KFLC Index utility in multiple sclerosis diagnosis: Further confirmation.

The Multiple Sclerosis (MS) diagnosis is based on dissemination of focal lesions in time and space. The free light chains (FLCs) determination might be a sensitive alternative to oligoclonal bands assay. The study aim was to redefine sensitivity, specificity of the kFLC Index cut-off. We analyzed serum and cerebrospinal fluid of 176 patients, with different neurological disorders. We obtained a cut off of 12,3 for kFLC Index with a sensitivity and specificity of 93% and 100% respectively. Our data confirm that the kFLC Index is a valid tool in the diagnosis of MS.

Bioactivation of calcium deficient hydroxyapatite with foamed gelatin gel. A new injectable self-setting bone analogue.

An alternative approach to bone repair for less invasive surgical techniques, involves the development of biomaterials directly injectable into the injury sites and able to replicate a spatially organized platform with features of bone tissue. Here, the preparation and characterization of an innovative injectable bone analogue made of calcium deficient hydroxyapatite and foamed gelatin is presented. The biopolymer features and the cement self-setting reaction were investigated by rheological analysis. The porous architecture, the evolution of surface morphology and the grains dimension were analyzed with electron microscopy (SEM/ESEM/TEM). The physico-chemical properties were characterized by X-ray diffraction and FTIR analysis. Moreover, an injection test was carried out to prove the positive effect of gelatin on the flow ensuing that cement is fully injectable. The cement mechanical properties are adequate to function as temporary substrate for bone tissue regeneration. Furthermore, MG63 cells and bone marrow-derived human mesenchymal stem cells (hMSCs) were able to migrate and proliferate inside the pores, and hMSCs differentiated to the osteoblastic phenotype. The results are paving the way for an injectable bone substitute with properties that mimic natural bone tissue allowing the successful use as bone filler for craniofacial and orthopedic reconstructions in regenerative medicine.

Novel biomimetic thermosensitive ß-tricalcium phosphate/chitosan-based hydrogels for bone tissue engineering.

Among the less invasive surgical procedures for tissue engineering application, injectable in situ gelling systems have gained great attention. In this contest, this article is aimed to realize thermosensitive chitosan-based hydrogels, crosslinked with ß-glycerophosphate and reinforced via physical interactions with ß-tricalcium phosphate. The kinetics of sol-gel transition and the composite hydrogel properties were investigated by rheological analysis. The hydrogels were also characterized by Fourier transform infrared study, X-ray diffraction, scanning electron microscopy, transmission electron microscopy analysis, and thermal and biological studies. The hydrogels exhibit a gel-phase transition at body temperature, and a three-dimensional network with typical rheological properties of a strong gel. The presence of the inorganic phase, made up of nanocrystals, provides a structure with chemico-physical composition that mimics natural bone tissue, favoring cellular activity. These findings suggest the potential of the materials as promising candidates for hard tissue regeneration.

Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes.

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55-67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7-9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32-39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and ß-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log(10)CFU/g) and lactic acid bacteria (3 log(10) CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.

Erythrocyte glutathione transferase: a new biomarker for hemodialysis adequacy, overcoming the Kt/V(urea) dogma?

Kt/V(urea) ratio is commonly used to assess the delivered dose of dialysis in maintenance hemodialysis (MHD) patients. This parameter only reflects the efficacy of dialytic treatments in removing small toxins, but not middle and protein-bound toxins. Erythrocyte glutathione transferase (e-GST), an enzyme devoted to cell depuration against a lot of large and small toxins, is overexpressed in uremic patients. Aim of the present study is to verify whether e-GST may represent a novel biomarker to assess the adequacy of different dialytic techniques complementary to Kt/V(urea) parameter. Furthermore, it will be investigated whether e-GST could reflect the 'average' adequacy of multiple dialytic sessions and not of a single one treatment as it occurs for Kt/V(urea). One hundred and three MHD patients and 82 healthy subjects were tested. Fourty four patients were treated with standard bicarbonate hemodialysis (HD) and 59 patients were on online hemodiafiltration (HDF). In all MHD patients e-GST activity was 60% higher than in healthy controls. In HDF, e-GST activity was lower than in HD subgroup (8.2±0.4 versus 10.0±0.4 U/g(Hb), respectively). Single-pool Kt/V(urea) and total weekly Kt/V(urea) were higher in HDF than in HD, but no correlation was found between e-GST activity and Kt/V(urea) data. e-GST, whose level is stable during the erythrocyte life-span, provides information on the long-term depurative efficacy of dialysis treatments.

