RESUMEN
OBJECTIVE: Human colorectal cancer (CRC) is characterized by a sequence of biological events that determine its induction and progression. Gut microbiota has an important role in this multistep model of carcinogenesis, as well as constitutive activation of Signal Transducer and Activator Factors 3 (p-STAT3) and Protein Inhibitor of Activated STAT3 (PIAS3), which negatively controls STAT3. It has been reported that a liver growth factor, the Augmenter of Liver Regeneration (ALR), an anti-apoptotic, anti-metastatic factor, exerts protective/cell survival and anti-metastatic activities and has been detected highly expressed in neoplastic cells. PATIENTS AND METHODS: To evaluate, by immunohistochemistry, p-STAT3, PIAS3 and ALR expression in neoplastic human tissues from CRC patients, grouping the data in accordance with the histological alterations (G1, G2 and G3) and metastasis presence. Western blot (WB) analysis of ALR was also determined in neoplastic and surrounding tissues. Finally, cell proliferation (Ki-67) and apoptosis (Bcl-2) were determined. RESULTS: Colon cancer tissue samples showed: (1) ALR and p-STAT3 strongly over-expression in 100% of G1 tissue samples, reducing in G2 and G3 tissue samples; (2) PIAS3 immunological determination was poorly expressed in G1 tissue samples and highly expressed in the 100% of colorectal tissues from group G2 and G3. Ki-67 progressively increases with the importance of the anatomic-pathological alterations and Bcl-2 resulted higher in G3 tissue samples compared to G1 neoplastic tissues. WB data evidenced, in neoplastic tissues, compared to the tumour-surrounding tissues, ALR over-expressed in G1 neoplastic tissues and down-expressed in G3 neoplastic tissues. CONCLUSIONS: Our data demonstrate a different dynamism of the investigated factors in relation to the severity of CRC histological findings. We hypothesize that the positive expression of ALR and p-STAT3 in the neoplastic tissue samples from CRC G1 group, associated to the absence of PIAS3, could be useful marker to identify an early stage of the disease. Based on these data and on our previous studies on gut microbiota in precancerous intestinal lesions, we are confident that, after microbial priming, a cascade of molecular events is started. So, the detectable molecules acting in these initial steps should be considered for the study of CRC progression and therapy.
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Neoplasias Colorrectales/genética , Regeneración Hepática/genética , Chaperonas Moleculares/genética , Proteínas Inhibidoras de STAT Activados/genética , Factor de Transcripción STAT3/genética , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Proliferación Celular , Neoplasias Colorrectales/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/análisis , Proteínas Inhibidoras de STAT Activados/análisis , Factor de Transcripción STAT3/análisisRESUMEN
Polaritonic devices exploit the coherent coupling between excitonic and photonic degrees of freedom to perform highly nonlinear operations with low input powers. Most of the current results exploit excitons in epitaxially grown quantum wells and require low-temperature operation, while viable alternatives have yet to be found at room temperature. We show that large single-crystal flakes of two-dimensional layered perovskite are able to sustain strong polariton nonlinearities at room temperature without the need to be embedded in an optical cavity formed by highly reflecting mirrors. In particular, exciton-exciton interaction energies are shown to be spin dependent, remarkably similar to the ones known for inorganic quantum wells at cryogenic temperatures, and more than one order of magnitude larger than alternative room temperature polariton devices reported so far. Because of their easy fabrication, large dipolar oscillator strengths, and strong nonlinearities, these materials pave the way for realization of polariton devices at room temperature.
