Erythrocyte viscoelastic recovery after liver transplantation in a cirrhotic patient affected by spur cell anaemia.

In physiological conditions, red blood cells (RBCs) are capable of dramatic deformations when passing through the microvasculature. This extreme deformability is closely related to the RBC biconcave shape, to the fluidic nature of the haemoglobin and the cell membrane structure, primarily consisting of a phospholipid bilayer with an underlying two-dimensional spectrin network. In many pathological and inflammatory conditions, the shape and the extreme deformability of erythrocytes appear to be significantly altered. These findings have stimulated intense research towards the search and validation of novel erythrocyte-based mechanical biomarkers, useful for disease diagnosis and therapy monitoring. In this study, we investigated with Atomic Force Microscopy (AFM) the mechanical properties of erythrocytes obtained from a 68 years old cirrhotic man diagnosed with spur cell anaemia and cold agglutinated disease, before and after liver transplantation. Mechanical changes are compared with ultrastructural alterations as studied by scanning electron microscopy and discussed according to confocal fluorescence microscopy results, showing possible alterations induced by the cirrhotic environment at the level of the RBCs cytoskeletal organisation and lipidic composition. Taken together, the results here presented show that liver transplantation not only contributes to restoring the proper RBC morphology, but it also induces recovery of the physiological viscous behaviour of cells, further stressing the relevance of viscous and dissipative forces in determining the RBC biomechanical response.

A simple cell proliferation assay and the inflammatory protein content show significant differences in human plasmas from young and old subjects.

Some studies showed a "rejuvenating" effect of exposing aging tissues to a young environment. In mouse heterochronic parabiosis experiments, in response to young organisms, old animals lived longer than isochrony old age-matched conjoint animals. Comparable "rejuvenating" effects were obtained by injecting young plasma in old mice. This raised great hopes of slowing down the senescence process in humans by the injection of young plasma, as well as to prevent or cure age-related diseases. Some clinical trials are currently being performed or were recently completed. However, these studies are small and of limited duration, and we still lack convincing evidence to support the effectiveness of young plasma injection. It is urgent to perform additional investigations, including the development of an assay to measure the cell proliferation induction capability of different human plasmas, before one can seriously think of a large-scale treatment of humans. We adopted a simple method to measure the potential of different plasmas in supporting cell line proliferation, regardless of the co-presence of a platelet lysate. By comparing plasmas from young and old subjects, we observed a decreased activity in plasmas from old individuals. The young plasma effect may be attributed to specific proteins and growth factors more abundant in younger individuals that could decrease with age. Alternatively, or at the same time, the reduced cell proliferation support could be due to inhibitors present in the old plasma. Studying the different protein content of young and old plasmas was out of the scope of this article. Such differences should be adequately investigated by proteomics using many samples. However, a preliminary study of the different protein content of young and old plasmas was part of the assay validation using a commercially available cytokine array for parallel determination of the relative levels of 105 selected human proteins. We could show the existence of specific differences between young and old plasmas and that plasmas from old individuals presented a higher concentration of "inflammatory" proteins.

VMAT-like plans for magnetic resonance guided radiotherapy: Addressing unmet needs.

PURPOSE: VMAT delivery technique is currently not applicable to Magnetic Resonance-guided radiotherapy (MRgRT) hybrid systems. Aim of this study is to evaluate an innovative VMAT-like (VML) delivery technique. MATERIAL AND METHODS: First, planning and dosimetric evaluation of the MRgRT VML treatment have been performed on 10 different disease sites and the results have been compared with the corresponding IMRT plans. Then, in the second phase, 10 of the most dosimetrically challenging locally advanced pancreas treatment plans have been retrospectively re-planned using the VML approach to explore the potentiality of this new delivery technique. Finally, VML robustness was evaluated and compared with the IMRT plans, considering a lateral positioning error of ± 5 mm. RESULTS: In phase one, all VML plans were within constraint for all OARs. When PTV coverage is considered, in the 50% of the cases VML PTV coverage is equal or higher than in IMRT plan. In the remaining 50%, the highest target under coverage difference in comparison with IMRT plan is -1.71%. The mean and maximum treatment time differences (VML-IMRT) is 0.2 min and 3.1 min respectively. In phase two, the treatment time variation (VML-IMRT), shows a mean, maximum and minimum variations of 1.3, 4.6 and -0.6 min respectively. All VML plans have a better target coverage if compared with IMRT plans, keeping in any case the OARs constraints within tolerance. VML doesn't increase plan robustness. CONCLUSION: VMAT-like treatment approach appeared to be an efficient planning solution and it was decided to clinically implement it in daily practice, especially in the frame of hypo fractionated treatments.

