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1.
Euro Surveill ; 22(4)2017 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-28181902

RESUMEN

Since early November 2016, the number of laboratory-confirmed norovirus infections reported in Germany has been increasing steeply. Here, we report the detection and genetic characterisation of an emerging norovirus recombinant, GII.P16-GII.2. This strain was frequently identified as the cause of sporadic cases as well as outbreaks in nine federal states of Germany. Our findings suggest that the emergence of GII.P16-GII.2 contributed to rising case numbers of norovirus gastroenteritis in Germany.


Asunto(s)
Infecciones por Caliciviridae/epidemiología , Brotes de Enfermedades , Gastroenteritis/virología , Genotipo , Norovirus/clasificación , Norovirus/genética , Infecciones por Caliciviridae/virología , Niño , Preescolar , Diarrea/epidemiología , Diarrea/virología , Notificación de Enfermedades/estadística & datos numéricos , Gastroenteritis/epidemiología , Variación Genética , Alemania/epidemiología , Humanos , Lactante , Norovirus/aislamiento & purificación , Filogenia , ARN Viral/genética , Estaciones del Año , Análisis de Secuencia de ADN
2.
J Gen Virol ; 97(8): 1865-1876, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27147296

RESUMEN

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.


Asunto(s)
Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/sangre , Linfocitos T/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Línea Celular , Células Cultivadas , Hepacivirus/genética , Hepatocitos/virología , Humanos , Ratones Endogámicos BALB C , Ratones Transgénicos , Microscopía Electrónica , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Proteínas del Núcleo Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Virosomas/genética , Virosomas/inmunología , Virosomas/metabolismo , Virosomas/ultraestructura
3.
J Viral Hepat ; 22(1): 55-63, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24548489

RESUMEN

Hepatitis D virus (HDV) infection is acquired as a co- /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10-20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty-one HDV isolates in Vietnamese HBsAg-positive patients. HDV subgenomic and full-length genome sequences were obtained using newly established HDV-specific RT-PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full-length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg-positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro-epidemiology of HDV and subsequent pathophysiology in chronic HBV- /HDV-related liver diseases.


Asunto(s)
Variación Genética , Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B/complicaciones , Hepatitis D/virología , Virus de la Hepatitis Delta/clasificación , Virus de la Hepatitis Delta/genética , Adulto , Anciano , Pueblo Asiatico , Femenino , Genotipo , Virus de la Hepatitis Delta/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Recombinación Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia , Adulto Joven
4.
J Viral Hepat ; 21(10): 744-50, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24329913

RESUMEN

Acute hepatitis B virus (aHBV) infection can lead to fulminant liver failure, which likely is prevented by early lamivudine therapy. Even nonfulminant but severe acute hepatitis B can lead to significant morbidity and impaired quality of life. Therefore, lamivudine was evaluated in patients with severe aHBV in a placebo-controlled trial. Patients with severe aHBV infection (ALT >10× ULN, bilirubin >85 µm, prothrombin time >50%) were prospectively treated with lamivudine 100 mg/day or with placebo within 8 days after the diagnosis. The primary end point was time to bilirubin <34.2 µm. Secondary end points were time to clear HBsAg and HBV-DNA, development of anti-HBs and normalization of ALT. Eighteen cases were randomized to lamivudine, 17 to placebo. 94% of patients were hospitalized. No individual progressed to hepatic failure; all but one patient achieved the primary end point. Due to smaller than expected patient numbers, all study end points did not become statistically significant between treatment arms. Median time end points [in days] were bilirubin <34.2 µm (26.5 vs 32), ALT normalization (35 vs 48) and HBsAg clearance (48 vs 67) referring to earlier recovery under lamivudine, in contrast to loss of HBV-DNA (62 vs 54) and development of anti-HBs (119 vs 109). In all but two patients (one in every group), HBsAg clearance was reached in the study. Adverse events occurred more frequently during lamivudine therapy, but did not reach statistical significance. Lamivudine may ameliorate severe aHBV infection, but limited patient numbers prevented definite conclusions.


