RESUMO
BACKGROUND/AIMS: Drugs with antivascular endothelial growth factor A (anti-VEGF-A) action are under clinical evaluation with encouraging results in advanced hepatocellular carcinoma (HCC). The relative VEGF-A protein expression in non-advanced HCC and in the cirrhotic non-tumoral tissue in the same patient, a variable that could be important for treatment efficacy, has been investigated with conflicting results, only using the cirrhotic tissue surrounding the neoplasm (CS). METHODS: We measured, for the first time, VEGF-A expression in non-advanced HCC and in the respective CS and cirrhotic tissue at a distance from the tumour (CD), in 24 patients who underwent liver transplantation. RESULTS: VEGF-A protein was more expressed (P<0.05) in HCC than in CD, while no difference was found between HCC and CS. In HCC patients with a serum alpha-fetoprotein (AFP) higher than 20 ng/ml, VEGF-A protein expression in HCC was higher than in the corresponding CD in 83% of cases and AFP and serum VEGF-A corrected for the platelet count positively correlated with the differential VEGF-A protein expression between HCC and CD. CONCLUSION: Our data provide a rationale for clinical trials involving anti-VEGF-A treatments in patients with non-advanced HCC, and suggest that serum AFP and VEGF-A are variables to be taken into account in these studies.
Assuntos
Carcinoma Hepatocelular/sangue , Expressão Gênica , Cirrose Hepática/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , alfa-Fetoproteínas/metabolismo , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Itália , Transplante de Fígado , Masculino , Pessoa de Meia-IdadeRESUMO
A 'locally acting' IGF1 (insulin-like growth factor 1) isoform has been recently identified in the skeletal muscle and neural tissues where it accelerates injury repair. No information exist on the expression and function of IGF1 isoforms in the liver. We investigated IGF1 isoforms in rat hepatocytes and cholangiocytes and evaluated their involvement in cell proliferation or damage induced by experimental cholestasis (bile duct ligation, BDL) or hydrophobic bile salts. IGF1 isoforms were analyzed by real-time PCR by using beta-actin as internal reference. In both hepatocytes and cholangiocytes, the 'locally acting' IGF1 isoform (XO6108) and 'circulating' IGF1 isoform (NM_178866) represented respectively 44 and 52% of the total IGF1. Basal mRNAs for both 'locally acting' and 'circulating' IGF1 isoforms were higher (P<0.05) in hepatocytes than cholangiocytes. After BDL for 3 h, the 'locally acting' IGF1 isoform decreased threefold (P<0.05) in hepatocytes but remained stable in cholangiocytes with respect to sham-controls. After 1 week of BDL, hepatocytes displayed a further fivefold decrease of 'locally acting' IGF1 mRNA. In contrast, cholangiocytes showed an eightfold increase of the 'locally acting' IGF1 mRNA. The effect of 3 h of BDL on IGF1 isoforms was reproduced in vitro by incubation with glycochenodeoxycholate (GCDC). The cytotoxic effects (inhibition of proliferation and induction of apoptosis) of GCDC on isolated cholangiocytes were more pronounced after selective silencing (SiRNA) of 'locally acting' than 'circulating' IGF1 isoform. Rat hepatocytes and cholangiocytes express the 'locally acting' IGF1 isoform, which decreased during cell damage and increased during cell proliferation. The 'locally acting' IGF1 was more active than the 'circulating' isoform in protecting cholangiocytes from GCDC-induced cytotoxicity. These findings indicate that, besides muscle and neural tissues, also in liver cells the 'locally acting' IGF1 isoform is important in modulating response to damage.