Dosimetric accuracy of dual isocenter irradiation in low magnetic field resonance guided radiotherapy system for extended abdominal tumours.

PURPOSE: Due to limited field size of Magnetic Resonance Linear Accelerators (MR-Linac), some treatments could require a dual-isocenter planning approach to achieve a complete target coverage and thus exploit the benefits of the online adaptation. This study evaluates the dosimetric accuracy of the dual-isocenter intensity modulated radiation therapy (IMRT) delivery technique for MR-Linac. MATERIAL AND METHODS: Dual-isocenter multi leaf collimator (MLC) and couch accuracy tests have been performed to evaluate the delivery accuracy of the system. A mono-isocenter plan delivered in clinical practice has then been retrospectively re-planned with dual-isocenter technique. The dual-isocenter plan has been re-calculated and delivered on a 3-dimensional (3D) ArcCHECK phantom and 2-dimensional (2D) films to assess its dosimetric accuracy in terms of gamma analysis. Clinical and planning target volume (CTV and PTV respectively) coverage robustness was then investigated after the introduction of ± 2 mm and ± 5 mm positioning errors by shifting the couch. RESULTS: MLC and couch accuracy tests confirmed the system accuracy in delivering a dual-isocenter irradiation. 2D/3D gamma analysis results occurred always to be above 95% if considered a gamma criteria 1%/2 mm and 1%/1 mm respectively for the 2D and 3D analysis. The mean variations for CTV D98% and PTV V95% were 0.2% and 1.1% respectively when positioning error was introduced separately in each direction, while the maximum observed variations were 0.9% (CTV) and 3.7% (PTV). CONCLUSION: The dosimetric accuracy of dual-isocenter irradiation has been verified for MR-Linac, achieving accurate and robust treatment strategy and improving dose conformality also in presence of targets whose extension exceeds the nominal maximum field size.

When your MR linac is down: Can an automated pipeline bail you out of trouble?

PURPOSE: The unique treatment delivery technique provided by magnetic resonance guided radiotherapy (MRgRT) can represent a significant drawback when system fail occurs. This retrospective study proposes and evaluates a pipeline to completely automate the workflow necessary to shift a MRgRT treatment to a traditional radiotherapy linac. MATERIAL AND METHODS: Patients undergoing treatment during the last MRgRT system failure were retrospectively included in this study. The core of the proposed pipeline was based on a tool able to mimic the original MR linac dose distribution. The so obtained dose distribution (AUTO) has been compared with the distribution obtained in the conventional radiotherapy linac (MAN). Plan comparison has been performed in terms of time required to obtain the final dose distribution, DVH parameters, dosimetric indices and visual analogue scales scoring by radiation oncologists. RESULTS: AUTO plans generation has been obtained within 10 min for all the considered cases. All AUTO plans were found to be within clinical tolerance, showing a mean target coverage variation of 1.7% with a maximum value of 4.3% and a minimum of 0.6% when compared with MAN plans. The highest OARs mean variation has been found for rectum V60 (6.7%). Dosimetric indices showed no relevant differences, with smaller gradient measure in favour of AUTO plans. Visual analogue scales scoring has confirmed comparable plan quality for AUTO plans. CONCLUSION: The proposed workflow allows a fast and accurate generation of automatic treatment plans. AUTO plans can be considered equivalent to MAN ones, with limited clinical impact in the worst-case scenario.

Population receptive field tuning properties of visual cortex during childhood.