RESUMEN
Psoriasis is a chronic inflammatory skin disease with systemic involvement that might predispose to many psoriasis-related comorbidities, such as metabolic syndrome and cardiovascular disorders. Clusterin (Clu), also known as apolipoprotein J (ApoJ), is a highly conserved disulfide-linked heterodimeric glycoprotein implicated in a great variety of physiological and pathophysiological processes including lipid transportation, tissue remodeling, senescence, cell interaction, stress response, inflammation, apoptosis, diabetes mellitus and metabolic syndrome. Serum levels of Clu were assessed in 15 patients with moderate-to-severe psoriasis defined by the presence of a Psoriasis Area and a Severity Index (PASI) value of 10 or more. It was found that the Clu value was significantly higher in patients than in healthy subjects (p <0.001). Our data confirm that the association of psoriatic disease with some comorbidities, especially metabolic and cardiovascular disease, might support the correlation with increased circulating Clu. In particular, it should be pointed out that, according to the recent literature, the Clu could also have a protective role in the comorbidity of psoriasis patients. In addition, it has been published that Clu protects cardiomyocytes against ischemic cell death and is a potential therapeutic agent in the treatment of myocardial infarction; therefore it can be assumed that an artificial enhancement of Clu in the blood could limit the severity of damage also in respect to skin lesions. Although the increase in serum level of Clu was found in all patients with psoriasis, more studies on a larger cohort of patient samples is necessary to confirm the significance of high serum levels of clusterin/ApoJ and to suggest the use of this glycoprotein as an additional new marker in psoriasis pathogenesis. It could be a possibility to improve the prognosis in patients with psoriasis.
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Clusterina/sangre , Psoriasis/sangre , Anciano , Anciano de 80 o más Años , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/complicaciones , Femenino , Humanos , Masculino , Enfermedades Metabólicas/sangre , Enfermedades Metabólicas/complicaciones , Persona de Mediana Edad , Psoriasis/complicacionesRESUMEN
The mammalian growth factor erv1-like (GFER) gene encodes a sulfhydryl oxidase enzyme, named Augmenter of Liver Regeneration (ALR). Recently it has been demonstrated that ALR supports cell proliferation acting as an anti-apoptotic factor. This effect is determined by ALR ability to support the anti-apoptotic gene expression and to preserve cellular normoxic conditions. We recently demonstrated that the addition of recombinant ALR (rALR) in the culture medium of H(2)O(2)-treated neuroblastoma cells reduces the lethal effects induced by the hydrogen peroxide. Similar data have been reported in the regenerating liver tissue from partially hepatectomized rats treated with rALR. The purpose of the present study was to evaluate the effect of the GFER inhibition, via the degradation of the complementary mRNA by the specific siRNA, on the behaviour of the apoptosis (apoptotic gene and caspase expression and apoptotic cell number) and of the oxidative stress-induced parameters (reactive oxygen species (ROS), clusterin expression and mitochondrial integrity) in T98G glioma cells. The results revealed a reduction of (i) ALR, (ii) clusterin and (iii) bcl-2 and an increase of (iv) caspase-9, activated caspase-3, ROS, apoptotic cell number and mitochondrial degeneration. These data confirm the anti-apoptotic role of ALR and its anti-oxidative properties, and shed some light on the molecular pathways through which ALR modulates its biological effects.
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Apoptosis , Reductasas del Citocromo/metabolismo , Regulación de la Expresión Génica , Glioma/patología , Estrés Oxidativo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Clusterina/metabolismo , Reductasas del Citocromo/antagonistas & inhibidores , Glioma/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.
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Epítopos/química , Epítopos/inmunología , Hepacivirus/química , Hepacivirus/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Anticuerpos/inmunología , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Proteoma/química , Proteoma/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: An abnormal intestinal permeability could contribute to establish an altered sensitivity to food-allergen. AIM: To evaluate the intestinal permeability in subjects with adverse reactions to food on allergen-free diet. SUBJECTS: Twenty-one patients with food allergy and 20 with food hypersensitivity on allergen-free diet were enrolled and divided in four groups according to the seriousness of their referred clinical symptoms when they were on a free diet. METHODS: Intestinal permeability was evaluated by Lactulose/Mannitol ratio urinary detection determined by anion-exchange chromatography. RESULTS: Statistically significant different Lactulose/Mannitol ratio was evidenced in subjects with food allergy (p=0.003) or hypersensitivity (p=0.0008) compared to control patients. The correlation between Lactulose/Mannitol ratio and the seriousness of clinical symptoms, by using Spearman test, was statistically significant for food allergy (p=0.0195) and hypersensitivity (p=0.005) patients. CONCLUSIONS: The present data demonstrate that impaired intestinal permeability, measured in our conditions, is present in all subjects with adverse reactions to food. In addition, for the first time, we report a statistically significant association between the severity of referred clinical symptoms and the increasing of Intestinal Permeability Index. These data reveal that intestinal permeability is not strictly dependent on IgE-mediated processes but could better be related to other mechanisms involved in early food sensitisation, as breast-feeding, or microbial environment that influence the development of oral tolerance in early infancy.
