The cell membrane is the main target of resveratrol as shown by interdisciplinary biomolecular/cellular and biophysical approaches.

One of the research lines developed in our laboratory is focused on the study of the bioactivity of natural substances. Resveratrol (RV) is a polyphenol nonflavonoid compound present in a number of plant species but mainly in the berries of the red grape Vitis vinifera. The powerful antioxidant action of this molecule is well documented. In this work we evaluated the effects of this substance by adopting diverse experimental strategies. In particular, we studied the effects on cell vitality and cycle by MTT and cytofluorimetric assays. In addition, we explored the action of RV on the cell membrane by a well-consolidated biophysical approach: electrorotation. This technique allows assessment of the structure/function of the cell membrane. The results presented here demonstrate that RV shows a modest effect on the biological properties of the cell in terms of cytotoxicity and cell cycle alterations. On the contrary, a significant effect on the membrane structure/function was observed, consisting of an enhanced intramembrane ion transport. The implications and interpretation of these membrane alterations are discussed.

Inositol activity in oligoasthenoteratospermia--an in vitro study.

BACKGROUND: Oligoasthenoteratospermia, a reduction in motilty and number of spermatozoa and a change in their morphology, is one of the most relevant causes of infertility in men. One of the factors, which may influence male infertility is linked to the production of reactive oxygen species (ROS) by morphologically altered spermatozoa. Spermatozoa are more susceptible than other cell species to the detrimental activity of these chemical compounds. In particular ROS can affect motility, morphology and DNA stability of spermatozoa. AIM: In the present in vitro study the role of a natural substance, inositol, has been investigated as a possible antioxidant agent both for the systemic treatment of male infertility and for the improvement in the in vitro quality of the sperm used for the fertilization applied to medically assisted reproductive procedures. MATERIALS AND METHODS: The collected samples, belonging to subjects suffering from oligoasthenoteratospermia and of healthy subjects were submitted to phase constrast microscopy in order to evaluate spermatozoa motility, treated with inositol 2 mg/ml and then submitted to scansion electron microscopy (SEM) and to transmission electron microscopy (TEM). SEM allowed to study both the surface morphology of the biological samples and the changes induced on them by the treatment with inositol. TEM allowed to study ultrastructural details of the biological samples. RESULTS: In the samples of subjects suffering from oligoasthenoteratospermia the spermatozoa appear entirely covered with an amorphous fibrous material, that gives an excessive viscosity to the seminal fluid, and reduces or avoids cell mobility. The micrographs of these samples show that the mitochondria, in their intermediate tract, appear to be altered with markedly damaged cristae. After treatment with inositol the pathologic samples clearly shows the absence of the amorphous material, perhaps due to a variation in seminal fluid pH. Furthermore, they show the presence of mitochondria morphologically more similar to control specimen mitochondria, with less damage involving mitochondrial cristae. CONCLUSIONS: These preliminary data appear to suggest that inositol, on account of its antioxidant activity, could preferentially aim at the mitochondrium. Further studies are requested to the purpose of better defining the combination between ROS values of the samples, inositol in vitro treatment and oligoasthenoteratospermia.

Intratumoral vs systemic administration of meta-tetrahydroxyphenylchlorin for photodynamic therapy of malignant gliomas: assessment of uptake and spatial distribution in C6 rat glioma model.

Malignant gliomas, with an incidence of 5 cases per 100,000 population per year, represent the most common primary brain tumour. They have an overall survival length of less than 2 years. Many different adjuvant therapies have been developed. Among them, Photodynamic Therapy (PDT), that is based on photochemical reactions between light and tumoral tissue selectively labelled with exogenous photosensitizing agents. Among photosensitizers, m-THPC (Temoporfin), seems to be the most promising one for the treatment of brain tumors, but, unfortunately, it causes problems of high skin photosensitivity. To by-pass this problem, we devised an intratumoral route of administration of this photosensitizer. The aim of this study is to investigate and compare the uptake of m-THPC in brain tumor and normal tissue after systemic and intratumoral administration of the drug. 30 female Wistar rats received m-THPC 12 days after C6 tumor implantation. Temoporfin was administered intratumorally in 24 rats at two different concentrations. 6 rats constituted the control group and received m-THPC by means of an intraperitoneal injection. The brains were extracted at 4 h, 24 h and 96 h after Temoporfin injection. The samples were examined with a confocal laser scanning microscope. All samples showed high fluorescence emission exclusively in the tumour area, without appreciable differences between the samples taken at the different times of sacrifice and the two routes of administration. No fluorescence whatsoever was detected among normal brain tissue surrounding the tumour. The intratumoral route appears to give comparable results to the systemic one, regarding intracellular uptake efficiency and tumour--normal tissue ratio, with the advantage of a much shorter time needed to reach optimal intratumoural concentration--that is just four hours from m-THPC injection.