Asunto(s)
Antivirales/administración & dosificación , Hepatitis B/tratamiento farmacológico , Lamivudine/administración & dosificación , Placebos/administración & dosificación , Adulto , Alanina Transaminasa/sangre , Antivirales/efectos adversos , Bilirrubina/sangre , ADN Viral/sangre , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Lamivudine/efectos adversos , Persona de Mediana Edad , Estudios Prospectivos , Resultado del Tratamiento
5.
J Viral Hepat ; 20(10): 687-98, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24010643

RESUMEN

Hepatitis B virus infection is a high-risk factor for hepatocellular carcinoma. The human major histocompatibility complex class I chain-related gene A (MICA) is a ligand of the NKG2D receptor that modulates the NK and T-cell-mediated immune responses and is associated with several diseases. This study determined the effects of MICA polymorphisms during HBV infection and HBV-induced HCC. We conducted a case-controlled study in a Vietnamese cohort and genotyped ten functional MICA polymorphisms including the microsatellite motif in 552 clinically classified hepatitis B virus patients and 418 healthy controls. The serum soluble MICA levels (sMICA) were correlated with MICA variants and liver enzyme levels. We demonstrated a significant contribution of MICA rs2596542G/A promoter variant and nonsynonymous substitutions MICA-129Met/Val, MICA-251Gln/Arg, MICA-175Gly/Ser, triplet repeat polymorphism and respective haplotypes with HBV-induced HCC and HBV persistence. The circulating sMICA levels in HBV patient groups were elevated significantly compared with healthy controls. A significant contribution of studied MICA variants to sMICA levels was also observed. The liver enzymes alanine amino transferase (ALT), aspartate transaminase (AST), total bilirubin and direct bilirubin were positively correlated with sMICA levels suggesting sMICA as a biomarker for liver injury. We conclude that MICA polymorphisms play a crucial role in modulating innate immune responses, tumour surveillance and regulate disease susceptibility during HBV infection.


Asunto(s)
Carcinoma Hepatocelular/genética , Predisposición Genética a la Enfermedad , Virus de la Hepatitis B/inmunología , Hepatitis B/complicaciones , Hepatitis B/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Vietnam , Adulto Joven
6.
J Clin Virol ; 111: 4-11, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30580015

RESUMEN

BACKGROUND: Viruses are the leading cause of acute gastroenteritis in children worldwide. Understanding of the occurrence and genetic diversity of these viruses can help to prevent infections. OBJECTIVES: The present study describes the presence, genetic diversity and possible recombination of five enteric viruses in children with gastroenteritis in Southwestern Nigeria. STUDY DESIGN: From August 2012 to December 2013, stool samples and sociodemographic data of 103 diarrheic children <5 years were collected to detect and characterize rotavirus A, norovirus, human astrovirus, aichivirus and sapovirus using PCR techniques followed by sequencing and phylogenetic analyses. RESULTS: At least one virus was identified in 58.3% (60/103) of the stool samples. Rotavirus, norovirus and astrovirus were detected in 39.8% (41/103), 10.7% (11/103), and 6.8% (7/103) respectively. Notably, aichivirus was detected for the first time in Nigeria (1/103; 0.97%). Sapovirus was not detected in the study. Co-infections with rotavirus were observed in eight samples either with norovirus or astrovirus or aichivirus. Phylogenetic analyses of different genome regions of norovirus positive samples provided indication for recombinant norovirus strains. A novel astrovirus strain closely related to canine astrovirus was identified and further characterized for the first time. CONCLUSIONS: Viruses are the common cause of acute gastroenteritis in Nigerian infants with rotavirus as most frequently detected pathogen. New norovirus recombinants and a not yet detected zoonotic astrovirus were circulating in Southwestern Nigeria, providing new information about emerging and unusual strains of viruses causing diarrhea.


Asunto(s)
Infecciones por Astroviridae/epidemiología , Astroviridae/clasificación , Infecciones por Caliciviridae/epidemiología , Gastroenteritis/epidemiología , Kobuvirus/clasificación , Norovirus/clasificación , Animales , Astroviridae/aislamiento & purificación , Preescolar , Diarrea/virología , Heces/virología , Femenino , Gastroenteritis/virología , Variación Genética , Humanos , Lactante , Recién Nacido , Kobuvirus/aislamiento & purificación , Masculino , Nigeria/epidemiología , Norovirus/aislamiento & purificación , Filogenia , Infecciones por Picornaviridae/epidemiología , ARN Viral/genética , Virus Reordenados/clasificación , Rotavirus/genética , Rotavirus/aislamiento & purificación , Zoonosis/virología
7.
Pathologe ; 29 Suppl 2: 112-7, 2008 Nov.
Artículo en Alemán | MEDLINE | ID: mdl-18820926