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Hipersensibilidad a los Alimentos/metabolismo , Alimentos/efectos adversos , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Intestinos/fisiopatología , Lactulosa/orina , Masculino , Manitol/orina , Persona de Mediana Edad , PermeabilidadRESUMEN
BACKGROUND: Augmenter of Liver Regeneration is an important secondary hepatic growth factor. Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver-resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown. AIMS: To define the enzymatic activity of augmenter of liver regeneration protein. The carboxy terminus of augmenter of liver regeneration protein contains a special CXXC motif characteristic for redox proteins and with faint homologies to the redox-active site of sulfhydryl oxidases. Tests were, therefore, carried out to establish whether isolated augmenter of liver regeneration protein can also function in the formation of sulfur bridges. METHODS: Purified augmenter of liver regeneration proteins from rat and human were tested in enzyme assays for the ability to introduce disulfide bonds into protein substrates. The isolated proteins were tested for the formation of dimers and the presence of bound FAD was investigated spectroscopically. The function of the conserved CXXC motif was investigated by in vitro mutagenesis experiments and subsequent enzyme assays. RESULTS: In this study, we demonstrate that rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates. A flavin moiety is firmly but not covalently attached to the protein. In human cell cultures augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers. The active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human Q6 growth regulator protein. In vitro mutagenesis of one cysteine residue in the CXXC motif results in loss of enzymatic function and the mutated protein no longer binds FAD. CONCLUSIONS: For the first time, these data assign an enzymatic activity to the important hepatic growth factor augmenter of liver regeneration protein. The finding that augmenter of liver regeneration protein acts as a FAD-linked sulfhydryl oxidase is essential to identify the molecular targets inside liver cells and to elucidate the precise role of mammalian augmenter of liver regeneration protein in hepatic cell growth, liver disease and regeneration.
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Proteínas de Unión al ADN/biosíntesis , Sustancias de Crecimiento/metabolismo , Regeneración Hepática/fisiología , Mitocondrias/fisiología , Oxidorreductasas/metabolismo , Proteínas , Animales , Secuencia de Bases , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Sensibilidad y Especificidad , Especificidad de la Especie , Análisis EspectralRESUMEN
BACKGROUND: The mammalian augmenter of liver regeneration gene encodes a protein involved in the unique process of liver regeneration. The augmenter of liver regeneration respective protein stimulates hepatocyte proliferation in hepatectomized rats and inhibits cytotoxic activity of liver-derived Natural Killer cells from intact rats. Augmenter of liver regeneration protein shares homology with a Saccharomyces Cerevisiae protein essential for the viability, oxidative phosphorylation and cell-division cycle. AIMS: To demonstrate if augmenter of liver regeneration protein, like the homologous in the yeast, plays a role in the regulation of biogenesis of mitochondria. METHODS: Augmenter of liver regeneration protein was injected in intact rats and, in the hepatic tissue, the expression of two genes located in two different regions of the mitochondrial genome, mitochondrial ATPase 6/8, and ND1 subunit, and of a nuclear gene, mitochondrial Transcription Factor A, were considered. In addition, cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria were evaluated. RESULTS: The augmenter of liver regeneration protein administration induces an increase in the mitochondrial gene expression and enhances cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria. CONCLUSIONS: The present data demonstrate a comparable role in the regulation of mitochondria biogenesis in the eukaryotic cell like the yeast protein. This phenomenon could be part of the complex mechanism through which augmenter of liver regeneration regulates hepatocyte proliferation.
