RESUMO
The aim is to describe the molecular epidemiology and perform a genomic characterization of hepatitis B virus (HBV) circulating in Mar del Plata and to identify the origin and diversification patterns of the most prevalent genotype. The S gene and the region encompassing the X gene, basal core promoter (BCP), and precore (preC) was analyzed in 56 samples. They were genotyped as: 80% F1b, 9% A2, 7% D3, and 2% D1. A recombinant F4/D2 genome was detected. The double substitution G1764A/A1762T at the BCP (reduced HBeAg expression) was found in 20% F1b, 2% A2, 2% D1, and 2% D3 samples. A unique D3 presented the G1896A substitution at the preC (HBeAg negative phenotype). A 13% of the samples showed mutations at the HBsAg "a" immunodeterminant (escape from neutralizing antibodies). Mutations at the polymerase (antiviral resistance) were found in 52% of the samples. Coalescent analysis of subgenotype F1b, the most prevalent in the city, showed that viral diversification in Mar del Plata started by year 2000. F1b was the most prevalent genotype detected, being a characteristic of actual HBV infections in Mar del Plata. Local HBV exhibit clinically relevant mutations, but a minority of them was shown to be associated to potential vaccination escape or antiviral resistance. Nevertheless, further studies are needed to determine whether any of these mutants could pose a threat to prevention, diagnosis, or treatment.
Assuntos
Evolução Molecular , Genótipo , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B/virologia , Adulto , Idoso , Argentina/epidemiologia , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Regiões Promotoras Genéticas , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias , Adulto JovemRESUMO
BACKGROUND: HBV-X protein is associated with the pathogenesis of HBV related diseases, specially in hepatocellular carcinomas of chronic patients. Genetic variability of the X gene includes genotypic specific variations and mutations emerging during chronic infection. Its coding sequence overlaps important regions for virus replication, including the basal core promoter. Differences in the X gene may have implications in biological functions of the protein and thus, affect the evolution of the disease. There are controversial results about the consequences of mutations in this region and their relationship with pathogenesis. The purpose of this work was to describe the diversity of HBV-X gene in chronic hepatitis patients infected with different genotypes, according to liver disease. METHODS: HBV-X gene was sequenced from chronic hepatitis B patient samples, analyzed by phylogeny and genotyped. Nucleotide and aminoacid diversity was determined calculating intragenetic distances. Mutations at 127, 130 and 131 aminoacids were considered in relation to liver disease. RESULTS: The most prevalent genotype detected in this cohort was F (F1 and F4), followed by D and A. Most of the samples corresponding to genotypes A and F1 were HBeAg(+) and for genotypes D and F4, HBeAg(-) samples were represented in a higher percentage. Intragenetic distance values were higher in HBeAg(-) than in positive samples for all genotypes, and lower in overlapped regions, compared to single codification ones. Nucleotide and aminoacid diversities were higher in HBeAg(-), than in HBeAg(+) samples. CONCLUSIONS: Independently of the infecting genotypes, mutations at any of 127, 130 and/or 131 aminoacid positions and HBeAg(-) status were associated with mild liver disease in this cohort.
