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Biomedical subjects

Mauro Picardo

Publications and source records attributed to Mauro Picardo.

36 records · Page 2Linked to original sources
PubMed2004

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry.

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.

Ceramides↗
PubMed2004

Analysis of APAF-1 expression in human cutaneous melanoma progression.

APAF-1 plays a pivotal role in mitochondria-dependent apoptosis, binding to cytochrome c and favoring activation of caspase-9. It has been shown that epigenetic silencing of the APAF-1 gene is a common event in several metastatic melanoma cells in vitro. We determined, by Western blot, variation in the level of expression of APAF-1 in several human melanoma cell lines and, by immunohistochemistry, in a group of 106 histological samples including benign and malignant melanocytic lesions. We observed APAF-1 down-regulation or loss of expression in two metastatic melanoma cell lines, compared to primary melanoma cell lines. The immunohistochemical analysis revealed a significant difference in APAF-1 staining between nevi and melanomas. In addition, we found a significant negative correlation between APAF-1 expression level and tumor thickness and between primary melanomas and metastases. We conclude that loss of APAF-1 expression can be considered as an indicator of malignant transformation in melanoma.

Apoptosis↗
PubMed2003

UVB-induced activation and internalization of keratinocyte growth factor receptor.

Ultraviolet irradiation of mammalian cells induces several events that include activation of growth factor receptors and triggering of signal transduction pathway. Most of the UV responses are mediated by the production of reactive oxygen species (ROS) and can be blocked by antioxidants. In this study, we analysed the effect of UVB irradiation at physiologic doses and that of the pro-oxidant agent cumene hydroperoxide (CUH) on the activation of the receptor for keratinocyte growth factor (KGF), a key mediator of epithelial growth and differentiation. Exposure to both UVB (30-150 mJ/cm(2)) and CUH (200 microM of NIH3T3 KGFR (KGF receptors) transfectants caused a rapid tyrosine phosphorylation and activation of KGFR similar to that induced by KGF, and internalization of the activated receptor. The KGFR expression appeared unmodified by the treatments. Ultrastructural observations of both UVB- and CUH-treated cells showed a normal morphology of the plasma membranes and intracellular organelles. The antioxidant N-acetylcysteine inhibited UVB-induced receptor phosphorylation. The generation of an intracellular oxidative stress was detected as a decrease of catalase activity and of vitamin E, and reduced glutathione levels, whereas superoxide dismutase activity was not significantly modified. A peroxidation of polyunsaturated fatty acids of cell membranes was observed after both treatments, associated with the intracellular oxidative stress. Similar biochemical events were observed on NIH3T3 untransfected control cells, suggesting that KGFR activation follows intracellular generation of ROS and is not associated with a scavenging effect. Taken together our results demonstrate that exposure to UVB and to oxidant stimuli induces a rapid intracellular production of ROS, which in turn are capable of triggering KGFR activation and internalization, similar to those induced by KGF.

3T3 Cells↗
PubMed2003

Ni2+ enhances Fe2+/peroxide-induced oxidation of arachidonic acid and formation of geno/cytotoxic 4-hydroxynonenal: a possible contributory mechanism in nickel toxicity and allergenicity.

Ni(2+), a toxic, carcinogenic and allergenic agent, affected both the kinetic and chemical courses of the Fe(2+)-induced oxidation of arachidonic acid (AA) in 0.05 M phosphate buffer (pH 7.4) and at 37 degrees C. At 10 microM concentration, Ni(2+) decreased the rate of oxidation of peroxide-free AA (200 microM) promoted by 50 microM Fe(2+), as determined by measurement of thiobarbituric acid reactive species (TBARS) and 1H NMR analysis. However, in the presence of low levels of peroxides (e.g. 2%), Ni(2+) exerted a significant stimulatory effect on Fe(2+)-induced AA oxidation and TBARS formation. 1H NMR analysis showed that Ni(2+) (10 microM) enhanced formation of genotoxic alkenals including 4-hydroxy-2-nonenal (4-HNE, GC/MS evidence) by Fe(2+)-promoted degradation of both AA and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) methyl esters. The observed stimulatory effects of Ni(2+) on peroxide breakdown and cytotoxic aldehyde formation provide an attractive explanation to the enhanced sensitization capacity of nickel in inflammatory states compared to normal states.