Chemical composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil from Cynomorium coccineum L.

Cynomorium coccineum L. is a non-photosynthetic plant, spread over Mediterranean countries, amply used in traditional medicine. We investigated the composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil obtained from dried stems of the plant. Oil isolation has been performed by supercritical fractioned extraction with CO2. 13C NMR spectroscopy has been used to study the molecular composition of oil lipids; fatty acid composition was identified using GC and HPLC techniques. The fixed oil was composed mainly by triacylglycerols and derivates. The main fatty acids were 18:1 n-9 (38%), 18:2 n-6 (20%), 16:0 (15%), and 18:3 n-3 (10.8%). The oil showed a significant in vitro inhibitory effect on the growth of colon cancer undifferentiated Caco-2 cells. Moreover, cell viability, lipid composition, and lipid peroxidation were measured in intestinal epithelial cells (differentiated Caco-2 cells) after 24 h incubation with fixed oil. The oil did not show a toxic effect on colon epithelial cell viability but induced a significant change in fatty acid composition, with a significant accumulation of the essential fatty acids 18:3 n-3 and 18:2 n-6. The results showed remarkable biological activity of Maltese mushroom oil, and qualify it as a potential resource for food/pharmaceutical applications.

Modifications of the 1H NMR metabolite profile of processed mullet (Mugil cephalus) roes under different storage conditions.

(1)H NMR spectroscopy was employed to study the modifications over time of the water-soluble low molecular weight metabolites extracted from samples of salted and dried mullet (Mugil cephalus) roes (mullet bottarga) stored at different conditions. Samples of grated mullet bottarga were stored for 7 months at -20 °C, at 3 °C, and at room temperature in the presence and in the absence of light and then timely extracted and analyzed by NMR. Principal component multivariate data analysis applied to the spectral data indicated that samples stored at -20 °C maintained similar features over time whereas, along PC1, samples stored at room temperature in the presence and in the absence of light showed, over time, marked metabolite modifications. The comparative analysis of the integrated areas of the selected regions of the (1)H NMR spectra indicated that the major compositional changes due to storage conditions were (i) the increase of the derivatives of the breakdown of phosphatidylcholine (choline, phosphorylcholine, and glycerol), (ii) the breakdown of nucleosides, (iii) the decrease of methionine, tryptophan, and tyrosine, and (iv) the cyclization of creatine. These changes were observed at different storage conditions, with more pronounced trends in the samples stored at room temperature. The role of metabolites in food aging is discussed.

Extraction and separation of volatile and fixed oils from seeds of Myristica fragrans by supercritical CO2: chemical composition and cytotoxic activity on Caco-2 cancer cells.

UNLABELLED: Isolation of volatile and fixed oils from nutmeg have been obtained by supercritical fractioned extraction with carbon dioxide. Extraction experiments were carried out at pressures of 90 and 250 bar and temperature of 40 °C. The extraction step performed at 90 bar produced a volatile fraction mainly formed by myristicin (32.8%), sabinene (16.1%), α-pinene (9.8%), ß-pinene (9.4%), ß-phellandrene (4.9%), safrole (4.1%) and terpinen-4-ol (3.6%). The oil yield relative to this step of the process was 1.4% by weight of the charge. The last extraction step at 250 bar produced a butter-like material (nutmeg butter). The yield of this step was 14.4% by weight. The most represented fatty acids of fixed oil from nutmeg were 14:0 (79.2%), 18:1 n-9 (7.4%) and 16:0 (6.1%), and in particular the unsaturated fatty acids 18:1 n-9 averaged 32.96 µg/mg of oil. The level of myristicin in the nutmeg essential and fixed oils was also directly quantified by reversed HPLC-DAD. Moreover, the essential oil obtained from nutmeg, as well as myristicin, showed a significant in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells). PRACTICAL APPLICATION: In this study, the chemical characterization and the anticancer activity of nutmeg oils obtained by supercritical extraction with carbon dioxide were investigated. This is important for their potential application in food and pharmaceutical industries.

