Guanosine protects human neuroblastoma cells from oxidative stress and toxicity induced by Amyloid-beta peptide oligomers.

Amyloid-beta (Abeta) peptide aggregation forms such as soluble oligomers (O) have a causal role in neuronal dysfunction and death associated with Alzheimer?s Disease (AD). The main efforts for the development of neuroprotective drugs are therefore focused on preventing Abeta production, aggregation or downstream neurotoxic events. We therefore investigated the effect of guanosine (GUO), a guanine based purine, that exerts neurotrophic and neuroprotective effects. The GUO showed the ability to reduce neuronal death in terms of apoptosis, but not necrosis, elicited by Abeta1-42O in human neuroblastoma SH-SY5Y cells. The neuroprotective effect was recorded only when the GUO was added simultaneously to treatment of the SH-SY5Y cells with Abeta1-42O. By contrast, the GUO treatment of SH-SY5Y cells before and after the appearance of beta1-42O toxicity had no neuroprotective effects. The employment of specific inhibitors showed the involvement of neuronal survival pathways, such as PI3K?Akt and MAPK-ERK for the GUO anti-apoptotic effects observed. In parallel, the SH-SY5Y cells treated with GUO, in experimental conditions similar to those adopted to evaluate neuronal death, showed a marked decrease of the early reactive oxygen species formation induced by Abeta1-42O and pro-oxidant H2O2. In the same neuronal model, GUO was also shown to inhibit the extra- and intra-cellular Abeta1-42 release as well as the beta-secretase activity evoked by H2O2 pro-oxidant action. Based on these findings, GUO and other guanine based purines appear to be a promising class of compounds with neuroprotective properties that may play an important role in the therapy of AD.

Influence of cultivation system on bioactive molecules synthesis in strawberries: spin-off on antioxidant and antiproliferative activity.

Strawberries (Fragaria ananassa L., cv. favette) were studied to investigate the influence of cultivation practices (biodynamic, conventional) on the synthesis of bioactive molecules (ascorbic acid, ellagic acid, anthocyanins, flavonols) and to evaluate their antioxidant activity. Additionally, the in vitro bioactivity, in terms of antioxidant and antiproliferative activity, of the same strawberry samples in human colon carcinoma (Caco-2) cells was also studied. Compared to conventional strawberries, biodynamic fruits had a significantly higher content of ascorbic acid (P < 0.01), pelargonidin-3-glucoside (P < 0.05), cyanidin-3-glucoside (P < 0.01), ellagic acid (P < 0.01), quercetin, and kaempferol (both P < 0.01). Antioxidant activity of biodynamic strawberry crude extract was significantly higher than that of the conventional one (P < 0.05); in addition, while the antioxidant activity of water-soluble fraction was very similar in both biodynamic and conventional strawberries, that of water-insoluble fraction of biodynamic fruits was significantly higher (P < 0.05). The same crude extract of biodynamic strawberry samples effectively corresponded to an increase of bioactivity, in terms of both cellular antioxidant activity and antiproliferative activity, in Caco-2 cells differentiated to normal intestinal epithelia and in undifferentiated Caco-2, respectively. Further studies are needed to confirm whether the practice of biodynamic agriculture is likely to increase the bioactivity of other varieties of fruits and vegetables.

Antioxidant effectiveness of organically and non-organically grown red oranges in cell culture systems.

