Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

BackgroundPUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells.MethodsTwo lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane.ResultsWe have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR.N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa.ConclusionsOur results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants.

In recent years, there has been a growing interest in natural antioxidants as replacements of synthetic compounds because of increased safety concerns and worldwide trend toward the usage of natural additives in foods. One of the richest sources of natural antioxidants, nowadays largely studied for their potential to decrease the risk of diseases and to improve oxidative stability of food products, are edible brown seaweeds. Nevertheless, their antioxidant mechanisms are slightly evaluated and discussed. The aims of this study were to suggest possible mechanism(s) of Fucus vesiculosus antioxidant action and to assess its bioactivity during the production of enriched rye snacks. Chemical and cell-based assays indicate that the efficient preventive antioxidant action of Fucus vesiculosus extracts is likely due to not only the high polyphenol content, but also their good Fe 2+ -chelating ability. Moreover, the data collected during the production of Fucus vesiculosus-enriched rye snacks show that this seaweed can increase, in appreciable measure, the antioxidant potential of enriched convenience cereals. This information can be used to design functional foods enriched in natural antioxidant ingredients in order to improve the health of targeted consumers.

Wound healing is a very complex process that allows organisms to survive injuries. It is strictly regulated by a number of biochemical and physical factors, mechanical forces included. Studying wound healing in space is interesting for two main reasons: (i) defining tools, procedures, and protocols to manage serious wounds and burns eventually occurring in future long-lasting space exploration missions, without the possibility of timely medical evacuation to Earth; (ii) understanding the role of gravity and mechanical factors in the healing process and scarring, thus contributing to unravelling the mechanisms underlying the switching between perfect regeneration and imperfect repair with scarring. In the study presented here, a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) has been used to evaluate the effect of unloading conditions on the healing process and the effectiveness of platelet rich plasma (PRP) as a countermeasure. The results reveal that microgravity caused a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. Moreover, investigating the effects of microgravity and PRP on an in vitro wound healing model, it was found that PRP is able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. This could be one of the mechanisms underlying the effectiveness of PRP in preventing healing impairment in unloading conditions.

Although several studies have aimed to identify mare's milk proteins, only the major whey proteins and some caseins have yet been characterized. Incomplete sequencing of the equine genome and the difficulty of recovering highly hydrophobic proteins mean that little is known to date about the proteins associated with milk fat globules, which have been shown to play an important role in newborns' defense mechanisms. The fat fraction, in particular the distribution of unsaturated fatty acids, has been more extensively studied, but complex lipids are only partially elucidated. This study reports a 2-DE approach combined with a powerful method for de novo protein sequencing, and quali-quantitative data on complex lipid composition determined by high performance TLC (HPTLC) and GC. The presence in mare's milk of long-chain highly unsaturated fatty acids, and the evidence of close similarity between equine and human milk fat globule membrane proteins, support the use of mare's milk for human nutrition.

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