Effect of storage conditions on lipid components and color of Mugil cephalus processed roes.

UNLABELLED: The salted and semidried mullet (Mugil cephalus) ovary product (bottarga) is proposed as an important source of the long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid, and docosahexaenoic acid. In this work, we investigated the extent of lipid oxidation and browning of grated bottarga samples during 7 mo of storage at -20 °C, 2 to 3 °C in the absence of light, and at room temperature in the presence or absence of light. Modifications of the levels of total choline (as index of phospholipid breakdown), total sugars, and free amino acids such as lysine, methionine, and tryptophan (involved in nonenzymatic browning) were also studied at different storage conditions. Storage of bottarga did not significantly affect the n-3 PUFA and cholesterol levels with respect to the control; nevertheless, a significant hydroperoxide increase was observed during 7 mo in bottarga samples at all the storage conditions, while low malondialdeyde levels were measured. Samples placed at room temperature in the absence and in the presence of light showed over time a marked browning process, lipid breakdown, a sensible decrease in the levels of total sugars, tryptophan, and methionine with respect to control and samples stored at -20 °C and 2 to 3 °C. The resistance against the oxidation of the isolated bottarga lipids was also assessed in dry state at several temperatures (37, 75, and 140 °C). PRACTICAL APPLICATION: We evaluated the change in lipid compounds and color of dried and salted mullet roes under different storage conditions. The obtained results suggest the importance of the low temperatures to preserve the nutritional properties of this fish product during long storage.

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage.

Hydroxytyrosol (2-(3',4'-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3'-O-ß-d-glucuronide and 4'-O-ß-d-glucuronide derivatives and 2-(3',4'-dihydroxyphenyl)ethanol-1-O-ß-d-glucuronide, in comparison with the parent compound, to inhibit H(2)O(2) induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H(2)O(2) treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.

[Exclusive intra-operative radiation therapy (IORT) for early stage breast cancer: pilot study of feasibility]. / Radioterapia intraoperatoria (IORT) esclusiva nel carcinoma iniziale della mammella: studio pilota di fattibilità.

AIM: To evaluate feasibility, tolerability and cosmetic outcome of intra-operative radiation therapy (IORT) as an exclusive post-surgery treatment of early stage breast cancer. PATIENTS AND METHODS: From October 2008 to October 2009 30 patients underwent wide breast cancer excision or quadrantectomy followed by IORT on tumor bed with accelerated electrons at the dose of 21Gy. The characteristics of the patients were: ductal breast cancer or invasive lobular cT1, cT2 ≤ 2,5 cm, cN0, G1-2, age over 35 years, M0. RESULTS: The average age was 51.7 (range 38 - 75) with an average follow up of 11.7 months (range 6 - 18). The pathologic stage of the lesions resulted pT1 in 29 cases (96,6%), in particular: one case pT1a (3,3%), 21 cases pT1b (70,0%) and 7 cases pT1c (23,3%). One case (3,3%) was pT2 with a diameter of 2.5 cm. The grading was G2 in 20 cases (66,6%) and G1 in 10 cases (33,3%). The toxicity, evaluated according to the EORTC-RTOG criteria, was G0 (33.3%) in 10 cases, G1 (63,3%) in 19 cases, G2 in one case (3,4%); there was no G3 toxicity. The time needed for a complete healing of the wound was less than 10 days in 96,7% of the cases, with one case of limphocele (3,3%). There were no infections of the surgical wound nor any mastitis, neither in the treated quadrant nor in the other ones. We observed a light fibrosis in 5 cases (16,6%), moderate in 2 cases (6,6%) but never severe. Cosmetics, evaluated in four levels, according to Danoff et al., was excellent in 3 cases (43,3%), good in 15 cases (50%), sufficient in 2 cases (6,7%), never insufficient. As regards local control, there was no local relapse. The global survival was 100%. CONCLUSIONS: The IORT in early breast cancer, at the doses used in this study, proved itself as a secure technique, repeatable, with limited complications. The advantages of its use are the possibility of a direct control, by the surgeon and the radiotherapist, of the structures to treat and those to protect; the absence of time needed for cellular repopulation between surgery and radiotherapy; a good cosmetic outcome; and logistic advantages. It is necessary to have a long term follow up to evaluate the efficacy in terms of long term cosmetic and local control.