Assuntos
Sistema Biliar/metabolismo , Colestase/prevenção & controle , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Isoenzimas/fisiologia , Animais , Apoptose , Sequência de Bases , Sistema Biliar/citologia , Western Blotting , Proliferação de Células , Células Cultivadas , Primers do DNA , Inativação Gênica , Hepatócitos/citologia , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We describe a quantitative assay of dsDNA based on real-time PCR measurement of fluorescence due to the interaction of PicoGreen dye with dsDNA. An aliquot of 1 to 5 ml of the sample is mixed with 45 ml of diluted PicoGreen reagent within an optical PCR tube. This is placed into the real-time apparatus set to read SYBR Green I dye at the end of three cycles of 94 degrees C for 30 s and 65 degrees C for 30 s. The averaged fluorescence value is converted into DNA amount using a calibration curve prepared with lambda-DNA standard. The calibration curve has a dynamic linear range from 0.20 to 50 ng and a standard deviation variability below 5.0%. In conclusion, this method allows reliable determinations on minimal amounts of DNA from biological samples and PCR products in clinical applications of molecular biology.
Assuntos
Sistemas Computacionais , DNA/análise , Corantes Fluorescentes/análise , Fluorometria/métodos , Reação em Cadeia da Polimerase/métodos , Bacteriófago lambda/genética , Calibragem , DNA/isolamento & purificação , DNA Viral/análise , Éxons/genética , Fluorometria/normas , Humanos , Compostos Orgânicos , Kit de Reagentes para Diagnóstico , Receptores de LDL/genética , Padrões de Referência , TemperaturaRESUMO
Nicastrin is a protein recently discovered associated to presenilins and involved in the production of amyloid beta peptide that accumulates in Alzheimer's disease (AD) brain. In this study the nicastrin gene was examined for unknown mutations and polymorphisms in 104 patients with familial AD (52 early-onset and 52 late-onset), 174 sporadic AD and 191 healthy neurological controls of Italian origin. The scanning of the nicastrin gene identified a missense mutation (N417Y) in two patients with sporadic AD, in an early-onset familial AD and in a young control subject. Furthermore, we found two silent mutations and four intronic polymorphisms, three of them co-segregating in a single haplotype. We found some differences in the distribution of genotype alterations in the AD population compared to the controls. However, our data together with other evidence did not support the pathological role of missense mutation N417Y.
Assuntos
Doença de Alzheimer/genética , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/classificação , Secretases da Proteína Precursora do Amiloide , Apolipoproteína E4 , Apolipoproteínas E/genética , Asparagina/genética , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tirosina/genéticaRESUMO
The 8344C/T polymorphism of the apoB gene was genotyped by an original modification of PCR allele-specific amplification consisting in a single amplification reaction double-primed by two opposite allele-specific oligonucleotides nested in a larger amplified fragment. This method was used to genotype 200 randomly selected healthy individuals (113 males, 87 females). The frequency of the rare allele in this random Italian population was 0.240, i.e. not far from the 0.282 frequency observed in hypocholesterolemic Norwegians and suggestive of a moderating effect on LDL levels of our population. However, we did not find any significant cholesterol-lowering effect of this polymorphism either by comparing the frequency of mutant alleles in the population stratified for its plasma lipoprotein levels or by studying the association between ApoB genotype and the different lipoproteins. In conclusion this ApoB polymorphism appeared to have a secondary role in LDL- and HDL-cholesterol variations of our population.
Assuntos
Apolipoproteínas B/genética , Colesterol/sangue , Adulto , Idoso , Alelos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To assess a technology-aided programme for promoting leisure engagement and communication in a man with amyotrophic lateral sclerosis (ALS). METHOD: The programme involved a laptop computer equipped with a Clicker 5 software package, an optic microswitch and an interface device. The participant could choose between two leisure options (i.e. songs and videos), could write requests and general messages through a virtual keyboard and a microswitch and could have the written text read out to caregivers and staff. RESULTS: The use of the programme increased the mean frequency of words written to about 15 per 20-minute session during the second intervention phase. Those words were used by the participant for formulating a mean of over two requests/messages per session. The participant also listened to songs and watched videos. CONCLUSION: A simple technology-aided programme may allow ALS patients to manage leisure engagement and communication.
