lectins Ulex europaeus I and Arachis hypogaea, respectively. Labeling of the monocyte surface with several lectins also increased with the cultivation time. Thrombocytes showed the formation of a net with actin involvement

Coculture of chicken thrombocytes and monocytes: morphological changes and lectin binding

Chicken leukocytes were separated from blood on a Percoll cushion following adherence on coverslips, resulting in a coculture of thrombocytes and monocytes. This system was characterized by light microscopy, scanning and transmission electron microscopy, by lectin binding and actin localization in thrombocytes. During the first 24 hours nuclear condensation, cytoplasmic shrinkage, detachment and death of thrombocytes were observed. In contrast, monocytes gradually increased their spreading capacity, specially after thrombocyte detachment from the coverslips. During culture, a large number of fucose and beta-galactose residues were expressed on the surface of thrombocytes, revealed by the lectins Ulex europaeus I and Arachis hypogaea, respectively. Labeling of the monocyte surface with several lectins also increased with the cultivation time. Thrombocytes showed the formation of a net with actin involvement

Coculture of chicken thrombocytes and monocytes: morphological changes and lectin binding.

Chicken leukocytes were separated from blood on a Percoll cushion following adherence on coverslips, resulting in a coculture of thrombocytes and monocytes. This system was characterized by light microscopy, scanning and transmission electron microscopy, by lectin binding and actin localization in thrombocytes. During the first 24 hours nuclear condensation, cytoplasmic shrinkage, detachment and death of thrombocytes were observed. In contrast, monocytes gradually increased their spreading capacity, specially after thrombocyte detachment from the coverslips. During culture, a large number of fucose and beta-galactose residues were expressed on the surface of thrombocytes, revealed by the lectins Ulex europaeus I and Arachis hypogaea, respectively. Labeling of the monocyte surface with several lectins also increased with the cultivation time. Thrombocytes showed the formation of a net with actin involvement.