Comparing in vivo biodistribution with radiolabeling and Franz cell permeation assay to validate the efficacy of both methodologies in the evaluation of nanoemulsions: a safety approach.

The Franz cells permeation assay has been performed for over 25 years. However, the advent of nanotechnology created a whole new world, especially with regard to topical products. In this new global scenario an increasing number of nanostructure-based delivery systems (NDSs) have emerged and a global warning relating to the safety of these NDSs is arising. This work studied the efficacy of the Franz cells assay, comparing it with the radiolabeling biodistribution test. For this purpose a formulation of sunscreen based on an NDS was developed and characterized. The results demonstrated both that the NDS did not present in vitro cytotoxicity and that the radiolabeling biodistribution test is more precise for the evaluation of NDS cosmetics than the Franz cells assay, since it detected the permeation of the NDS at a picogram order. Due to this fact, and considering all the concerns related to NDSs and nanoparticles in general, more precise methods must be used in order to guarantee the safe use of these new classes of products.

An endocytic process in HEp-2 cells induced by enteropathogenic Escherichia coli.

Infection of HEp-2 cells by enteropathogenic Escherichia coli (EPEC) was examined by transmission and scanning electronmicroscopy. EPEC strains of serogroups O111:K58 and O55:K59 recently isolated from human patients did not exhibit enterotoxic activity, as judged by the Vero-cell and suckling-mouse assays, or invasive ability as judged by the Sereny test. These strains attached to and penetrated HEp-2 cells. Transmission electronmicroscopy showed bacteria in close contact with cell membranes 15 min after infection; later, intense swelling and budding of membranes and penetration of EPEC into the cell cytoplasm occurred. Intracellular bacteria were enclosed in membrane-bound vacuoles in the cell cytoplasm underlying localised adherence sites observed by light microscopy. Scanning electronmicroscopy showed morphologically altered membranes only at the sites of bacterial attachment. Bacteria inactivated by ultraviolet light were not internalised and cytochalasin B (greater than or equal to 10 mg/L) markedly inhibited uptake. These observations suggest that penetration of EPEC into HEp-2 cells occurs by an endocytic process in metabolically active bacteria.

Effect of monensin on Mayaro virus replication in monkey kidney and Aedes albopictus cells.

The effect of a cationic ionophore, monensin, on the replication of Mayaro virus in monkey kidney TC7 and Aedes albopictus cells has been studied. Treatment of these cells with 1 micromol/l monensin during infection did not affect the virus protein synthesis but inhibited severely the virus replication. Electron microscopy of the cells infected with Mayaro virus and treated with monensin revealed that the morphogenesis of Mayaro virus was impaired in TC7 but not in A. albopictus cells.

Damage induction by direct electric current in tumoural target cells.

Damage induction to tumour target cells (P815) by direct electric current (DC) was investigated. A 6 min treatment of P815 cells with DC generated decreased levels of cell viability and proliferation. The ultrastructural analysis of DC-treated cells revealed the presence of blebs, loss of cell surface filopodia, and ruptures in cell membrane. Mitochondrial alterations, swelling of cells, cytoplasmic matrix rarefaction, and cellular debri formation were also observed. The study shows that tumoural target cells can be damaged by direct electric current and this approach may provide means to understand the mechanism of tumour regression induced by electrochemical therapy.

Phytochemical and antioedematogenic studies of commercial copaiba oils available in Brazil.

The composition of eight samples of commercial copaiba oils, used in the Amazonian region as antiinflammatory agents and available in popular markets, were analysed by gas chromatography/mass spectrometry (HRGC-MS). Major differences were observed in their chemical composition and some adulterations were pointed out. When tested in vivo oils 1 and 3, and to a lesser extent oil 6, significantly inhibited bradykinin-induced oedema formation. The other tested oils had no effect. When assessed in carrageenan-induced oedema formation, oils 1, 2 and 6, but not oil 3, significantly attenuated the oedema formation. The other tested oils failed to affect carrageenan-induced paw oedema. Oils 1 and 6 were further fractionated and several sesquiterpenes and diterpenes were detected. It is suggested that the naturally occurring sesquiterpenes present in the copaiba oils seem to be responsible for the antiinflammatory action reported in the folk medicine. Furthermore, our results clearly show an adulteration in copaiba oils available in Brazil.

Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells.

Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems. In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy. DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells. In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability. Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied. The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells. These results were confirmed by flow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment. Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed.

Activation-induced CD4+ T cell death by apoptosis in experimental Chagas' disease.

Infection of mice with Trypanosoma cruzi, the causative protozoan agent of human Chagas' disease, leads to immunosuppression of the T cell compartment and to chronic cardiac inflammation which resembles the human infection. Recently, reinduction of programmed cell death by apoptosis in mature T cells has been demonstrated. It has been suggested that mature T cell apoptosis could play a role in immunosuppression caused by virus infection. In this report, we have investigated the occurrence of mature T cell apoptosis in murine experimental Chagas' disease. Infection with T. cruzi metacyclic forms led to a relative accumulation of CD8 T cells over CD4 T cells in the spleens of infected mice. Splenic T cells from T. cruzi-infected donors, but not from control littermates, died in vitro upon stimulation with T cell mitogens Con A and anti-TCR-alpha beta mAb in a dose-dependent fashion. DNA fragmentation into nucleosome-sized bands was detected in the supernatants of CD4+ T cells from infected origin, after stimulation with the T cell mitogen Con A. Upon in vitro stimulation with either anti-TCR-alpha beta or Con A, CD4+ T cells were susceptible to elimination, whereas CD8+ T cells were not. Splenic T cells from infected donors were markedly unresponsive to anti-TCR mAb in proliferative assays and underwent apoptosis in vitro, as assessed by electron microscopy. Apoptosis also occurred in vivo in the course of acute infection, as seen by DNA fragmentation in freshly explanted splenic cells and purified T cell subsets. The data indicate that activation-induced CD4+ T cell death by apoptosis is a prominent feature of experimental infection with T. cruzi, and could play a role in immunosuppression and parasite persistence in infected hosts.

Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis.

Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked alterations in vital structures of P815 cells. Alterations varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modifications. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-specific lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors.