Microvesicles and exosomes in metabolic diseases and inflammation.

Metabolic diseases are based on a dysregulated crosstalk between various cells such as adipocytes, hepatocytes and immune cells. Generally, hormones and metabolites mediate this crosstalk that becomes alterated in metabolic syndrome including obesity and diabetes. Recently, Extracellular Vesicles (EVs) are emerging as a novel way of cell-to-cell communication and represent an attractive strategy to transfer fundamental informations between the cells through the transport of proteins and nucleic acids. EVs, released in the extracellular space, circulate via the various body fluids and modulate the cellular responses following their interaction with the near and far target cells. Clinical and experimental data support their role as biomarkers and bioeffectors in several diseases includimg also the metabolic syndrome. Despite numerous studies on the role of macrophages in the development of metabolic diseases, to date, there are little informations about the influence of metabolic stress on the EVs produced by macrophages and about the role of the released vesicles in the organism. Here, we review current understanding about the role of EVs in metabolic diseases, mainly in inflammation status burst. This knowledge may play a relevant role in health monitoring, medical diagnosis and personalized medicine.

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy.

Rose Bengal acetate photodynamic therapy (RBAc-PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.

In vitro study of the interaction of polyalkilimide and polyvinyl alcohol hydrogels with cells.

Hydrogels are a class of polymers that in the last decade have had a great development and application for soft tissue augmentation, due to their similarity to this tissue for their high water content. The in vitro effects of polyalkylmide hydrogel (pAI) and polyvinyl alcohol hydrogel (pVOH) on human lymphocytes and U937 cells viability, apoptosis and cell shape were investigated. Cell viability was always higher than 70%, thus showing the hydrogels were not cytotoxic for both cell lines. Some differences were, however, found. At short time, lymphocytes were very sensitive to the hydrogels incubation, while at long time, U937 cells were the most sensitive cells. Other differences on cell viability were related to the time of incubation, to the type of hydrogel and to the polymers concentration. Cell viability decreased only at the longest time of incubation and with the highest hydrogel concentration. Accordingly, cell death by apoptosis increased; necrosis was never observed in the cultures. Concentration- and hydrogel-dependent modifications of cell shape (bigger cell volume, elongations of cells) were observed in a few percentage of viable cells. In conclusion, the very high in vitro degree of biocompatibility shown by both hydrogels encourages their use as dermal fillers.

In vitro and in vivo biocompatibility evaluation of a polyalkylimide hydrogel for soft tissue augmentation.

Injectable fillers are commonly used in Plastic and Reconstructive Surgery to correct serious and slight aesthetic defects due to their low invasiveness and an easy implant technique procedure. Synthetic hydrogels are proposed as filler materials for their similarity with soft tissue and to avoid many disadvantages of naturally derived materials such as short persistence, allergenicity, and immunogenicity. Our studies are focused on the biocompatibility evaluation of a polyacrylic hydrogel containing alkylimide-amide groups and pyrogen free water (96%) (Bio-Alcamid by means of the in vitro cytotoxicity and mutagenicity assays and the in vivo skin irritation, sensitization test, and subcutaneous implant. All tests conducted on Bio-Alcamid showed no toxicity. It is a substance easy to inject and remove; it does not migrate, and its safety allows it to be a suitable filler for the correction of slight and also very serious aesthetic defects.

Hepatic clearance of apoptotic lymphocytes: simply removal of waste cells?

The in situ liver recognition of apoptotic lymphocytes was studied by using different sources of lymphocytes (i.e. human, rat and mouse) and animal models (i.e. rat and mouse). Lymphocytes were induced to apoptosis using 10(-2)M cycloheximide for up to 24 hours; three types of apoptosing lymphocytes, corresponding to different stages in the apoptotic process, were described: type 1 or early apoptosis, type 2 or mature apoptosis and type 3 or late/necrotic apoptosis. When livers were in situ injected with apoptotic lymphocytes enriched for type 1 (early), 2 (mature) or 3 (late/necrotic) apoptosis, they recognized and internalized apoptosing cells, with an efficiency directly dependent on the stage of the apoptotic process. The highest recognition rate, which was, in all cases, mediated by galactose- and mannose-specific receptors, was obtained with homologous apoptotic cells (i.e. rat lymphocytes and rat liver). Moreover, the drastically reduced efficiency of recognition of human or mouse apoptotic lymphocytes when injected into rat liver, suggested the involvement also of species-specific antigens.

Lymphocytes apoptosis: young versus aged and humans versus rats.

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.

Cell shape and plasma membrane alterations after static magnetic fields exposure.

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.