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Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Regeneración Hepática/fisiología , Mitocondrias/fisiología , Proteínas Mitocondriales , Proteínas Nucleares , Proteínas , Adenosina Trifosfatasas/biosíntesis , Animales , Proteínas de Unión al ADN/biosíntesis , Masculino , Fosforilación Oxidativa , Ratas , Ratas Endogámicas F344 , Factores de Transcripción/biosíntesisRESUMEN
BACKGROUND: We have shown that the administration of exogenous Augmenter of Liver Regeneration protein in intact rats i) regulates mitochondrial gene expression by inducing the transcription and translation of the nuclear-encoded mitochondrial transcription factor A, and ii) inhibits the lytic activity of liver-resident Natural Killer cells. AIMS: The present investigation was carried out to study the effect, in intact rats, of exogenous administration of Augmenter of Liver Regeneration protein on Interferon-gamma, a cytokine produced by activated Natural Killer cells and known to control the expression of mitochondrial transcription factor A, a nuclear gene responsible for mitochondrial metabolism. METHODS: Interferon-gamma was measured as messenger RNA in liver-derived mononuclear leukocytes and as protein in liver-derived Natural Killer cells after a single injection of Augmenter of Liver Regeneration protein. RESULTS: The data obtained demonstrate that: i) in intact rats, Augmenter of Liver Regeneration protein administration induces a reduction of Interferon-gamma in the liver-resident Natural Killer cells and ii) the administration of Interferon-gamma in 70% hepatectomized rats is followed by a significant reduction both of the mitochondrial transcription factor A expression and of liver regeneration. CONCLUSIONS: These data demonstrate the pivotal role of Augmenter of Liver Regeneration as Growth Factor and as immunoregulator by controlling, through Interferon-gamma levels, the mitochondrial transcription factor A expression and the lytic activity of liver-resident Natural Killer cells.
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Sustancias de Crecimiento/farmacología , Hepatocitos/citología , Interferón gamma/metabolismo , Regeneración Hepática/inmunología , Proteínas Mitocondriales , Proteínas Nucleares , Proteínas , ARN Mensajero/genética , Animales , Western Blotting , División Celular/efectos de los fármacos , Cartilla de ADN/química , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Interferón gamma/genética , Interferón gamma/farmacología , Células Asesinas Activadas por Linfocinas/metabolismo , Regeneración Hepática/efectos de los fármacos , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The purpose of this review is to bring to the attention of the reader the latest developments in research on an important new emerging gene family. The respective genes are found in eukaryotes from yeast to man and even on the genome of some doubled-stranded DNA viruses. They have essential functions in the biogenesis of mitochondria, the cell division cycle and, in higher eukaryotes, in the development of organs like liver and testis. The most important medical implication is their probable role in liver regeneration that will, therefore, be addressed in detail. Aspects of molecular biology, medical implications and problems of developmental biology reflect the complexity of the functions of these proteins and the subjects of the respective research. This is just the beginning of an interdisciplinary effort directed towards the elucidation of the precise function of these essential factors inside the eukaryotic cell. In the general part of this review, we will concentrate on the history of the discovery of these genes and on a summary of their characteristic features. In the more specialized section, the specific role as augmenter of liver regeneration will be addressed in detail.
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Genes Fúngicos/genética , Regeneración Hepática/genética , Mitocondrias Hepáticas/genética , Mitocondrias/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Genes Esenciales/genética , Humanos , Ratones , Datos de Secuencia Molecular , RatasRESUMEN
The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.