Visuospatial abilities such as contrast sensitivity and Vernier acuity improve until late in childhood, but the neural mechanisms supporting these changes are poorly understood. We tested to which extent this development might reflect improved spatial sensitivity of neuronal populations in visual cortex. To do this, we measured BOLD-responses in areas V1-V4 and V3a, whilst 6- to 12-year-old children and adults watched large-field wedge and ring stimuli in the MRI scanner, and then fitted population receptive field (pRF) tuning functions to these data (Dumoulin and Wandell, 2008). Cortical magnification and pRF tuning width changed with eccentricity at all ages, as expected. However, there were no significant age differences in pRF size, shape, cortical magnification, or map consistency in any visual region. These findings thus strongly suggest that spatial vision in late childhood is not substantially limited by the spatial tuning of neuronal populations in early visual cortex. Instead, improvements in performance may reflect more efficient read-out of spatial information in early visual regions by higher-level processing stages, or prolonged tuning to more complex visual properties such as orientation. Importantly, this in-depth characterisation of the pRF tuning profiles across childhood, paves the way for in-vivo-testing of atypical visual cortex development and plasticity.

Effect of coffee drinking on platelets: inhibition of aggregation and phenols incorporation.

Epidemiological studies indicate a J-shaped relationship linking coffee consumption and cardiovascular risk, suggesting that moderate coffee consumption can be beneficial. Platelet aggregation is of critical importance in thrombotic events, and platelets play a major role in the aetiology of several CVD. The aim of this study was to evaluate the effect of coffee drinking on platelet aggregation ex vivo, using caffeine as control. A crossover study was performed on ten healthy subjects. In two different sessions, subjects drank 200 ml coffee, containing 180 mg caffeine, or a capsule of caffeine (180 mg) with 200 ml water. Platelets were separated from plasma at baseline and 30 and 60 min after coffee drinking. Platelet aggregation was induced with three different agonists: collagen, arachidonic acid and ADP. Coffee drinking inhibited collagen (P < 0.05 from baseline at time 30 min) and arachidonic acid (P < 0.05 from baseline at time 60 min) induced platelet aggregation. Caffeine intake did not affect platelet aggregation induced by the three agonists. Coffee consumption induced a significant increase of platelet phenolic acids (likely present as glucuronate and sulphate derivatives), caffeic acid, the principal phenolic acid in coffee, raising from 0.3 (SEM 0.1) to 2.4 (SEM 0.6) ng/mg (P < 0.01). Caffeine was not detectable in platelets. Coffee drinking decreases platelet aggregation, and induces a significant increase in phenolic acid platelet concentration. The antiplatelet effect of coffee is independent from caffeine and could be a result of the interaction of coffee phenolic acids with the intracellular signalling network leading to platelet aggregation.

Alpha/beta hydrolase fold enzymes: the family keeps growing.

The alpha/beta hydrolase fold is a typical example of a tertiary fold adopted by proteins that have no obvious sequence similarity, but nevertheless, in the course of evolution, diverged from a common ancestor. Recently solved structures demonstrate a considerably increased variability in fold architecture and substrate specificity, necessitating the redefinition of the minimal features that distinguish the family.

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.

Interaction of pantetheinase with sulfhydryl reagents and disulfides.

The effect of many thiol reagents and disulfides on pantetheinase (E.C. 3.5.1.-; pantetheine hydrolase) was studied in the presence or absence of S-pantetheine-3-pyruvate as substrate. Iodoacetamide, iodoacetate, bromopyruvate and N-ethylmaleimide irreversibly inactivate the enzyme at very different rates. Inactivation constants, corrected for the different reactivity of halogeno derivatives with non-protein thiols, suggest the presence of an essential sulfhydryl group in the enzyme and a negatively charged environment near this group. p-Chloromercuribenzoate is the most effective inhibitor; 2-nitro-5-thiocyanobenzoate, o-iodosobenzoate and hydrogen peroxide give a biphasic inhibition pattern, indicating the existence of two sulfhydryl groups whose modification affects activity. Organic arsenicals decrease activity to about 50%. Neutral and positively charged disulfides are effective inhibitors. Substrate protects the enzyme from inactivation, except in the case of negatively charged disulfides, where the presence of substrate enhances the inhibitory effect. Titration with Ellman's reagent or 4,4'-dithiodipyridine under various experimental conditions demonstrated the existence of two sulfhydryls and three disulfides in the fully active enzyme. Pantetheinase may become inactive during purification with concomitant loss of one titrable sulfhydryl group.