Effect of aqueous and lipophilic mullet (Mugil cephalus) Bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells.

The importance of n-3 polyunsaturated fatty acid (n-3 PUFA) intake has long been recognized in human nutrition. Although health benefits, n-3 PUFA are subject to rapid and/or extensive oxidation during processing and storage, resulting in potential alteration in nutritional composition and quality of food. Bottarga, a salted and semi-dried mullet ( Mugil cephalus ) ovary product, is proposed as an important source of n-3 PUFA, having high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we investigated the extent of lipid oxidation of grated bottarga samples during 7 months of storage at -20 °C and room temperature under light exposure. Cell viability, lipid composition, and lipid peroxidation were measured in intestinal differentiated Caco-2 cell monolayers after 6-48 h of incubation with lipid and hydrophilic extracts obtained from bottarga samples at different storage conditions. The storage of bottarga did not affect the n-3 PUFA level, but differences were observed in hydroperoxide levels in samples from different storage conditions. All tested bottarga extracts did not show a toxic effect on cell viability of differentiated Caco-2 cells. Epithelial cells incubated with bottarga oil had significant changes in fatty acid composition but not in cholesterol levels with an accumulation of EPA, DHA, and 22:5. Cell hydroperoxides were higher in treated cells, in relation to the oxidative status of bottarga oil. Moreover, the bottarga lipid extract showed an in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells).

Protective role of arzanol against lipid peroxidation in biological systems.

This study examines the protective effect of arzanol, a pyrone-phloroglucinol etherodimer from Helichrysum italicum subsp. microphyllum, against the oxidative modification of lipid components induced by Cu(2+) ions in human low density lipoprotein (LDL) and by tert-butyl hydroperoxide (TBH) in cell membranes. LDL pre-treatment with arzanol significantly preserved lipoproteins from oxidative damage at 2h of oxidation, and showed a remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol levels, inhibiting the increase of oxidative products (conjugated dienes fatty acids hydroperoxides, 7ß-hydroxycholesterol, and 7-ketocholesterol). Arzanol, at non-cytotoxic concentrations, exerted a noteworthy protection on TBH-induced oxidative damage in a line of fibroblasts derived from monkey kidney (Vero cells) and in human intestinal epithelial cells (Caco-2), decreasing, in both cell lines, the formation of oxidative products (hydroperoxides and 7-ketocholesterol) from the degradation of unsaturated fatty acids and cholesterol. The cellular uptake and transepithelial transport of the compound were also investigated in Caco-2 cell monolayers. Arzanol appeared to accumulate in Caco-2 epithelial cells. This phenol was able to pass through the intestinal Caco-2 monolayers, the apparent permeability coefficients (P(app)) in the apical-to-basolateral and basolateral-to-apical direction at 2h were 1.93±0.36×10(-5) and 2.20±0.004×10(-5)cm/s, respectively, suggesting a passive diffusion pathway. The results of the work qualify arzanol as a potent natural antioxidant with a protective effect against lipid oxidation in biological systems.

Protective effect of simple phenols from extravirgin olive oil against lipid peroxidation in intestinal Caco-2 cells.

Complex polyphenols present in extravirgin olive oil are not directly absorbed, but undergo gastrointestinal biotransformation, increasing the relative amount of tyrosol (TYR) and hydroxytyrosol (HT) entering the small and large intestine. We investigated the capacity of TYR and HT to inhibit the insult of dietary lipid hydroperoxydes on the intestinal mucosa, using cultures of Caco-2, a cell line with enterocyte-like features, and studying the effect of tert-butyl hydroperoxide (TBH) treatment on specific cell membrane lipid targets. The effect of homovanillic alcohol (HVA), metabolite of HT in humans and detected as metabolite of HT in Caco-2 cells, was also evaluated. Exposure to TBH induced a significant increase of the level of MDA, the formation of fatty acid hydroperoxides and 7-ketocholesterol and the loss of α-tocopherol. Pretreatment with both HT and HVA protected Caco-2 cells from oxidative damage: there was no significant detection of oxidation products and the level of α-tocopherol was preserved. Noteworthy, TYR also exerted a protective action against fatty acids degradation. In vitro trials, where the simple phenols were tested during linoleic acid and cholesterol oxidation, gave evidence of a direct scavenging of peroxyl radicals and suggested a hydrogen atom-donating activity.