BACKGROUND: Consumers consider plant food products from organic origin healthier than the corresponding conventional plant foods. Clear experimental evidence supporting this assumption is still lacking. AIM OF THE STUDY: To determine if the organic red oranges have a higher phyto-chemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and in vitro bioactivity, in terms of protective effect against oxidative damage at cellular level, than nonorganic red oranges. METHODS: Total phenolics were measured using the Folin Ciocalteau assay, while total anthocyanins and ascorbic acid levels were determined by spectrophotometric and HPLC analysis, respectively. In addition, the total antioxidant activity of red orange extracts was measured by the ABTS(*+) test. The ability of red orange extracts to counteract conjugated diene containing lipids and free radical production in cultured rat cardiomyocytes and differentiated Caco-2 cells, respectively, was assessed. RESULTS: Organic oranges had significantly higher total phenolics, total anthocyanins and ascorbic acid levels than the corresponding non-organic oranges (all p < 0.05). Moreover, the organic orange extracts had a higher total antioxidant activity than non-organic orange extracts (p < 0.05). In addition, our results indicate that red oranges have a strong capacity of inhibiting the production of conjugated diene containing lipids and free radicals in rat cardiomyocytes and differentiated Caco-2 cells, respectively. Statistically higher levels of antioxidant activity in both cell models were found in organically grown oranges as compared to those produced by integrated agriculture practice. CONCLUSIONS: Our results clearly show that organic red oranges have a higher phytochemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and bioactivity than integrated red oranges. Further studies are needed to confirm whether the organic agriculture practice is likely to increase the antioxidant activity of other varieties of fruits and vegetables.

Photomutagenic properties of terfenadine as revealed by a stepwise photostability, phototoxicity and photomutagenicity testing approach.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm(-2) UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm(-2) UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity--carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Interleukin-12 induces efficient lysis of natural killer-sensitive and natural killer-resistant human osteosarcoma cells: the synergistic effect of interleukin-2.

Previously we demonstrated that some osteosarcoma cell lines varied greatly in their susceptibility to natural killer (NK) cell lysis in vitro. The expression of CD54 and CD58 adhesion molecules on their surface appeared to influence their vulnerability, and the tumour necrosis factor-alpha (TNF-alpha)-induced positive modulation of CD54 increased osteosarcoma susceptibility in vitro. This study investigated whether peripheral blood mononuclear cells from normal healthy donors could be activated by interleukin (IL)-12 and IL-2, separately or in combination, to lyse osteosarcoma cell lines in vitro, as evaluated by using a microcytotoxicity test. In addition, we analysed (by flow cytometry) whether this function correlated with modifications of the CD2, CD11a, CD11b and CD18 molecules, which are involved in the adhesion of effector cells to the counter-receptors (CD54 and CD58) on osteosarcomas. This study demonstrates that incubation with IL-12 and/or IL-2 triggered NK cell cytolytic activity against osteosarcoma targets and that cytolytic activity was enhanced to a greater extent when lymphocytes were incubated simultaneously with a combination of IL-12 and IL-2. The density of CD18 and CD2 molecules involved in NK adhesion was also up-modulated following cytokine incubation. These changes in the density of adhesion molecules can be involved in the increased lytic activity of effector lymphocytes and in the modification of their binding capacity to osteosarcoma target cells.

Vitamin D, thyroid hormones and muscle mass influence natural killer (NK) innate immunity in healthy nonagenarians and centenarians.

Increasing evidence has demonstrated that the immune system closely interacts with other physiological systems, whose communications are mediated by circulating cytokines and hormones. The aim of our study was to test whether the number and cytolytic activity of NK cells in a group of relatively healthy Italian nonagenarians and centenarians were affected by the modifications of endocrine, metabolic and functional parameters that occur during ageing. Because of the extreme age of the study population, a cross-sectional analysis was performed. This study revealed that the group of oldest subjects with the highest number of NK cells and the best preserved cytolytic function also presented a preserved metabolism of thyroid hormones and vitamin D and integrity of muscle mass. In fact, the NK cell number and/or cytolytic activity of healthy subjects > 90 years old was positively associated with serum levels of vitamin D, while T3, FT4, i-PTH hormones and lean body mass were associated only with NK cell number. In conclusion, our results stress the paramount importance of nutritional evaluation in the clinical assessment of elderly people.

Age-dependent decreases of NK cell phosphoinositide turnover during spontaneous but not Fc-mediated cytolytic activity.

