Browsing by Author "Vital, AL"
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- Amplified and Homozygously Deleted Genes in Glioblastoma: Impact on Gene Expression LevelsPublication . Crespo, I; Tão, H; Nieto, AB; Rebelo, O; Domingos, P; Vital, AL; Patino, MC; Barbosa, MD; Lopes, MC; Resende de Oliveira, C; Orfão, A; Tabernero, MDBACKGROUND: Glioblastoma multiforme (GBM) displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown. METHODOLOGY: Single-nucleotide polymorphism (SNP)-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46), and to evaluate the impact of copy number alterations (CNA) on mRNA levels of the genes involved. PRINCIPAL FINDINGS: Recurrent amplicons were detected for chromosomes 7 (50%), 12 (22%), 1 (11%), 4 (9%), 11 (4%), and 17 (4%), whereas homozygous deletions involved chromosomes 9p21 (52%) and 10q (22%). Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2), while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1-q15 and 4q12, respectively). Despite homozygous del(9p21) and del(10q23.31) included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene. CONCLUSIONS: Overall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.
- Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-kappaB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line.Publication . Vital, AL; Gonçalo, Margarida; Cruz, MT; Figueiredo, A; Duarte, CB; Lopes, MCAIMS: Nitric oxide (NO) has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in a mouse fetal skin dendritic cell line. METHODS: NO production was assessed by the method of Griess. Expression of the inducible isoform of nitric oxide synthase (iNOS) protein was evaluated by western blot analysis and immunofluorescence microscopy. Western blot analysis was also performed to evaluate cytosolic IkappaB-alpha (IkappaB-alpha) protein levels. The electrophoretic mobility shift assay was used to evaluate the activation or inhibition of nuclear factor kappa B (NF-kappaB). RESULTS: GM-CSF induced iNOS expression and NO production, and activated the transcription factor NF-kappaB. Dexamethasone inhibited, in a dose-dependent manner, NO production induced by GM-CSF. Addition of dexamethasone to the culture, 30 min before GM-CSF stimulation, significantly inhibited the cellular expression of iNOS. Dexamethasone also inhibited GM-CSF-induced NF-kappaB activation by preventing a significant decrease on the IkappaB-alpha protein levels, thus blocking NF-kappaB migration to the nucleus. CONCLUSIONS: The corticosteroid dexamethasone inhibits GM-CSF-induced NF-kappaB activation, iNOS protein expression and NO production. These results suggest that dexamethasone is a potent inhibitor of intracellular events that are involved on NO synthesis, in skin dendritic cells.
- Intratumoral patterns of clonal evolution in gliomasPublication . Vital, AL; Tabernero, MD; Crespo, I; Rebelo, O; Tão, H; Gomes, F; Lopes, MC; Orfão, AFew studies have explored the patterns of clonal evolution in gliomas. Here, we investigate the cytogenetic patterns of intratumoral clonal evolution of gliomas and their impact on tumor histopathology and patient survival. Cytogenetic analysis of 90 gliomas was performed in individual tumor cells (>200 cells/tumor) using multicolor (N = 16 probes) interphase-FISH. Overall, chromosome gains were more frequent than chromosome losses. Gains of chromosome 7 and/or EGFR amplification were detected in 91% of the cases, whereas del(9p21) (77%) and del(10q23) (78%) were the most frequent chromosome losses. Virtually, all cases (99%) showed >/=2 tumor cell clones, with higher numbers among high- versus low-grade gliomas (p = 0.001). Nine different cytogenetic patterns were found in the ancestral tumor clones. In most gliomas, ancestral clones showed abnormalities of chromosome 7, 9p, and/or 10q and cytogenetic evolution consisted of acquisition of additional abnormalities followed by tetraploidization. Conversely, early tetraploidization was associated with low-grade astrocytomas-2/3 pilocytic and 3/6 grade II diffuse astrocytomas-and combined loss of 1p36/19q13 with oligodendrogliomas, respectively; both aberrations were associated with a better patient outcome (p = 0.03). Overall, our results support the existence of different pathways of intratumoral evolution in gliomas.
- The sensitizers nickel sulfate and 2,4-dinitrofluorobenzene increase CD40 and IL-12 receptor expression in a fetal skin dendritic cell linePublication . Vital, AL; Gonçalo, Margarida; Cruz, MT; Figueiredo, A; Duarte, CB; Lopes, MCDendritic cells (DCs) are antigen-presenting cells (APCs) capable of capturing haptens and to process and present them to T lymphocytes. In order to sensitize T cells for contact hypersensitivity (CHS), skin DCs suffer a maturation process with modifications on their surface molecules. The aim of this work was to evaluate changes induced by two contact sensitizers, 2,4-dinitrofluorobenzene (DNFB) and nickel sulfate (NiSO4), and a non-sensitizer 2,4-dichloronitrobenzene (DCNB), on the protein levels of two activation markers, CD40 and IL-12 receptor (IL-12R), in a mouse skin dendritic cell line (FSDC). The expression of CD40 and IL-12R proteins was evaluated by western blot assay and direct immunofluorescence microscopy. The results showed that CD40 and IL-12R expression increased significantly after cell exposure to NiSO4 and DNFB, although DNFB exhibited a stronger activity. There was no effect with DCNB. The epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), also used in the experiments, slightly increased the expression of both CD40 and IL-12R and when tested together with the sensitizers the effect was partially additive. The results suggest that the sensitizers DNFB and NiSO4 are directly involved on the changes of the surface markers CD40 and IL-12R in skin DCs, during the sensitization phase of CHS, and this effect may be enhanced by GM-CSF. In contrast, no effect was observed with DCNB.