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Proteínas Fúngicas/metabolismo , Sustancias de Crecimiento/metabolismo , Proteínas Mitocondriales , Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Proteínas Fúngicas/genética , Expresión Génica , Prueba de Complementación Genética , Ingeniería Genética , Vectores Genéticos , Proteínas Fluorescentes Verdes , Sustancias de Crecimiento/genética , Humanos , Proteínas Luminiscentes/genética , Mitocondrias , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Ratas , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , TransfecciónRESUMEN
Fine balanced sequential changes of the levels of circulating hepatotrophic factors are essential for normal liver regeneration. Our recent studies have indicated that liver-resident natural killer (NK) cells are important regulators of liver regeneration and have raised the possibility that hepatotrophic factors might mediate their activities through NK cells. In the present study, we assessed the effects of in vivo administration of three hepatotrophic factors (augmenter of liver regeneration [ALR], insulin-like growth factor-II [IGF-II], and hepatocyte growth factor [HGF]) on NK cells in normal rats. Each of the three, given over a 1-day period in doses known to produce hepatotrophic activity, induced inhibition of NK cell cytotoxic activities in the population of mononuclear leukocytes (MNL) in the liver, but not in MNL from the spleen or peripheral blood. In contrast to these results obtained by the whole animal treatment, the three molecules had no effect on NK cell functions when added to cultures of MNL from the livers, spleens, or blood of untreated rats. These data support and extend our previously advanced hypothesis that ALR and other hepatotrophic factors play an important role in liver regeneration by regional regulation of NK cells through some as-yet-unknown intermediary mechanism.
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Sustancias de Crecimiento/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Factor II del Crecimiento Similar a la Insulina/farmacología , Células Asesinas Naturales/efectos de los fármacos , Proteínas , Animales , Hepatectomía , Células Asesinas Naturales/fisiología , Masculino , Monocitos/efectos de los fármacos , Monocitos/fisiología , Ratas , Ratas Endogámicas F344 , Organismos Libres de Patógenos EspecíficosRESUMEN
Helicobacter pylori (HP) infection is the main etiopathogenetic agent responsible for inflammatory and ulcerative changes in gastroduodenal mucosa and the basis for both intestinal and diffuse types of gastric carcinoma. In this latter case, intestinal metaplasia is the intermediary between gastritis and cancer. In this study we describe the proliferative activity of gastric epithelium in the progressive stages of HP infection. The expression of proliferating cell nuclear antigen (PCNA), which has proven to be a reliable method for this evaluation, was used as a marker. The study was performed on endoscopic biopsies of the gastric antrum of 40 patients, who were divided into five groups, eight in each group: normal histology and endoscopy, HP-; histological HP+ gastritis with normal endoscopy; histological HP+ gastritis with endoscopic evidence of chronic erosions; complete and incomplete intestinal metaplasia in a HP+ stomach. PCNA was detected by immunohistochemistry and expressed as labeling index, ie, percentage of positive nuclei either in the whole or upper third of foveolae. Our data show a progressive increase of epithelial proliferation in the successive stages of HP infection ranging from gastritis alone to the development of incomplete intestinal metaplasia, a well-known precancerous condition. The proliferative pattern tended to expand towards the upper foveolar third, which in normal conditions does not represent a site of epithelial renewal. These alterations may be related to the development of neoplastic transformations of gastric epithelium. It is well known that genetic mutations are facilitated in proliferating cells. Therefore, our results indicate that the high epithelial turnover, expressed by PCNA LI, may be an indicator of increased risk of neoplastic changes in long-standing untreated HP+ chronic gastritis.