Assuntos
Variação Genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Transativadores/genética , Substituição de Aminoácidos , DNA Viral/química , DNA Viral/genética , Genótipo , Hepatite B Crônica/patologia , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Filogenia , Análise de Sequência de DNA , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide, associated with a high mortality rate. HCC patient's prognosis remains unfavorable, mainly due to late diagnosis and treatment instauration delay. Thus, there is an urgent requirement for the development of new therapeutical options, particularly for advanced HCC patients. Polyphenols are specialized plant metabolites, which have been broadly characterized for their beneficial effects on human health. Potato is one of the main sources of these compounds in human diet, due to its high consumption. The objectives of this study were to: characterize the polyphenolic composition, analyze the antioxidant properties, evaluate the cytotoxic activity in human tumoral hepatocytes, and describe the molecular mechanisms involved in cell death, of potato polyphenolic extracts (PPEs) from the pigmented Andigena cultivar CL658. The results showed that phenolic acids were the main group of polyphenols in the extracts, chlorogenic acid being the major compound, followed by caffeic acid. HPLC-DAD profile showed pelargonidin as the principal anthocyanin. The treatment of HCC cell lines with the cytotoxic concentration 50 (CC50 ) of the PPEs resulted in cytotoxicity, evidenced by increases in the percentage of dead cells (5-10%) after treatments. This cytotoxic effect was mainly due to apoptosis activation, evidenced by modulation of the Bcl-2 family proteins expression, in favor of pro-apoptotic members over anti-apoptotic in the final balance. PPEs also induced autophagy, indicated by increased LC3 expression (122-274%) and autophagosomes, and reduced p62 expression (32-48%), in variable magnitudes according to the treated cell line. Overall, these results support the potential use of PPEs from the cultivar CL658 as a source of bioactive compounds for novel treatments against HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Solanum tuberosum , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Apoptose , Autofagia , Polifenóis/farmacologia , Antineoplásicos/farmacologia , Extratos Vegetais/farmacologia , Linhagem Celular TumoralRESUMO
In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes.
Assuntos
Apoptose , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Mutação , Regiões Promotoras Genéticas , Replicação Viral , Linhagem Celular , Vírus da Hepatite B/genética , Hepatócitos/fisiologia , HumanosRESUMO
Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.
Assuntos
Autofagia/genética , Genótipo , Vírus da Hepatite B/genética , DNA Viral/genética , Vírus da Hepatite B/patogenicidade , Hepatócitos/virologia , Humanos , Lisossomos/genética , Lisossomos/virologia , Mutação , Regiões Promotoras Genéticas/genética , Proteólise , Replicação Viral/genéticaRESUMO
The hepatitis B virus X protein (HBV-X) is a multifunctional regulatory protein associated with the pathogenesis of liver disease in chronic HBV infection. Basal core promoter mutations (BCP), associated with the clinical course of chronic HBV infection, affect HBV-X at 130-131 positions. The role of these mutations on HBV-X biological activity remains largely unknown. The aim of this study was to analyze the impact of the presence of different amino acids at 130-131 positions of HBV-X on the biological activity of the protein. Transient expression of wild type and mutant F1b and F4 HBV-X increased cell mortality by the induction of apoptosis in human hepatoma cells. The wild type and mutant HBV-X differentially modulate the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-X) regulatory proteins of the Bcl-2 family. Furthermore, the expression of HBV-X variants of both subgenotypes induced autophagy of human tumoral hepatocytes. In conclusion, HBV-X variants of the Latin American HBV F genotype promotes human hepatocytes death by the induction of apoptosis and autophagy. The results of this work describe some of the molecular mechanisms by which HBV-X variants contribute to the pathogenesis of liver diseases in the infected liver and help to the biological characterization of genotype F, responsible of the majority of HBV infections in Argentina.
Assuntos
Apoptose , Autofagia , Vírus da Hepatite B/fisiologia , Hepatócitos/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Transativadores/metabolismo , Linhagem Celular Tumoral , Genótipo , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
AIM: To study the mechanism of cytotoxicity of a new active 5-alkyl resorcinol [1, 3-dihydroxy-5- (tridec-4', 7'-dienyl) benzene] isolated from Lithraea molleoides leaves on liver tumor cells. METHODS: Human hepatocarcinoma cell lines (HepG2 and Hep3B) in culture were treated with inhibitory concentrations, 50% of the compound, for 24 h. The induction of apoptosis was detected in treated cells by analysis of DNA fragmentation, DNA content, and acridine orange and propidium iodide staining. RESULTS: After 24 h of 5-alkyl resorcinol treatment, both cell lines showed: (1) the typical morphological alterations of apoptosis; (2) DNA fragmentation, detected by laddering and appearance of a subG0 population by flow cytometry; and (3) condensed and fragmented nuclei by acridine orange-propidium iodide staining. CONCLUSION: Based on the results, this compound exerts its cytotoxic effect in both hepatocellular cell lines through apoptotic cell death. For Hep3B, cells with mutated p53 and Fas, apoptosis would proceed by p53- or Fas-independent pathways.