RESUMEN

Molecular biological methods have confirmed the pathogenetic role of enteroviruses, primarily coxsackieviruses of group B (CVB), in the induction and maintenance of inflammatory cardiomyopathy. More recently, adenoviruses, various herpes viruses, and increasingly parvovirus B19 (B19) have been identified as potential cardiotropic agents. While cardiac myocytes are target cells for enterovirus and adenovirus infections with virus-induced cytolysis, B19-associated inflammatory cardiomyopathy is characterized by infection of intracardiac endothelial cells of small arterioles and veins, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells, and secondary myocyte necrosis. Recent observations showed that B19 is involved in intracellular calcium regulation by the viral phospholipase. B19-induced caspase activation can lead to proinflammatory/proapoptotic processes through dysregulation of STAT signaling. These cellular interactions may contribute to mechanisms by which B19 establishes persistent infection in endothelial cells and play a critical role in viral pathogenesis of inflammatory cardiomyopathy.


Asunto(s)
Miocarditis/patología , Infecciones por Parvoviridae/patología , Virosis/patología , Apoptosis/fisiología , Calcio/metabolismo , Cardiomiopatías/patología , Caspasas/metabolismo , Vasos Coronarios/patología , Endotelio Vascular/patología , Humanos , Microcirculación/fisiología , Miocitos Cardíacos/patología , Necrosis , Parvovirus B19 Humano/patogenicidad , Factores de Transcripción STAT/fisiología , Transducción de Señal/fisiología , Virulencia
8.
Int J Infect Dis ; 66: 5-13, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29097248

RESUMEN

BACKGROUND: People who inject drugs (PWID) are at high risk of hepatitis B virus (HBV) infection by sharing needles and drug use paraphernalia. In Germany, no routine surveillance of HBV prevalence and vaccination coverage among PWID exists. METHODS: Socio-demographic and behavioural data were collected between 2011 and 2014 through face-to-face interviews, during a bio-behavioural survey of PWID recruited in eight German cities. Dried blood spots (DBS) prepared with capillary blood were tested for HBV markers. Factors associated with past/current HBV infection and vaccination status were analysed by univariable and multivariable analysis using logistic regression. The validity of self-reported HBV infection and vaccination status was analysed by comparison to the laboratory results. RESULTS: Among 2077 participants, the prevalence of current HBV infection was 1.1%, of past HBV infection was 24%, and of vaccine-induced HBV antibodies was 32%. No detectable HBV antibodies were found in 43%. HBV infection status was significantly associated with study city, age, years of injecting, use of stimulants, migration status, and homelessness; HBV vaccination status was significantly associated with study city, age, and level of education. Correct infection status was reported by 71% and correct vaccination status by 45%. CONCLUSIONS: HBV seroprevalence among PWID was about five times higher than in the general population in Germany, confirming PWID as an important risk group. Targeted information campaigns on HBV and HBV prevention for PWID and professionals in contact with PWID need to be intensified. Routinely offered HBV vaccination during imprisonment and opioid substitution therapy would likely improve vaccination rates among PWID.


Asunto(s)
Hepatitis B/etiología , Abuso de Sustancias por Vía Intravenosa/complicaciones , Adulto , Ciudades , Susceptibilidad a Enfermedades , Femenino , Alemania/epidemiología , Hepatitis B/complicaciones , Hepatitis B/epidemiología , Anticuerpos contra la Hepatitis B/análisis , Vacunas contra Hepatitis B/administración & dosificación , Humanos , Modelos Logísticos , Masculino , Compartición de Agujas , Prevalencia , Factores de Riesgo , Asunción de Riesgos , Estudios Seroepidemiológicos
9.
Circulation ; 111(7): 879-86, 2005 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-15710767

RESUMEN

BACKGROUND: The etiology of left ventricular (LV) isolated diastolic dysfunction often remains unclear. In the present study, we report a strong association between parvovirus B19 (PVB19) genomes and isolated LV diastolic dysfunction. METHODS AND RESULTS: In 70 patients (mean+/-SD age, 43+/-11 years) admitted with exertional dyspnea and/or reduced exercise tolerance despite preserved LV systolic contractility (ejection fraction=68%), isolated diastolic dysfunction was clinically suspected. Patients with classic risk factors for diastolic dysfunction such as hypertension, coronary heart disease, diabetes mellitus, or pulmonary disease had been excluded. Diastolic function was assessed by echocardiography and LV and RV catheterization. Endomyocardial biopsies (EMBs) were analyzed for the presence of storage or infiltrative diseases or myocarditis, including molecular screening for cardiotropic virus genomes. In a substudy of 24 patients who reported atypical angina, coronary endothelial function was additionally investigated with a coronary Doppler flow-wire technique. In 37 of 70 patients (53%), isolated diastolic dysfunction was confirmed as the cause of their clinical symptoms. No evidence for cardiac storage or infiltrative diseases was found in these cases, but in 35 of 37 of these patients (95%), cardiotropic virus genomes were detected in EMBs (P<0.001). PVB19 was the most frequent pathogen in 31 of 37 patients (84%). In a subgroup of 10 patients with diastolic dysfunction and coexisting endothelial dysfunction, all 10 (100%) were PVB19 positive. CONCLUSIONS: PVB19 genomes were predominant in patients with unexplained, isolated diastolic dysfunction. A strong association with the incidence of endothelial dysfunction was obvious, consistent with the hypothesis that PVB19-induced endothelial dysfunction may be a possible pathomechanism underlying diastolic dysfunction.


