Biomedical subjects

Mauro Picardo

Publications and source records attributed to Mauro Picardo.

At least 19 recordsLinked to original sources

PubMed2006

Rab33A: characterization, expression, and suppression by epigenetic modification.

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

Amino Acid Sequence↗

PubMed2006

Peroxidated squalene induces the production of inflammatory mediators in HaCaT keratinocytes: a possible role in acne vulgaris.

In order to investigate whether products derived from the oxidation of sebum can be responsible for the induction of inflammatory processes, HaCaT keratinocytes were treated with peroxidated squalene. NF-kappaB activation, secretion, and expression of IL-6, as well as peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA and protein levels, were measured at the end of the treatment and after 24 and 48 hours of recovery. Squalene peroxidation products were administered in amounts able to elicit significant hyperproliferation and to induce lipoxygenase (LOX) activity. The results showed an early activation of NF-kappaB followed by an increase in PPARalpha mRNA and protein levels. Moreover, squalene peroxides induced an initial upregulation of IL-6 production and secretion that was counteracted by PPARalpha activation, as suggested by the subsequent decrease of NF-kappaB nuclear translocation and IL-6 levels. Inflammatory processes play an important role in the development of acne vulgaris. In combination with our own previous findings, which indicated an association between LOX stimulation and increased percentage of proinflammatory lipids in acne as well as a correlation between increased cytokine levels in the infundibulum, pilosebaceous duct hyperkeratinization, and augmented sebogenesis, the present data further support the involvement of lipid peroxides, in particular squalene peroxides, in establishing an inflammatory process in acne.

Acne Vulgaris↗

PubMed2006

Activating PTPN11 mutations play a minor role in pediatric and adult solid tumors.

The PTPN11 gene encodes SHP-2, a widely expressed cytoplasmic protein tyrosine phosphatase functioning as a signaling transducer. Germ-line PTPN11 mutations cause Noonan syndrome (NS), a developmental disorder characterized by an increased risk of malignancies. Recently, a novel class of activating mutations in PTPN11 has been documented as a somatic event in a heterogeneous group of leukemias. Because of the relatively higher prevalence of certain solid tumors in children with NS and the positive modulatory function of SHP-2 in RAS signaling, a wider role for activating PTPN11 mutations in cancer has been hypothesized. Here, we screened a number of solid tumors, including those documented in NS or in which deregulated RAS signaling occurs at significant frequency, for PTPN11 mutations. No disease-associated mutation was identified in rhabdomyosarcoma (n = 13), neuroblastoma (n = 32), melanoma (n = 50), thyroid (n = 85), and colon (n = 48) tumors; a novel missense change, promoting an increased basal phosphatase activity of SHP-2, was observed in one glioma specimen. Our data document that deregulated SHP-2 function does not represent a major molecular event in pediatric and adult tumors, further supporting our previous evidence indicating that the oncogenic role of PTPN11 mutations is cell-context specific.

Adult↗

PubMed2006

Ferritin light chain down-modulation generates depigmentation in human metastatic melanoma cells by influencing tyrosinase maturation.

Recently, after the identification of ferritin light chain (L-ferritin) gene and protein over-expression in human metastatic melanoma cells, we engineered, starting from the LM metastatic melanoma cell line, clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. The present investigation started from the observation that L-ferritin down-regulated LM cells displayed a less pigmented phenotype, confirmed by a major decrease of total melanin, when compared to control LM cells. This finding was accompanied by a dramatic decrease in tyrosinase activity, which was not paralleled by a concomitant reduction of the amount of tyrosinase specific mRNA. Western blot analysis of tyrosinase in control LM cells displayed a pattern, which corresponds to the progressive glycosylation of the native protein up to the 80 kDa form, considered the functional one. Tyrosinase pattern assayed in L-ferritin down-regulated LM cells showed the remarkable absence of the 80 kDa form and a prevalence of endoglycosidase H (endo H)-sensitive immature (70 kDa) tyrosinase, accumulated in the endoplasmic reticulum (ER), as confirmed by confocal microscopy analysis. These results demonstrate that, in a human metastatic melanoma cell line, the stress condition promoted by L-ferritin down-modulation, can substantially influence proper maturation of tyrosinase.