Assuntos
Anacardiaceae/química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Resorcinóis/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Citotoxinas/análise , Citotoxinas/farmacologia , Citotoxinas/uso terapêutico , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Mutação , Extratos Vegetais/análise , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
The recent history of the hepatitis C virus (HCV) subtypes 1a and 1b in the central region of Argentina is hypothesized by phylogeographic reconstruction using coalescent based Bayesian analyses. Direct partial E2 sequences from HCV 1a and 1b infected patients attending different health-care centers of the country were analyzed. The inferred date of the most recent common ancestor (tMRCA) for HCV-1a was: 1962 (between 1943 and 1977) and for HCV-1b was earlier: 1929 (between 1895 and 1953). Diverse ancestral populations were inferred from both subtypes in Córdoba and in Buenos Aires cities and after that, HCV spread within and between larger cities and to other smaller cities. The analyses suggested that HCV-1b was dispersed first and it is currently in a stationary phase whereas HCV-1a was dispersed latter and it is still in a growth phase. Finally, as it was observed in the developed countries, while the transmission of HCV-1b appears to have been somehow prevented, the HCV-1a may still represent a concern in the public health. Further work should be carried out to address their current transmission rate (and its main transmission route) in the Argentinean population.
Assuntos
Genótipo , Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/virologia , Argentina/epidemiologia , Geografia , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C/história , História do Século XX , História do Século XXI , Humanos , Dados de Sequência Molecular , Filogenia , Filogeografia , Estudos Retrospectivos , Análise de Sequência de DNA , Proteínas do Envelope Viral/genéticaRESUMO
The aim of this work was to describe the current molecular epidemiology and genetic diversity of HBV in Mar del Plata, an important Argentinean touristic city. The phylogenetic analysis of 29 HBV DNA positive serum samples showed that F1b was the predominant subgenotype (sgt, 62.1%), followed by sgt A2 (13.8%) and sgt F4, gt D and gt G (6.9% each). Among anti-HBc IgM positive samples, 75.0% were sgt F1b, followed by sgt F4 (12.5%), sgt A2 (6.25%) and sgt D (6.25%). Three recombinant full length genomes were found: two G/F1b (some of the first gt G detected in Argentina) and one F4/D2. The circulation of clinical important mutations in the city was described. Mutations at the HBsAg were detected in 34.5% of the analyzed samples, associated with laboratory diagnosis and antiviral treatment failures, immune escape and hepatocellular carcinoma. Most of the samples presented wild type BCP/PC sequences. Coalescence analysis for the most prevalent sgt F1b estimated that the diversification mainly occured during mid '90s and the tMRCA was estimated in 1987. Finally, the high presence of the autochthonous sgt F1b, associated with the anti-HBc IgM positive infection and its present-day diversification process, shows the strong impact of internal human migratory movements into the current population of Mar del Plata.
Assuntos
Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B/virologia , Sequência de Aminoácidos , Argentina/epidemiologia , DNA Viral/sangue , DNA Viral/genética , Hepatite B/epidemiologia , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Humanos , Dados de Sequência Molecular , Filogenia , Recombinação Genética/genética , Alinhamento de SequênciaRESUMO
The global epidemiology of hepatitis C virus (HCV) may be roughly described by two groups of genotypes: the worldwide distributed ones (subtypes 1a, 1b, 2a and 3a, among others) and the endemic ones (subtypes 4a, 5a, 6a, among others). Epidemiological and population dynamic studies of the worldwide distributed genotypes have shown that subtypes 1a and 3a are common among intravenous drug users (IDUs) and that they are also in expansion in some countries. The molecular survey of HCV provides some clues about the epidemiological status of the infections in a local scale and the phylogenetic and demographic reconstruction analyses complement this study by inferring whether the infections of certain subtypes are in a steady state or expanding. Here, a molecular survey of the HCV variants that circulate in the touristic city of Mar del Plata (Buenos Aires, Argentina) was performed in samples obtained from 42 patients. The subtypes detected were 1a (32 patients), 3a (8 patients) and 1b (2 patients). The demographic history of subtype 1a inferred using the sequence data showed an exponential growth in the 1990's. The period of viral expansion was delayed compared with that observed for the same genotype in other countries where the transmission was associated with IDUs. Also, the phylogeographic analysis of HCV-1a showed a statistically significant association between the location of the samples and the phylogeny, which may be the result of the local transmission of HCV in the city. The molecular analysis helped in the description of the complex epidemiological context of a touristic city, and pointed out that some sanitary measures should be taken in order to reduce the transmission of HCV (and maybe of HIV) among IDUs.