Bovine brain ketimine reductase.

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase.

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).

Detection of 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine) in the bovine brain by a fluorometric assay.

A new sulfur imino acid, 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine), has been detected in the bovine brain by means of fluorometric and HPLC procedures. The fluorometric assay is based on the fluorescent property of the copper-ketimine interaction product at pH 11.5. Other ketimines do not fluoresce in these conditions. The fluorophore exhibits an excitation maximum at 353 nm and an emission at 462 nm and is stable for at least 24 h. In the test conditions the fluorescence is proportional to the ketimine concentration from 1 to 200 microM. Detection of endogenous lanthionine ketimine has been performed after a simple enrichment procedure (brain deproteinization and extraction with diethyl ether) which minimizes degradative by-reactions of the unstable ketimine. The concentration of this new sulfur imino acid in the brain ranges from 0.5 to 1 nmol/g in three different samples. Identification and quantitations were confirmed by an HPLC procedure which takes advantage of the selective absorption at 380 nm of the phenylisothiocyanate-ketimine adduct. The identification of lanthionine ketimine in nervous tissues may have important metabolic and physiological implications.

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.

Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study.

Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site".

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene.

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

Directed evolution of an enantioselective lipase.

BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity. RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions. CONCLUSIONS: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.

Modulation of ceramide-induced NF-kappaB binding activity and apoptotic response by caffeic acid in U937 cells: comparison with other antioxidants.

Ceramide acts as second messenger in the signal transduction triggered by a variety of stress stimuli and extracellular agents. Stress response through ceramide is involved in the development of many human diseases, such as atherosclerosis, inflammation, neurodegenerative disorders, and acquired immunodeficiency syndrome. Dietary polyphenols have been reported to exert a beneficial effect on the onset and development of most of these human chronic-degenerative pathologies. However, the mechanisms underlying this beneficial effect are mostly not understood at the present. To investigate the ability of polyphenols in modulating fundamental cellular functions, we studied the effect of caffeic acid, a widespread phenolic acid largely present in human diet, in the modulation of ceramide-induced signal transduction pathway leading to apoptosis in U937 cells, in comparison with other established antioxidants of nutritional interest (N-acetylcysteine, d-alpha-tocopherol acetate and ascorbic acid). Our results indicate that caffeic acid efficiently inhibits both ceramide-induced NF-kappaB binding activity and apoptosis at micromolar concentration. Other antioxidants tested are totally ineffective in inhibiting apoptosis, although affecting NF-kappaB activation. Caffeic acid was found to inhibit protein tyrosine kinase activity, suggesting that this mechanism can be on the basis of the inhibition of apoptosis. Our results suggest that dietary caffeic acid might modulate ceramide-induced signal transduction pathway and NF-kappaB activation through either antioxidant and nonantioxidant mechanisms.

Inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivatives.

The antioxidant activity of the major phenols derived from hydroxycinnamic acid (caffeic, ferulic, and p-coumaric acids) on in vitro LDL oxidation was screened, using Cu2+ as catalyst. The presence of the second phenolic hydroxy group enhanced the inhibitory effect of these compounds. In fact, at 5 microM concentration, only caffeic acid completely protected LDL from modification as measured as conjugated dienes formation and apo B-100 fragmentation, also preserving alpha-tocopherol. The effect of caffeic acid in inhibiting LDL oxidative modification induced by three different oxidant systems was tested. Using both Cu2+ and 2,2'-azobis (2-amidinopropane)-hydrochloride (AAPH), the inhibitory effect of caffeic acid was dose-dependent. Yet, the better protection was achieved in the metal-ion dependent system. Also the murine macrophages-mediated LDL oxidation was efficiently inhibited by 5 microM caffeic acid. UV-VIS spectra of caffeic acid incubated with cupric ions show the formation of a caffeic acid:copper complex, responsible for a transient chelating activity. This mechanism, coupled with its free radical scavenging property, accounts for the higher inhibitory activity exhibited by caffeic in Cu(2+)-catalyzed reaction.

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937.

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.