Manual orthodontic vs. oscillating-rotating electric toothbrush in orthodontic patients: a randomised clinical trial.

AIM: To compare the efficacy of manual and oscillating-rotating electric toothbrushes in removing plaque and reducing gingivitis in patients with fixed orthodontic appliances. STUDY DESIGN: Randomised clinical trial. MATERIALS AND METHODS: 20 subjects aged 10 to 14 years (8 males; 12 females) scheduled for fixed orthodontic treatment were enrolled, randomly divided into two groups, A and B, and later assigned electric or manual toothbrushes, respectively. Three months after orthodontic appliance bonding (T1), and again after a further 4 (T2) and 8 (T3) weeks, plaque levels (PI), gingival bleeding (GBI) and hypertrophy were assessed. RESULTS: Between the two toothbrush types, statistically significant differences in PI were detected from T2 to T3 (P value 0.010), but not from T1 to T2. In contrast, the GBI was significantly different from T1 to T2 (P value 0.025), but not from T2 to T3. CONCLUSION: With respect to the manual orthodontic toothbrush, the electric oscillating-rotating toothbrush was found to better improve both PI and GBI.

Involvement of ERK, Akt and JNK signalling in H2O2-induced cell injury and protection by hydroxytyrosol and its metabolite homovanillic alcohol.

The olive oil polyphenol, hydroxytyrosol (HT), is believed to be capable of exerting protection against oxidative kidney injury. In this study we have investigated the ability of HT and its O-methylated metabolite, homovanillic alcohol (HVA) to protect renal cells against oxidative damage induced by hydrogen peroxide. We show that both compounds were capable of inhibiting hydrogen peroxide-induced kidney cell injury via an ability to interact with both MAP kinase and PI3 kinase signalling pathways, albeit at different concentrations. HT strongly inhibited death and prevented peroxide-induced increases in ERK1/2 and JNK1/2/3 phosphorylation at 0.3 microM, whilst HVA was effective at 10 microM. At similar concentrations, both compounds also prevented peroxide-induced reductions in Akt phosphorylation. We suggest that one potential protective effect exerted by olive oil polyphenols against oxidative kidney cell injury may be attributed to the interactions of HT and HVA with these important intracellular signalling pathways.

Extra virgin olive oil phenolics: absorption, metabolism, and biological activities in the GI tract.

Olive oil, a typical ingredient of the Mediterranean diet, possesses many beneficial health effects. The biological activities ascribed to olive oil consumption are associated in part to its phenolics constituents, and mainly linked to the direct or indirect antioxidant activity of olive oil phenolics and their metabolites, which are exerted more efficiently in the gastrointestinal (GI) tract, where dietary phenolics are more concentrated when compared to other organs. In this regard, we present a brief overview of the metabolism, biological activities, and anticancer properties of olive oil phenolics in the GI tract.

Protective effect of hydroxytyrosol and tyrosol against oxidative stress in kidney cells.

Bioavailability studies in animals and humans fed with extravirgin olive oil demonstrated that hydroxytyrosol and tyrosol, the major simple phenolic compounds in extravirgin olive oil, are dose-dependently absorbed and excreted. Once absorbed, they undergo extensive metabolism; hydroxytyrosol and tyrosol concentrate mainly in the kidney, where they may exert an important role in the prevention of oxidative stress induced renal dysfunction. In this study we monitored the ability of hydroxytyrosol and tyrosol to protect renal cells (LLC-PK1) following oxidative damage induced by H2O2. Oxidative stress was evaluated by monitoring the changes of the membrane lipid fraction. Hydroxytyrosol exerted a significant antioxidant action, inhibiting the production of MDA, fatty acids hydroperoxides and 7-ketocholesterol, major oxidation products of unsaturated fatty acids and cholesterol, and thus protecting the cells from H2O2-induced damage. Tyrosol, instead, in this experimental model, did not exert any protective effect.