Assuntos
Hepatite C/epidemiologia , Hepatite C/virologia , Argentina/epidemiologia , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/patogenicidade , Humanos , Filogenia , Abuso de Substâncias por Via IntravenosaRESUMO
Recent evidences indicate new roles for the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in essential mammalian cell processes, such as apoptosis and proliferation. To clarify the involvement of this protein in growth and programmed cell death in the liver, cell models of hepatocytes in culture were used to study GAPDH expression, localization and enzymatic activity in hepatocyte proliferation and apoptosis. GAPDH expression in cell compartments was studied by Western blot. Nuclear expression of GAPDH increased in apoptosis, and cytoplasmic expression was elevated in apoptosis and proliferation. Subcellular localization was determined by GAPDH immunostaining and confocal microscopic analysis. Quiescent and proliferating hepatocytes showed cytoplasmic GAPDH, while apoptotic cells showed cytoplasmic but also some nuclear staining. The glycolytic activity of GAPDH was studied in nuclear and cytoplasmic cell compartments. GAPDH enzymatic activity increased in the nucleus of apoptotic cells and in cytoplasms of apoptotic and proliferating hepatocytes. Our observations indicate that during hepatocyte apoptosis GAPDH translocates to the nucleus, maintaining in part its dehydrogenase activity, and suggest that this translocation may play a role in programmed hepatocyte death. GAPDH over-expression and the increased enzymatic activity in proliferating cells, with preservation of its cytoplasmic localization, would occur in response to the elevated energy requirements of dividing hepatocytes. In conclusion, GAPDH plays different roles or biological activities in proliferating and apoptotic hepatocytes, according to its subcellular localization.
Assuntos
Apoptose , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hepatócitos/citologia , Hepatócitos/enzimologia , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Prothymosin alpha is an acidic protein, reported to be involved in cell proliferation and apoptosis, although its precise function in both processes are still unknown. Due to the importance of these processes in the pathogenesis of hepatic diseases and the need to understand the molecular mechanisms underlying these diseases we aimed to investigate the behavior of this protein in liver growth and apoptosis, in two models of hepatocytes in culture. Prothymosin alpha expression varied throughout the hepatocyte cell cycle, according to its progression. Proliferating hepatocytes showed increased expression of the protein, while apoptotic ones showed decreased levels. The subcellular location of prothymosin alpha differed according to the different phases of the cell cycle. Thus, it appeared with a stippled and widely dispersed pattern throughout the nucleus in quiescent and proliferating hepatocytes, while it became cytoplasmic in mitotic and late apoptotic cells. These results are in agreement with the idea that high levels of prothymosin alpha need to be present in the nucleus for proliferation, and programmed cell death requires low levels of prothymosin alpha outside of the nucleus. The differences in prothymosin alpha expression and localization during hepatocyte proliferation and apoptosis suggest that this protein may have a pleiotropic function that depends not only on its availability but also on its various localizations in different subcellular compartments.
Assuntos
Apoptose , Hepatócitos/citologia , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Masculino , Precursores de Proteínas/genética , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Timosina/genética , Timosina/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
La apoptosis es una forma de muerte celular que ocurre en una amplia variedad de enfermedades hepáticas. En ésta revisión se presentan generalidades del proceso y métodos de detección; la apoptosis como generadora de enfermedad en la hepatitis alcohólica, hepatitis virales, hepatocarcinoma, colestasis, rechazo de transplantes, enfermedad de Wilson y consideraciones sobre la posible implementación de terapéuticas que modulen la apoptosis en las enfermedades hepáticas