RESUMO
PURPOSE: To analyze the expression of c-Met, and to investigate correlations between the expression of c-Met, clinicopathologic variables, and survival in patients undergoing curative surgery followed by adjuvant chemoradiotherapy for extrahepatic bile duct (EHBD) cancer. METHODS: Ninety EHBD cancer patients who underwent curative resection followed by adjuvant chemoradiotherapy were enrolled. Expression of c-Met was assessed with immunohistochemical staining on tissue microarray. The correlation between clinicopathologic variables and survival outcomes was evaluated using Kaplan-Meier method and Cox proportional hazard model. RESULTS: On univariate analysis, 66 patients (76.7 %) showed c-Met expression. c-Met expression had a significant impact on 5-year overall survival (OS) (43.0 % in c-Met(+) vs. 25.0 % in c-Met(-), p = 0.0324), but not on loco-regional relapse-free survival or distant metastasis-free survival (DMFS). However, on multivariate analysis incorporating tumor location and nodal involvement, survival difference was not maintained (p = 0.2940). Tumor location was the only independent prognostic factor predicting OS (p = 0.0089). Hilar location tumors, nodal involvement, and poorly differentiated tumors were all identified as independent prognostic factors predicting inferior DMFS (p = 0.0030, 0.0013, and 0.0037, respectively). CONCLUSIONS: This study showed that c-Met expression was not associated with survival outcomes in EHBD cancer patients undergoing curative resection followed by adjuvant chemoradiotherapy. Further studies are needed to fully elucidate the prognostic value of c-Met expression in these patients.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/análise , Proteínas Proto-Oncogênicas c-met/biossíntese , Adulto , Idoso , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/terapia , Ductos Biliares Extra-Hepáticos/patologia , Quimiorradioterapia Adjuvante , Procedimentos Cirúrgicos do Sistema Digestório , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-met/análise , Análise Serial de Tecidos , Adulto JovemRESUMO
Peripheral-nerve injuries are a common clinical problem and often result in long-term functional deficits. Reconstruction of peripheral-nerve defects is currently undertaken with nerve autografts. However, there is a limited availability of nerves that can be sacrificed and the functional recovery is never 100% satisfactory. We have previously shown that gene therapy with vascular endothelial growth factor (VEGF) significantly improved nerve regeneration, neuronal survival, and muscle activity. Our hypothesis is that granulocyte colony-stimulating factor (G-CSF) synergizes with VEGF to improve the functional outcome after sciatic nerve transection. The left sciatic nerves and the adjacent muscle groups of adult mice were exposed, and 50 or 100 µg (in 50 µl PBS) of VEGF and/or G-CSF genes was injected locally, just below the sciatic nerve, and transferred by electroporation. The sciatic nerves were transected and placed in an empty polycaprolactone (PCL) nerve guide, leaving a 3-mm gap to challenge nerve regeneration. After 6 weeks, the mice were perfused and the sciatic nerve, the dorsal root ganglion (DRG), the spinal cord and the gastrocnemius muscle were processed for light and transmission electron microscopy. Treated animals showed significant improvement in functional and histological analyses compared with the control group. However, the best results were obtained with the G-CSF+VEGF-treated animals: quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers and blood vessels, and the number of neurons in the DRG and motoneurons in the spinal cord was significantly higher. Motor function also showed that functional recovery occurred earlier in animals receiving G-CSF+VEGF-treatment. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase, suggesting an improvement of reinnervation and muscle activity. These results suggest that these two factors acted synergistically and optimized the nerve repair potential, improving regeneration after a transection lesion.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/terapia , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Análise de Variância , Animais , Creatina Quinase/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Gânglios Espinais/ultraestrutura , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Locomoção/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Regeneração Nervosa/genética , Recuperação de Função Fisiológica/genética , Neuropatia Ciática/patologia , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Transplante , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca(2+) homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca(2+) distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca(2+) stores and selective Ca(2+) releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca(2+) stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca(2+) that precede Bax translocation. On the other hand, agents that mobilize Ca(2+) from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca(2+) release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. In addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca(2+) release may induce Bax transactivation and specifically control apoptosis.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Córtex Cerebral/metabolismo , Retículo Endoplasmático/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose , Células Cultivadas , Córtex Cerebral/citologia , Citometria de Fluxo , Microinjeções , Transporte Proteico , RatosRESUMO
AIMS: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. METHODS: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. RESULTS: Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. CONCLUSION: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.
Assuntos
Terapia Genética/métodos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/terapia , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Feminino , Camundongos , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologiaRESUMO
The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.
Assuntos
Animais , Humanos , Masculino , Camundongos , Sequência de Bases/genética , Hipóxia Celular/genética , Expressão Gênica/genética , /genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Vetores Genéticos/genética , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Plasmídeos/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.
Assuntos
Sequência de Bases/genética , Hipóxia Celular/genética , Expressão Gênica/genética , Vírus 40 dos Símios/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Reação em Cadeia da Polimerase , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
This study was aimed to examine cholesteryl ester transfer protein (CETP), apolipoprotein AI and CIII gene polymorphisms, and to verify whether these genetic determinants are associated with the prevalence of myocardial infarction (MI) or type 2 diabetes. The TaqIB restriction fragment length polymorphism (RFLP) in intron I of the CETP gene, the MspI in the third intron of the APOAI gene, and also SstI in the 3' untranslated region of the APOCIII gene were determined using standard methods. The prevalence of these polymorphisms was compared between diabetic (n = 119), and non-diabetic (n = 100) middle-aged individuals of both sexes. We found a higher prevalence of the B2B2 genotype of the CETP gene among diabetics than that observed in non-diabetics (P < 0.05), and a lower prevalence of this genotype among patients with previous MI (P < 0.02). The MspI polymorphisms of the APOAI gene showed that M1++ genotype was found mainly in diabetic patients (P < 0.04). Conversely, the SstI polymorphism of APOCIII gene was not significantly associated with either MI or diabetes. Therefore, among these genetic polymorphisms, TaqIB of CETP and MspI of apolipoprotein AI appeared to help significantly to identify diabetic individuals. In particular, the former may have an additional role in the primary prevention of coronary disease.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas C/genética , Proteínas de Transporte/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Glicoproteínas/genética , Infarto do Miocárdio/genética , Polimorfismo Genético , Apolipoproteína C-III , Estudos de Casos e Controles , Proteínas de Transferência de Ésteres de Colesterol , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization
Assuntos
Animais , Cricetinae , Ratos , Angiotensina II/fisiologia , Células CHO , Endocitose , Receptores de Angiotensina/fisiologia , Angiotensina II/antagonistas & inibidores , Northern Blotting , Membrana Celular , Endocitose/fisiologia , Ligantes , Microscopia Confocal , Transdução de Sinais , TransfecçãoRESUMO
A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.
Assuntos
Angiotensina II/fisiologia , Células CHO/metabolismo , Endocitose , Membrana Nuclear/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/antagonistas & inibidores , Antagonistas de Receptores de Angiotensina , Animais , Northern Blotting , Membrana Celular , Cricetinae , Endocitose/fisiologia , Ligantes , Microscopia Confocal , Ratos , Receptores de Angiotensina/fisiologia , Transdução de Sinais , TransfecçãoRESUMO
Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.