Aldehydes↗
PubMed2003

Alpha-tocopherol protects against cisplatin-induced toxicity without interfering with antitumor efficacy.

Our aim was 2-fold: to investigate the role of alpha-tocopherol supplementation on the antitumor activity of DDP and to evaluate the effect of alpha-tocopherol on the survival and neurotoxicity of DDP-treated mice. Experiments performed on the M14 human melanoma line demonstrated that alpha-tocopherol supplementation did not influence the efficacy of DDP; the inhibition of cell survival and of the in vivo tumor growth after treatment with alpha-tocopherol and DDP combination was similar to that observed after DDP alone. Conversely, alpha-tocopherol was also able to increase survival of mice treated with a high dose of DDP. While DDP alone produced death in about 70% of mice, the combination reduced deaths to about 30%. Analysis of oxidative stress markers and peroxidative damage in organs indicated that the protective effect of alpha-tocopherol was mainly related to its antioxidant activity. A significant increase in the concentration of TBARS and decreased PUFAs and catalase activity were observed after DDP treatment, while with alpha-tocopherol the levels of these markers were comparable to those observed in untreated mice. Histologic analysis performed on peripheral nerve revealed that alpha-tocopherol also protected mice from severe neurologic damage induced by DDP treatment. In conclusion, our results demonstrate that alpha-tocopherol protects against the systemic toxicity and neurotoxicity induced by DDP without interfering with its antitumor activity and suggest that this combination is a promising strategy to improve the therapeutic index of DDP-based chemotherapy.

Animals↗
PubMed2003

Neuroprotective effect of vitamin E supplementation in patients treated with cisplatin chemotherapy.

PURPOSE: The aim of this study is to evaluate the neuroprotective effect of antioxidant supplementation with vitamin E in patients treated with cisplatin chemotherapy. METHODS: Between April 1999 and October 2000, forty-seven patients were randomly assigned to either group one, which received vitamin E supplementation during cisplatin chemotherapy, or to group two, which received cisplatin chemotherapy alone. Alpha-tocopherol (vitamin E; 300 mg/d) was administered orally before cisplatin chemotherapy and continued for 3 months after the suspension of treatment. For preclinical studies, nude mice carrying the human melanoma tumor were treated with cisplatin alone or in combination with vitamin E. RESULTS: Twenty-seven patients completed six cycles of cisplatin chemotherapy: 13 patients in group one and 14 patients in group two. The incidence of neurotoxicity was significantly lower in group one (30.7%) than it was in group two (85.7%; P <.01). The severity of neurotoxicity, measured with a comprehensive neurotoxicity score based on clinical and neurophysiological parameters, was significantly lower in patients who were supplemented with vitamin E than in patients who were not supplemented with vitamin E (2 v 4.7, P <.01). The results of the preclinical studies showed that when cisplatin was combined with vitamin E, no differences were observed in tumor weight inhibition, tumor growth delay, or life span as compared with treatment with cisplatin alone. CONCLUSION: Supplementation of patients receiving cisplatin chemotherapy with vitamin E decreases the incidence and severity of peripheral neurotoxicity.

Adult↗
PubMed2003

Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expression.

Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5-bromo-2'-deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel-induced cell growth appeared enhanced when primary human keratinocytes were co-cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up-modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor-alpha (TGF-alpha) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up-modulation could be required for the nickel-induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment.

Cell Division↗
PubMed2003

Identification of genes down-regulated during melanoma progression: a cDNA array study.

In order to identify genes relevant for melanoma development, we carried out cDNA array experiments employing an in vitro model of human melanoma progression, consisting of two cell lines: one, LP, derived from a primary melanoma and the other, LM, from its metastatic supraclavicular lymph node. Basic cDNA array data identified 26 genes as down-regulated in the LM cell line. Northern blot analysis confirmed an effective transcriptional down-regulation for five out of 13 genes analyzed. The products of these five genes belong to different functional protein types, such as transcription and translation regulators (Edg-2, eIF-3 p110, and RNPL/RBM3), extracellular communicators (PRSS11) and members of the major histocompatibility complex (beta2-microglobulin). Some previously described differences in expression patterns, such as loss of HLA I, were confirmed by our array data. In addition, we identified and validated for the first time the reduced expression level of several genes during melanoma progression. In particular, reduced Edg-2 gene product expression was also confirmed in a group of 50 primary melanomas and unrelated metastases. In conclusion, comparative hybridization by means of cDNA arrays assisted in identifying a series of novel progression-associated changes in gene expression, confirming, at the same time, a number of previously described results.