Cell Line, Tumor↗

PubMed2006

Treatment of melasma.

Treatment of melasma involves the use of a range of topical depigmenting agents and physical therapies. Varying degrees of success have been achieved with these therapies. The Pigmentary Disorders Academy (PDA) undertook to evaluate the clinical efficacy of the different treatments of melasma in order to generate a consensus statement on its management. Clinical papers published during the past 20 years were identified through MEDLINE searches and methodology and outcome assessed according to guidelines adapted from the US Preventive Services Task Force (USPSTF). The consensus of the group was that first-line therapy for melasma should consist of effective topical therapies, mainly fixed triple combinations. Where patients have either sensitivity to the ingredients or a triple combination therapy is unavailable, other compounds with dual ingredients (hydroquinone plus glycolic acid) or single agents (4% hydroquinone, 0.1% retinoic acid, or 20% azelaic acid) may be considered as an alternative. In patients who failed to respond to therapy, options for second-line therapy include peels either alone or in combination with topical therapy. Some patients will require therapy to maintain remission status and a combination of topical therapies should be considered. Lasers should rarely be used in the treatment of melasma and, if applied, skin type should be taken into account.

Humans↗

PubMed2006

UVA-induced modification of catalase charge properties in the epidermis is correlated with the skin phototype.

The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products.

Antioxidants↗

PubMed2006

Expression of keratinocyte growth factor and its receptor in clear cell acanthoma.

The aetiopathogenic mechanism underlying clear cell acanthoma (CCA) is not completely clear and it has been postulated that CCA and psoriasis may have a similar pathogenesis because of the common features shared by the two diseases. As it has been recently demonstrated that in psoriatic lesions the paracrine epithelial growth factors [keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)-7 and FGF-10] are involved in promoting and sustaining the keratinocyte hyperproliferation, the aim of this study was to analyse the expression of KGF on CCA lesions and to search for a role of this growth factor in CCA pathogenesis. Immunohistochemical analysis showed an up-modulation of KGF in CCA, although the immunostaining was variable among the different samples collected. Positive immunoreactivity for KGF was detected mainly on dermal areas where the inflammatory infiltrate was more pronounced suggesting a relationship between lymphocyte activation and KGF up-modulation. Real-time quantitative RT-PCR assay performed on mRNA extracted from formalin-fixed paraffin-embedded CCA and normal skin (NS) samples further demonstrated the overexpression of the KGF/FGF-7 gene in all CCA samples compared with NS. Moreover, the evaluation by immunohistochemistry of KGF receptor distribution, the high-affinity tyrosine kinase receptor for KGF, showed a down-modulation of this receptor, as previously reported in the presence of increased levels of KGF. Taken together these results suggest the inflammatory nature of CCA and further support the hypothesis that this disease may represent, like psoriasis, an inflammatory dermatosis in which KGF up-modulation may be responsible for keratinocyte hyperproliferation and may represent a new common feature of both diseases.

Acanthoma↗

PubMed2006

A review and a new hypothesis for non-immunological pathogenetic mechanisms in vitiligo.

Vitiligo is an acquired depigmenting disorder characterized by the loss of functioning epidermal melanocytes because of multifactorial and overlapping pathogenetic mechanisms. Besides the immunological approach, the study of the metabolic deregulations leading to toxic damage of the melanocytes appears to be more and more relevant. It was only last year that the first in vitro evidence supporting the link and the temporal sequence between the immune response and the cellular oxidative stress was provided, suggesting that the intrinsic damage of the melanocytes is primitive. What can be the guide line of the multiple altered metabolisms? A compromised membrane could render the cell sensitive to the external and internal agents differently, usually ineffective on the cell activity and survival. The primitive altered arrangement of the lipids may affect the transmembrane housing of proteins with enzymatic or receptorial activities, also conferring on them antigenic properties.

Animals↗

PubMed2006

Hypopigmenting agents: an updated review on biological, chemical and clinical aspects.