The progressive increase in the number of peripheral NK cells found in the elderly does not correlate with a corresponding increase in lytic activity. On the contrary, a decreased function of circulating NK cells purified from old subjects was observed on a per cell basis. Most of the studies on NK cells have focused on late events such as lytic activity. In view of this, little is currently known about the modification of the early signalling pathways of NK cells in elderly people. This study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of these cells in the elderly. NK cells were negatively purified by immunomagnetic depletion from the peripheral blood of selected old and young healthy subjects. Hydrolysis of inositol phospholipids was measured following incubation with K562 target cells and/or CD16 mAb for different times. Our data show that there is a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover is preserved following Fc triggering (antibody-dependent cell-mediated cytotoxicity). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs and may aid the development of experimental and therapeutic strategies to counteract declines in cell mediated immune functions associated to advancing age.

Natural immunity and bone and muscle remodelling hormones in the elderly.

Increasing evidence has demonstrated that the immune system is closely integrated with two other physiological systems: endocrine and nervous. They communicate through circulating humoral factors such as cytokines, hormones and neurotransmitters. We undertook a cross-sectional analysis in a group of elderly subjects over 90 years to demonstrate that a functional relationship exists among the number and cytolytic activities of NK cells, bone and muscle remodelling hormones, anthropometric parameters and physical ability. Peripheral blood samples collected from 62, 90-106 years-old subjects underwent biochemical (bone and muscle remodelling hormone levels) and immunological determinations (Natural Killer cell distribution and activity), anthropometric and functional assessment. Significant associations were found among NK cell number and cytolytic activity and serum concentrations of vitamin D, anthropometric parameters, while functional independence in daily activity was only associated with NK cell number. In general a high level of physical ability was correlated with preserved body stores and vitamin D levels. In conclusion, our results stress the importance of nutritional evaluation in the clinical assessment of elderly people. The magnitude of the NK immune response, which constitutes the first line of defence against infected and neoplastic cells, is best preserved in oldest-old people with the best hormonal parameters and nutritional measures.

TNF-alpha but not IL-1 and IL-6 modifies the susceptibility of human osteosarcoma cells to NK lysis.

TNF-alpha-treated osteosarcoma cells have an enhanced susceptibility to NK lysis which mostly depends on the increased expression of CD54 molecules. Since IL-1 and IL-6 share overlapping biological properties with TNF-alpha, we investigated whether the treatment of osteosarcoma cells with these cytokines could modify their susceptibility to NK lysis and whether these modifications were related to a different distribution of CD54, CD56 and CD58 molecules. We demonstrated that the expression of CD54 and CD58 on osteosarcomas correlated positively with the susceptibility to NK lysis and that this susceptibility was enhanced by TNF-alpha treatment but not by IL-1 and IL-6 stimulation.

Expression of beta-galactoside alpha 2,6-sialyltransferase does not alter the susceptibility of human colon cancer cells to NK-mediated cell lysis.

The extent of processing of N-linked oligosaccharides and the sialylation of the target cell membranes has been positively correlated with resistance to lysis mediated by NK cells, but a conclusive evidence has never been reached. Colon cancer tissues express an increased activity of beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1, alpha 2,6ST), which catalyzed the addition of sialic acid in alpha 2,6-linkage to Gal beta 1,4GlcNAc (N-acetyllactosamine) sequences of glycoprotein N-linked chains. The resulting increased level of membrane alpha 2,6-sialylation appears to be related with a more invasive behavior of cancer cells. This phenomenon may depend on a decreased sensitivity of colon cancer cells to NK cells. To obtain conclusive evidence on the role played by sialylation of N-linked chains in determining the target cell susceptibility to NK-mediated lysis, human colon cancer cell lines not expressing sialyltransferases acting on N-linked chains were transfected with a rat alpha 2,6ST cDNA. Stable transfectants expressed different levels of alpha 2,6ST activity, were reactive with the Sambucus nigra lectin, specific for alpha 2,6-linked sialic acid, and compared with control transfectants, showed a remarkable decrease in the number of unsubstituted Gal beta 1,4GlcNAc terminal sequences. The NK susceptibility of these clones was found to be identical to that of control transfectants, either when unstimulated- or IL-2-stimulated lymphocytes were used as effectors. Neuraminidase treatment of target cells does not result in significant changes to NK susceptibility. Our data demonstrate that sialic acid alpha 2,6-linked to N-linked chains of target cell glycoproteins does not play a major role in recognition of the target by human NK cells.