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Mucosa Gástrica/patología , Gastritis/patología , Infecciones por Helicobacter/patología , Helicobacter pylori , Adulto , Anciano , División Celular , Enfermedad Crónica , Femenino , Mucosa Gástrica/inmunología , Gastritis/inmunología , Gastritis/microbiología , Gastroscopía , Infecciones por Helicobacter/inmunología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Antígeno Nuclear de Célula en Proliferación/análisis , Neoplasias Gástricas/patologíaRESUMEN
In this study, the stimulatory effect of bile salts (BS) was evaluated both in vitro, using hepatocyte primary cultures, and in vivo, in normal and 40% partially hepatectomized rats previously fed on BS-enriched diets for 4 weeks. In vitro results show that conjugated cholate (CA) and chenodeoxycholate (CDCA) augmented proliferative activity in rat hepatocytes cultured in absence of mitogens, whereas conjugated deoxycholate (DCA), and ursodeoxycholate (UDCA) did not have any significant effect. None of these BSs increased significantly the replicative response induced by submaximal concentrations of epidermal growth factor (EGF). In vivo, at the end of dietary treatment all animals fed on CA or DCA but not those fed on either CDCA, or UDCA, or tauroursodeoxycholate (TUDCA) developed cholestatic hepatitis and a burst of damage-induced hepatocyte proliferation. After 40% partial hepatectomy (PH), CA- and DCA-treated groups underwent a deterioration of cholestatic hepatitis. On the other hand, in CDCA-, and UDCA-, and TUDCA-treated groups liver histology, serum glutamic pyruvic transaminase (SGPT) and cholestasis indices did not change significantly compared with controls. As far as the proliferative activity, a significant increase was observed not only in CA and DCA but also in UDCA- and TUDCA-fed groups compared with controls, whereas a slight decrease was observed in CDCA-treated animals. In conclusion, our data indicate that conjugated BSs had only a modest stimulatory effect on hepatocyte proliferation in vitro. However, in vivo, in PH rats, UDCA or TUDCA treatment determined a further increase of hepatocellular proliferation not attributable to hepatotoxic effects. Our result suggest that modifications of bile acid pool could modulate hepatocellular proliferation.
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Ácidos y Sales Biliares/farmacología , Hígado/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Ácido Quenodesoxicólico/farmacología , Ácido Cólico , Ácidos Cólicos/farmacología , Ácido Desoxicólico/farmacología , Hepatectomía , Hígado/citología , Hígado/inmunología , Regeneración Hepática/efectos de los fármacos , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Endogámicas F344 , Ácido Tauroquenodesoxicólico/farmacología , Ácido Ursodesoxicólico/farmacologíaRESUMEN
To determine the role of NK cells in regulation of tissue growth, the phenotype and function of liver-resident NK cells were studied after 70% partial hepatectomy in rats. The process of liver regeneration was generally completed by day 14. In contrast, the number of liver-resident NK cells (NKR-P1bright) was restored as early as day 3 after partial hepatectomy. However, spontaneous functions of liver-resident NK cells, including killing of YAC-1 and P815 targets, Ab-dependent cellular cytotoxicity, and redirected killing via NKR-P1, were continuously suppressed throughout the entire period of liver regeneration (from 3 h to 14 days). Augmentation of NK cytotoxicity against P815 targets and induction of NK cell adherence to plastic following 24 h of IL-2 stimulation showed a similar pattern of suppression. However, IL-2-induced augmentation of YAC-1 killing, proliferation and generation of adherent NK cells, and LAK activity in 5- to 7-day cultures were found to be suppressed only during the first 24 h and increased between days 2 and 7 after hepatectomy. Sorted NK cells (> or = NKR-P1bright) from liver-resident mononuclear leukocytes 24 h after partial hepatectomy showed the same pattern of suppression as unsorted mononuclear leukocytes. In contrast to liver-resident NK cells, no significant changes were detected in peripheral blood or spleen NK cells of rats following partial hepatectomy. Of particular interest, in normal liver, hepatocytes were resistant to NK lysis, while resident NK cells were cytotoxic for various NK-sensitive targets. In contrast, during the early period of liver regeneration, when hepatocytes were sensitive to lysis by liver-resident NK cells of normal rats, NK cells obtained from regenerating liver tissues were unable to mediate cytotoxicity. At the final phase of liver regeneration (days 7-14 after hepatectomy), both resistance of hepatocytes to killing by NK cells and cytotoxicity of liver-resident lymphocytes against hepatocytes from regenerating liver were simultaneously restored. In vivo depletion of NK cells by injection of rats with anti-NKR-P1 mAb resulted in a significant augmentation of liver regeneration subsequent to partial hepatectomy. Our data suggest that liver-resident NK cells may be involved in regulation of the extent of liver regeneration.