An overview of agents causing hypopigmentation in human skin is presented. The review is organized to put forward groups of biological and chemical agents. Their mechanisms of action cover (i) tyrosinase inhibition, maturation and enhancement of its degradation; (ii) Mitf inhibition; (iii) downregulation of MC1R activity; (iv) interference with melanosome maturation and transfer; (v) melanocyte loss, desquamation and chemical peeling. Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as this enzyme catalyses the rate-limiting step of pigmentation. Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce effects in clinical trials. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their efficacy and safety. Successful treatments need the combination of two or more agents acting on different mechanisms to achieve a synergistic effect. In addition to tyrosinase inhibition, other parameters related to cytotoxicity, solubility, cutaneous absorption, penetration and stability of the agents should be considered. The screening test system is also very important as keratinocytes play an active role in modulating melanogenesis within melanocytes. Mammalian skin or at least keratinocytes/melanocytes co-cultures should be preferred rather than pure melanocyte cultures or soluble tyrosinase.

Animals↗

PubMed2005

Endocytic pathways and biological effects induced by UVB-dependent or ligand-dependent activation of the keratinocyte growth factor receptor.

UVB exposure of epidermal cells is known to trigger early and late molecular pathways dependent on receptor tyrosine kinases and reactive oxygen species (ROS). We have recently reported that UVB irradiation induces tyrosine phosphorylation, kinase activation, and internalization of the receptor for the keratinocyte growth factor (KGFR), a paracrine mediator of epithelial growth, differentiation, and survival. Here we analyzed in more detail the UVB-induced endocytic pathway of KGFR and the role of KGFR activation and internalization in regulating UVB-promoted apoptosis and cell cycle arrest. Immunogold electron microscopy and confocal analysis revealed that the UVB-induced endocytosis of KGFR occurs through clathrin-coated pits and that the internalized receptors are sorted to the degradative route and reach the lysosomal compartment with a timing similar to that induced by their ligand KGF. Treatment with the anti-oxidant N-acetylcysteine inhibited KGFR endocytosis, suggesting that the receptor internalization is mediated by the intracellular production of ROS. The ligand-independent KGFR endocytic pathway induced by UVB requires receptor kinase activity and tyrosine phosphorylation and involves transient receptor ubiquitination. Inhibition of KGFR activity reduces both the KGF-mediated proliferative response and the UVB-promoted apoptotic cell death, indicating a different effect of ligand-induced and UVB-induced KGFR triggering. In addition, receptor internalization leads to protection from apoptosis caused by UVB exposure. Finally, we compared directly the behavior of KGFR with that of the epidermal growth factor receptor (EGFR) upon UVB exposure. Surprisingly, biochemical and immunofluorescence analysis showed that EGFR, differently from KGFR, does not undergo UVB-induced tyrosine phosphorylation and internalization. Taken together, our results suggest a differential role of KGFR and EGFR in the response of epidermal cells to UVB possibly because KGFR endocytosis could be crucial for attenuation of survival signals in the suprabasal layers of human skin.

Animals↗

PubMed2005

Differential in vitro cellular response induced by exposure to synthetic vitreous fibers (SVFs) and asbestos crocidolite fibers.

In this study, we analyzed the effects of synthetic vitreous fibers (SVFs) on a mesothelial (MeT5A) and a fibroblast cell line (NIH3T3), compared to those exerted by crocidolite asbestos fibers. SVFs (glass wool, rock wools) do not induce significant changes in cell mortality, whereas crocidolite asbestos fibers caused a dose-dependent cytotoxicity. We investigated the correlation between the fiber-induced cytotoxicity and the extent and type of interaction of the fibers with the cell surface, and we observed that SVFs, unlike crocidolite asbestos fibers, establish few and weak interactions. Moreover, after internalization, crocidolite asbestos fibers are often found free in the cytoplasm, whereas glass wool fibers are mainly localized inside cytoplasmic vacuoles. After treatments, we also detected signs of oxidative stress, revealed by an increased reactive oxygen species (ROS) production and by an induction of superoxide dismutase (SOD) activity. The lipoperoxidative damage was characterized by a decrease in polyunsaturated fatty acids (PUFA), an increase in the content of thiobarbituric reactive species (TBARS) and a consumption of vitamin E, as a lipophilic antioxidant. Furthermore, we investigated the effect of fiber exposure on cell proliferation. and it was found that, unlike crocidolite asbestos fibers, SVFs did not induce a significant increase in DNA synthesis.

Animals↗

PubMed2005

Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress.

PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.