Assuntos
Esclerose Lateral Amiotrófica/reabilitação , Auxiliares de Comunicação para Pessoas com Deficiência , Comunicação , Pessoas com Deficiência/reabilitação , Atividades de Lazer , Cuidadores , Humanos , Masculino , Microcomputadores , Pessoa de Meia-Idade , Interface Usuário-ComputadorRESUMO
BACKGROUND/AIMS: We evaluated the role and mechanisms by which the GH/IGF1 axis modulates cholangiocyte proliferation. METHODS: GH-receptors (GH-R), IGF1, IGFBP3 (binding protein 3), IGF1-R and receptor substrates (IRS) were evaluated in cholangiocytes of normal or bile duct-ligated (BDL) rat livers. The effects of GH and IGF1 on proliferation of normal quiescent cholangiocytes and the transduction pathways involved were investigated. RESULTS: IGF1, GH-R, IGF1-R, IRS-1/2 were expressed in normal cholangiocytes and overexpressed in cholangiocytes proliferating after BDL which also secrete IGF1 in a higher amount than normal cells. IGFBP3, which may counter-regulate IGF1 effects, was decreased in BDL cholangiocytes. IGF1 promoted cholangiocyte proliferation in association with overexpression of p-IGF1R, IRS1, IRS-2, p-ERK1/2 and p-AKT. GH induced IGF1 expression and release in isolated cholangiocytes, and reproduced the effects of IGF1 but GH effects were abolished by IGF1-R blocking antibody, suggesting IGF1 as a mediator of GH. Finally, IGF1 and 17beta-estradiol reciprocally potentiated their proliferative effects on cholangiocytes, and by interacting at both receptor and post-receptor levels. CONCLUSIONS: Cholangiocytes respond to GH with production and release of IGF1 that modulates cell proliferation by transduction pathways involving IGF1-R, IRS1/2 and both ERK and PI3-kinase pathways. The biliary epithelium is a target of GH/IGF1 liver axis.
Assuntos
Ductos Biliares Intra-Hepáticos/anatomia & histologia , Proliferação de Células , Células Epiteliais/metabolismo , Epitélio/metabolismo , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/genética , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Wistar , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismoRESUMO
A variety of rearrangements in the low-density lipoprotein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.
Assuntos
Eletroforese em Microchip/métodos , Hiperlipoproteinemia Tipo II/genética , Mutação , Receptores de LDL/genética , Análise Mutacional de DNA/métodos , Primers do DNA , Éxons , Duplicação Gênica , Humanos , Reação em Cadeia da Polimerase/métodos , Deleção de SequênciaRESUMO
We developed a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) procedure based on the combination of competimer technology with microchip electrophoresis. The approach was applied to total RNA extracts from the human colon carcinoma cell line CaCo-2 cultured for 22 days onto tissue plate inserts that allow the polarized cell growth. At time of experiment these cells were incubated for 2 h with lipid micelles containing either cholesterol or a mixture cholesterol/beta-sitosterol or serum-free Dulbecco's modified Eagle medium (DMEM) alone. Total RNA was extracted from cell pellets by silica membrane spin columns and reverse-transcribed by a genetically engineered M-MuLV enzyme and an oligo (dT)(15) primer. The cDNA underwent PCR amplification for the following genes: apolipoprotein A1 (ApoAI), apolipoprotein E (ApoE), ATP-binding cassette A1 (ABCA1), liver X receptor alpha (LXRalpha) and farnesoid X receptor (FXR). The beta-actin gene, used as an endogenous reference, was coamplified with mixtures of primer and competimer at a ratio leading to a reference band of intensity comparable to that of the gene under analysis. Amplification products were separated and quantitated by microchip electrophoresis in order to determine, for each gene, the optimum cycle numbers and primer to competimer ratios for the beta-actin therefore used for evaluating the relative variations of gene expressions in the different experiments. The incubation with cholesterol micelles stimulated both LXR and FXR expression that was accompanied by an increased expression of ABCA1 and ApoAI genes (1.4- and 1.5-fold, respectively) and halved the APOE expression. The effect on ABCA1 and ApoAI expression was even stronger (5.7- and 2.6-fold, respectively) with beta-sitosterol-containing micelles. Similar changes are in line with previous findings and suggest that the short incubation with beta-sitosterol-enriched micelles stimulates a specific mechanism that, via ABCA1 activation, can increase the cholesterol and phytosterol basolateral efflux.