Contribution of electron microscopy to study in vitro inositol effects on human spermatozoa.

Infertility is a worldwide problem and male partner contributes to almost 30% of cases of infertility. The term oligoasthenoteratospermia is related to defective spermatogenesis and is characterized by a reduction of motility and number of spermatozoa and a change in their morphology. Electron microscopes are frequently used in order to evaluate sperm pathology and overall to establish a correlation between structural and functional deficiencies of altered sperm. High levels of reactive oxygen species endanger sperm function and viability. The correlation between male infertility, reactive oxygen species levels and the innovative therapeutic strategy employing inositol has been highlighted through analysis of literature data.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

Nanomedicines for antimicrobial interventions.

The development of new antimicrobial therapeutic tools addresses the emergence of multidrug-resistant micro-organisms or clones and the need for more effective antimicrobial strategies. Overcoming the hurdles in providing early diagnosis and intervention on hard-to-reach and/or resting bacteria (i.e. biofilm-embedded cells) represents a challenging task. In this review, we identify a set of organic, inorganic, and hybrid materials that might be used for prevention and control of healthcare-associated infections. We report the current knowledge on nano- and microparticle-based antimicrobial agents and describe the possible mode of their action.

Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma.

In this study we investigated the feasibility of mixed liposomes formed by dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and cationic gemini surfactant (Gemini 1) loaded with the chlorin m-tetrahydroxyphenylchlorin (m-THPC), in photodynamic therapy (PDT) for glioma. To this aim, an in vitro study was carried out by employing various human glioblastoma cell lines (A172, DBTRG, LN229, U118). The following liposomal formulations were tested: (i) DMPC and Gemini 1; (ii) m-THPC in DMPC in the absence or (iii) in the presence of Gemini 1 in the molar ratio 8:2; 7:3, and 6:4. The presence of Gemini 1 significantly increased the intracellular uptake of chlorin in all cell tested although with a different extent: LN229>U118>A172>DBTRG. The cytotoxicity of chlorin-loaded liposomes was then tested by cloning efficiency performed on different cultures, before and after irradiation with laser light at 652nm, at a Fluence Rate of 200mW/s for 100s, with a total Fluence of 20J/cm(-2). In the absence of irradiation, the different liposomal formulations induced a cytotoxicity in less than 30% of glioblastoma cells. On the contrary, irradiation induced total destruction of all cultures treated with m-THPC/DMPC+Gemini 1 in the ratios 8:2, or 7:3, or 6:4.

Invasive properties of multidrug resistant human melanoma cells.

Melanoma cells exhibit a high level of intrinsic or acquired resistance to the cytotoxic agents often associated with the over-expression of drug transporters such as P-glycoprotein (P-gp). In this in vitro study, we investigated the possible relationship between P-gp and CD44, the cell adhesion molecule involved in metastasis and tumor progression of melanoma cells. CD44 expression appeared to be similar in the parental sensitive M14 WT cells and in their resistant counterparts M14 ADR cells. Double-labeling of cryosectioned cells showed that P-gp and CD44 were transported from the synthesis loci to the cell periphery by different vesicles and began to coalesce in proximity of the plasma membrane; thus, P-gp and CD44 seemed to reach together the cell surface. Moreover, P-gp and CD44 appeared to be associated with ERM proteins. The invasive activities of both M14 WT and M14 ADR cells were analyzed by the "transwell chamber invasion" assay. M14 WT cells revealed low capacity to traverse the filters, both in the absence (motility) and in the presence (invasion) of a Matrigel coating. In comparison, M14 ADR cells displayed significantly higher motility and invasion. SEM observations showed that sensitive cells employed lamellar cytoplasmic extrusions to pass through the filter pores whereas resistant cells elongated along the hole through globular processes. In conclusion, the results herein reported suggest that drug resistance in melanoma cells appears associated with a more aggressive behaviour. P-gp and CD44 might cooperate to confer this more invasive phenotype.

Geriatric patients with depression. Improving outcomes using a multidisciplinary clinical path model.

Clinical pathways define multidisciplinary staff members' responsibilities, time lines, and patient outcomes. Although medical and surgical care settings frequently use them to improve quality, clinical, and fiscal outcomes for specific patient populations, staff members of psychiatric care settings have been hesitant to use clinical pathways because psychiatry emphasizes patient individuality. The authors describe the development and implementation of a psychiatric clinical pathway for geriatric patients with depression and identify common multidisciplinary interventions and a pattern of outcomes over the course of treatment for these patients. They also delineate quality and fiscal outcomes and future directions. Implementation of the pathway has been successful, and the development and implementation processes have applicability to other patient populations and care settings.