Animals↗

PubMed2005

Relationship among lipoperoxides, jasmonates and indole-3-acetic acid formation in potato tuber after wounding.

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0-30?min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC-MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.

Cyclopentanes↗

PubMed2005

Keratinocyte growth factor promotes melanosome transfer to keratinocytes.

Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Adult↗

PubMed2005

ATP2C1 gene mutation analysis in Italian patients with Hailey-Hailey disease.

Hailey-Hailey disease (HHD) is a rare autosomal dominant disorder characterized by recurrent skin lesions predominantly involving the body folds. It is caused by heterozygous mutations in the ATP2C1 gene, encoding the human secretory pathway Ca2+/Mn2+-ATPase protein 1 (hSPCA1). In this report we describe the molecular studies performed in eight HHD cases from Italy that led us to identify six different mutations scattered through the ATP2C1 gene in seven of eight cases. Four of the detected mutations were novel. Our results confirm the high allelic heterogeneity of the ATP2C1 gene and support the notion that HHD is a genetically homogeneous disorder. Furthermore, we created a table summarizing all previously reported ATP2C1 mutations, adapting the nomenclature, if needed, according to the guidelines of the Human Genome Variation Society.

Adult↗

PubMed2005

Polyunsaturated fatty acids partially reproduce the role of melanocytes in the epidermal melanin unit.

The incidence rate of melanoma is higher in fair-skinned than in dark-skinned individuals. In negroid skin there is more eumelanin which is present in all skin layers and fewer polyunsaturated fatty acids (PUFA) than in caucasoid skin. The western diet, which is rich in omega-6 polyunsaturated fatty acids, is associated with more proneness to cancer including cutaneous melanoma. To study the respective influence of omega-6 PUFA and low phototype melanocytes on redox status -basal and following UV irradiation-, we used epidermal reconstructs. The addition of polyunsaturated fatty acids as well as the presence of low phototype melanocytes affected basal status similarly except for catalase activity, which decreased significantly in polyunsaturated fatty acid-supplemented reconstructs. Following UV, polyunsaturated fatty acids and low phototype melanocytes increased lipid and protein oxidative damage without affecting direct DNA damage. However, polyunsaturated fatty acids increased epidermal apoptosis whereas low phototype melanocytes decreased it. Since our data suggest that an omega-6 PUFA rich-diet may increase oxidative damage in melanocytes without inducing apoptosis, the long-term net outcome could be cumulated mutations and an increased risk of skin cancer, especially melanoma.

Apoptosis↗

PubMed2005

Relevance of thyroiditis and of other autoimmune diseases in children with vitiligo.

BACKGROUND: Studies that clearly define the possible association of childhood vitiligo with autoimmune and/or endocrine diseases are lacking. OBJECTIVE: To examine the presence of autoimmune disorders, in particular of thyroid disease, in paediatric patients with vitiligo and investigate the utility of such screening in these patients. METHODS: One hundred and twenty-one paediatric patients (40 males, 81 females) with vitiligo were grouped in segmental and non-segmental vitiligo. All patients were screened for thyroid disease. RESULTS: 13 out of 121 patients had different degrees of thyroid parameter alterations. These patients were all affected by the non-segmental type while none of those with the segmental form presented thyroid alterations. CONCLUSION: In paediatric patients with non-segmental vitiligo, a significant incidence of thyroid dysfunction was found. Since vitiligo usually appears before the development of the thyroid disease, it may be useful to screen thyroid autoantibodies in all paediatric patients with non-segmental vitiligo who present symptoms related to thyroid disease.

Adolescent↗

PubMed2004

Analysis of corticosteroids by high performance liquid chromatography-electrospray mass spectrometry.

A high performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) method, for the detection of corticosteroids in cosmetics has been developed. A water-acetonitrile linear gradient on a C-18 reversed-phase column was found to be suitable in separating triamcinolone and its main derivatives, which greatly differ in lipophilicity. Detection was performed in negative electrospray ionisation mode. Good correlation between peaks areas and solutions concentration was found in the range 0.05-10.0 micro g ml(-1) and the detection limits resulted in the range of 20-45 pg injected. The method was successfully applied to the analysis of real samples of shampoo.

Adrenal Cortex Hormones↗