Human osteosarcoma cell susceptibility to natural killer cell lysis depends on CD54 and increases after TNF alpha incubation.

Osteosarcoma cell lines vary widely in their susceptibility to natural killer (NK) cell lysis in vitro although it is still unclear why this occurs. In this study we investigated the expression of some cell adhesion molecules on osteosarcomas to determine which of these can modify the susceptibility to NK lysis and we also attempted to modulate the cytolytic susceptibility of these targets with TNF alpha. We found that osteosarcoma lysis induced by NK cells correlates with different expression of the CD54 adhesion molecule on osteosarcomas and the increased susceptibility after TNF alpha treatment mostly depends on the expression of CD54 molecules on target cells.

Perforins in human cytolytic cells: the effect of age.

The ageing process is associated with a progressive increase in the number of circulating NK cells, together with a decreased lytic activity per cell. A similar decrease in activity was also found for CD8 lymphocytes. Cytotoxic T- and NK cells express cytoplasm granules containing cytolytic effector molecules (as perforins, studied here) which can recognize and destroy damaged, infected and/or mutated target cells. To investigate whether an altered distribution of perforins in cytolytic cells or a reduced number of cytolytic cells producing perforins underlies decreased cytolytic activity with advancing age, perforin expression was assessed at the single cell level in T- (CD4 and CD8) and NK (CD16) peripheral blood lymphocytes from elderly subjects by flow cytometry. Perforin distribution at the cellular level in CD8+ and CD16+ cell cytoplasm suggested a similar distribution during ageing and a similar number of cells producing perforins. In addition, perforin utilization was maintained in the generation of cytolytic activity against K562 target cells and perforin synthesis in culture following activation was unabated. These data stress the importance of other factors, such as defective signal transduction for granule exocytosis, that may account for the different pattern of lytic activity found in aged people.

Lytic activity of IL-2 and IL-12 stimulated NK cells against HOS osteosarcoma cell line.

NK cells are CD16, CD56 positive lymphocytes that spontaneously lyse tumor or virus infected cells. In this study we investigated whether IL-2 and/or IL-12 stimulated NK cells increased their lytic efficiency against HOS osteosarcoma cell line. Our results demonstrate that both 18 hour and 5 day incubation times enhanced the lytic activity of human PBL against HOS and K562 target cells and that IL-12 appears to be more efficient than IL-2 in augmenting NK cytotoxicity.

In vitro cytolytic activity of human NK cells against osteosarcoma cell lines.

NK lymphocytes present CD16, CD56 and lack CD3 surface molecules and are able to spontaneously lyse tumor or virus infected cells. In this study we evaluated the susceptibility of some human osteosarcoma cell lines to NK cytolytic activity and standardized the assay conditions. NK lymphocytes were used as effector cells in a cytotoxicity test against HOS, U-2 OS and Saos-2 osteosarcoma targets. While HOS cells were susceptible on the contrary U-2 OS and Saos-2 were osteosarcoma resistant lines. Our preliminary results support a model for the study of a possible interaction between the immune system and these tumors.

Aminopyrine: metabolism and effects in the rat after administration of inhibitors of hepatic monooxygenases.

The administration to the rat of the inhibitors of microsomal mixed function oxidase, SKF 525A and Oxine-5-sulphonic acid (OSA) caused a significant decrease of the hepatic aminopyrine N-demethylase activity, as well as an increase in the plasma levels and antipyretic activity of orally administered aminopyrine. The plasma concentrations of the aminopyrine metabolite 4-aminoantipyrine were reduced in SKF 525-A treated animals while they were slightly increased in those pretreated with OSA. These findings suggest that the in vivo changes of aminopyrine disposition and activity brought about by SKF 525-A were the result of an inhibited hepatic drug metabolism, while the effects produced by OSA were due to a more rapid intestinal absorption of aminopyrine.