Cell Line, Tumor↗
PubMed2003

Chemical and instrumental approaches to treat hyperpigmentation.

Many modalities of treatment for acquired skin hyperpigmentation are available including chemical agents or physical therapies, but none are completely satisfactory. Depigmenting compounds should act selectively on hyperactivated melanocytes, without short- or long-term side-effects, and induce a permanent removal of undesired pigment. Since 1961 hydroquinone, a tyrosinase inhibitor, has been introduced and its therapeutic efficacy demonstrated, and other whitening agents specifically acting on tyrosinase by different mechanisms have been proposed. Compounds with depigmenting activity are now numerous and the classification of molecules, based on their mechanism of action, has become difficult. Systematic studies to assess both the efficacy and the safety of such molecules are necessary. Moreover, the evidence that bleaching compounds are fairly ineffective on dermal accumulation of melanin has prompted investigations on the effectiveness of physical therapies, such as lasers. This review which describes the different approaches to obtain depigmentation, suggests a classification of whitening molecules on the basis of the mechanism by which they interfere with melanogenesis, and confirms the necessity to apply standardized protocols to evaluate depigmenting treatments.

Animals↗
PubMed2003

Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients.

The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients.

Adult↗
PubMed2003

In vitro effects of nickel-sulphate on immune functions of normal and nickel-allergic subjects: a regulatory role for zinc.

Nickel hypersensitivity represents a very common human disease state, mainly occurring in females, defined as allergic contact dermatitis. Ni is a transition metal whose activity may be modulated by congeners. Zinc, an essential component for living organisms, has been shown to counteract Ni effects in patients with Ni hypersensitivity. We analysed immune responses to both Ni and Zn in healthy subjects and patients with allergic contact dermatitis to Ni. Our in vitro results show that Ni modulates surface receptors expression, reduces phytohemagglutinin (PHA)-driven lymphoproliferation, and upregulates some proinflammatory cytokines production, including interferon (IFN)-gamma. Zn also induced CD4+ lymphocyte proliferation, but it abolished or reduced most Ni-mediated effects. Our data are consistent with the hypothesis that Zn and Ni, as part of the heavy transition metals, may exchange roles in immune-mediated phenomena leading to expression of allergic contact dermatitis.

Adult↗
PubMed2002

Analytical methods to investigate glutathione and related compounds in biological and pathological processes.

Reduced glutathione (GSH, gamma-L-glutamyl-L-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in studying oxidative stress. Many separative and non-separative methods have been developed and improved for the assay of GSH and related compounds. At present, high-performance liquid chromatography and capillary electrophoresis are the most used separative techniques to determine GSH and congeners. The review will deal with analytical methods developed over the last few years for the determination of GSH and related compounds, and with the procedures performed in sample pre-treatment in order to minimize analytical errors. Since GSH, GSSG, and related compounds lack of strong chromophores or fluorophores, it is advantageous, in many assays, to derivatize the compounds in order to improve the detection limit with UV-Vis and to allow fluorescence, thus the most commonly used labeling agents are also described.

Chromatography↗
PubMed2002

Separation of delta-, gamma- and alpha-tocopherols by CEC.

In this study capillary electrochromatography (CEC) was used for the separation of three tocopherols (TOHs), namely delta-, gamma- and alpha-TOH and the antioxidant compound, butylated hydroxytoluene (BHT). The CEC experiments were carried out using an octadecylsilica (ODS) stationary phase packed, in our laboratory, in a fused-silica capillary (100 microm I.D., 365 microm O.D. x 33 cm of total length and 24.6 or 8.4 cm effective length). The mobile phase was composed by a mixture of methanol (MeOH) and acetonitrile (ACN), at different concentrations and 0.01% (w/v) of ammonium acetate. Retention time (t(R)), retention factor (k), resolution (R(s)) of the three TOHs were strongly influenced by the organic solvent composition of the run buffer and by the effective length of the capillary. Optimum experimental conditions were found even employing the short effective length of the capillary achieving the baseline separation of the studied analytes in a relatively short time (less than 5 min). The optimized method was applied to the qualitative analysis of vitamin E (alpha-TOH) present in a human serum extract.