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Células Asesinas Naturales/inmunología , Regeneración Hepática/inmunología , Hígado/inmunología , Animales , Anticuerpos Monoclonales , Citotoxicidad Inmunológica , Hepatectomía , Tolerancia Inmunológica , Hígado/citología , Depleción Linfocítica , Masculino , Fenotipo , Ratas , Ratas Endogámicas F344 , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Despite numerous studies on the effects of bile salts therapy in chronic liver disease, there are no reports on the influence such therapy has on hepatocyte proliferation. The aim of this preliminary study was to evaluate the effect of TUDCA on hepatocyte proliferation in 5 patients with HCV-correlated chronic liver disease. All patients were treated with TUDCA (10-13 mg/day) for three months and the determination of PCNA (Proliferating Cell Nuclear Antigen) expression was used to assess the proliferative activity of hepatocytes at the beginning and at the end of treatment. TUDCA reduced both ALT and Knodell's score in the 5 patients in whom a significant increase of PCNA-LI (p < 0.05) was observed after treatment. TUDCA administration seems to stimulate hepatocyte proliferation in man.
Asunto(s)
Hepatopatías/patología , Ácido Tauroquenodesoxicólico/uso terapéutico , División Celular/efectos de los fármacos , Enfermedad Crónica , Femenino , Humanos , Hepatopatías/tratamiento farmacológico , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
The proliferative response of the rat liver was measured after temporary or permanent total biliary obstruction (BDO) and in different regions after selective ligation of the lobar ducts draining the right 60% of the hepatic mass. The results were compared with those after 70% partial hepatectomy (PH). Cell proliferation was assessed globally by measuring DNA synthesis and stratified to the separate cell populations with cytostaining techniques that allowed distinction of hepatocytes, duct cells, and nonparenchymal cells (NPCs). In selected experimental groups, gene expression was determined of transforming growth factor-beta 1 (TGF beta-1), prothrombin, c-erb-B2, transforming growth factor alpha (TGF alpha), human Cyclophilin (CyP), and 28S ribosomal RNA. The stimulation of a proliferative response to total BDO required obstruction for longer than 24 hours, but after this deligation did not switch off regeneration. In the first week after permanent BDO, there was progressive infiltration of NPCs, fibrous linkage of some portal areas, and a crescendo of DNA synthesis that was obvious at 24 hours, maximal at 48 hours, and back nearly to baseline at 6 days. At the 2-day mark, the bile duct cells had a 17-fold increase in proliferation, accompanied by a threefold to fourfold increase in hepatocyte renewal. Little or no increase in expression of TGF alpha or the hepatocyte-specific prothrombin gene was detectable in the first 48 hours, whereas levels of the oncogene c-erb-B2 that is associated with cholangiocarcinoma were expressed from 48 to 96 hours.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Conductos Biliares/patología , Colestasis/patología , Regulación de la Expresión Génica , Proto-Oncogenes , Animales , Bromodesoxiuridina/metabolismo , División Celular , Colestasis/metabolismo , ADN/biosíntesis , Hepatectomía , Masculino , Ratas , Ratas Endogámicas F344 , Receptor ErbB-2/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Subcutaneous injections of hormone triiodothyronine in rats resulted in peak blood levels at 24 hr with return to baseline by 96 hr. The injections stimulated a liver regeneration response that resembled in timing and in magnitude of DNA synthesis (peak, 24 hr) that induced by 40% hepatic resection. The principal proliferation was of hepatocytes. Although there were some temporal differences from the gene expression of transforming growth factor-alpha, transforming growth factor-beta, and c-Ha-ras that are known to follow partial hepatectomy, the overall profile of these changes was similar to those after partial resection. The effect was liver specific and could be reproduced three times with no diminution in response in the same animal with injections at 10-day intervals. No response was detected in kidney or intestine. This effect in intact animals contrasted with the minimal ability of triiodothyronine to stimulate hepatocytes in culture. However, when the culture medium was enriched with epidermal growth factor, there was a dose-related response to triiodothyronine. The totality of these experiments provides a preliminary basis for the creation with pharmacological techniques of an in vivo hyperplastic hepatic condition permissive of transfection of new genes, as an alternative to partial hepatectomy. Although triiodothyronine was the test agent used, other hepatic growth factors singly or in combination could be candidates for this purpose.