Antioxidants↗
PubMed2002

The endocytic pathway followed by the keratinocyte growth factor receptor.

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.

3T3 Cells↗
PubMed2002

Reactions of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoctadeca-9,11-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.

13(S)-hydroperoxy- and 13(S)-hydroxyoctadeca-9,11-dienoic acids (1a/b), 15(S)-hydroperoxy- and 15(S)-hydroxyeicosa-5,8,11,13-tetraenoic acids (2a/b), and their methyl esters reacted smoothly with NO2- in phosphate buffer at pH 3-5.5 and at 37 degrees C to afford mixtures of products. 1b methyl ester gave mainly the 9-nitro derivative 3b methyl ester (11% yield) and a peculiar breakdown product identified as the novel 4-nitro-2-oximinoalk-3-enal derivative 4 methyl ester (15% yield). By GC-MS hexanal was also detected among the products. Structures 3b and 4 methyl esters were secured by 15N NMR analysis of the products prepared from 1b methyl ester upon reaction with Na15NO2. 4 methyl ester (14% yield) was also obtained from 1a methyl ester along with the nitrated hydroperoxy derivative 3a methyl ester (10% yield). Under the same conditions, 2a/b methyl esters gave mainly the corresponding nitrated derivatives 5a/b, with no detectable breakdown products, whereas the model compound (E,E)-2,4-hexadienol (6) afforded two main nitrated derivatives identified as 7 and 8. A reaction pathway for 1a/b methyl esters was proposed involving conversion of nitronitrosooxyhydro(pero)xy intermediates which would partition between two competing routes, viz., loss of HNO2, to give 3a/b methyl esters, and a remarkably facile fission leading to 4 methyl ester and hexanal.

Annexins↗
PubMed2002

Epidermal reconstructs: a new tool to study topical and systemic photoprotective molecules.

In this study, we compared the effects of sunscreens and antioxidants on reconstructed epidermis made with or without melanocytes 24 h after UVB, UVA or UVA+B irradiation. For this purpose, we studied sunburn cells and cyclobutane pyrimidine dimer formation, protein and lipid oxidation, catalase and superoxide dismutase activities and vitamin E levels. Topical sunscreens protected against direct cell death and thymine dimer formation whereas their protective effect against protein and lipid oxidation and antioxidant depletion was less marked partly due to the difficulty of spreading the cream. Antioxidant molecules protected against direct cell death and protein oxidation but not against thymine dimer formation. Since topical sunscreens and systemic antioxidant protected the skin differently, we speculate that their association might protect more efficiently against UV-induced damage. This model is relevant to study systemic molecules but is less practical, due to the technical limitations of studying topical molecules.

Adult↗
PubMedDate not supplied

A comparative non randomized study of narrow-band (NB) (312 +/- 2 nm) UVB phototherapy versus sequential therapy with oral administration of low-dose Cyclosporin A and NB-UVB phototherapy in patients with severe psoriasis vulgaris.

Sequential therapy may improve the efficacy and safety of single treatments for severe psoriasis. We compared sequential Cyclosporin A and narrow-band UVB phototherapy versus narrow-band UVB phototherapy alone. A group of 30 patients with severe psoriasis received 3 mg/kg/day Cyclosporin A for 4 weeks. Afterwards, Cyclosporin A was rapidly tapered and phototherapy begun. An additional 30 patients received phototherapy alone. Treatments were given until psoriasis cleared or until partial improvement was achieved without further amelioration despite another week of treatment. Both treatments were highly effective and well tolerated but sequential therapy was more effective in lesions of UV-shielded body areas; itching disappeared more quickly. The cumulative narrow-band UVB dosages and number of exposures were lower. No difference was seen at follow-up extended up to 9 months. Sequential therapy was well tolerated and allowed for the reduction of narrow-band UVB dosage and exposures, quick relief of itching and improvement of UV-shielded lesions.

Administration, Oral↗