Asunto(s)
Diástole , Infecciones por Parvoviridae/complicaciones , Parvovirus B19 Humano , Disfunción Ventricular Izquierda/virología , Adulto , Biopsia , Angiografía Coronaria , Endotelio/patología , Endotelio/virología , Femenino , Genoma Viral , Corazón/fisiopatología , Corazón/virología , Humanos , Masculino , Persona de Mediana Edad , Parvovirus B19 Humano/genética , Prevalencia , Disfunción Ventricular Izquierda/epidemiología , Disfunción Ventricular Izquierda/etiología
10.
J Clin Pathol ; 59(10): 1114-5, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17021141

RESUMEN

A panel of five quasimonomorphic mononucleotide repeats that dispenses with the need to analyse corresponding germline DNA was proposed by Suraweera et al for the detection of high-frequency microsatellite instability (MSI) in colorectal cancer. Using this panel, a simplified and a more sensitive (compared with the original) algorithm (p<0.05) was developed to define the instability of each repeat by assessing the morphological shape of its plot and not its absolute length. 103 cases of colorectal tumours were investigated and the results compared with those obtained by the analysis of five consensus microsatellites (Bethesda reference panel). By the proposed method, a higher specificity, but no loss of sensitivity, was found. Thus, the use of the five mononucleotide repeats in combination with the modified assessment technique simplifies the assessment of MSI, while retaining the sensitivity of the Bethesda panel for the detection of high-frequency MSI.


Asunto(s)
Neoplasias Colorrectales/genética , Inestabilidad Genómica , Repeticiones de Microsatélite/genética , Algoritmos , ADN de Neoplasias/genética , Humanos , Reacción en Cadena de la Polimerasa/métodos , Estudios Retrospectivos , Sensibilidad y Especificidad
11.
Artículo en Inglés | MEDLINE | ID: mdl-16329659

RESUMEN

The human parvovirus B19 (PVB19), an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes such as hydrops fetalis, is the only currently known human pathogenic parvovirus. Recently, PVB19 has been identified as a causative agent of pediatric and adult inflammatory cardiac diseases. The first hints for a possible etiopathogenetic role of the PVB19 infection and the development of cardiac dysfunction were demonstrated by molecular biology methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR). In this regard, PVB19-associated inflammatory cardiomyopathy is characterized by infection of endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, and penetration of inflammatory cells in the myocardium.


Asunto(s)
Cardiomiopatías , Infecciones por Parvoviridae , Parvovirus B19 Humano , Cardiomiopatías/etiología , Cardiomiopatías/inmunología , Cardiomiopatías/virología , Endocardio/patología , Endocardio/virología , Genoma Viral , Humanos , Técnicas de Diagnóstico Molecular , Infecciones por Parvoviridae/complicaciones , Infecciones por Parvoviridae/inmunología , Parvovirus B19 Humano/clasificación , Parvovirus B19 Humano/genética , Parvovirus B19 Humano/inmunología , Parvovirus B19 Humano/fisiología
12.
J Mol Biol ; 246(4): 486-92, 1995 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-7877169

RESUMEN

Standard visualization of nucleic acids by electron microscopy requires the use of special spreading techniques. The most common method takes advantage of the formation of a complex between negatively charged nucleic acid molecules and a positively charged monolayer film of proteins or cationic agents. Here, we describe an alternative protocol for the rapid visualization of DNA by electron microscopy based on the complexes formed when nucleic acids are exposed to buffers containing polyamines in the presence of sodium chloride. This procedure has been devised for the detection and analysis of large DNA molecules, such as yeast artificial chromosomes, but can be applied to DNA molecules of small size as well. The formation of DNA-polyamine complexes stabilizes large DNA molecules in solution and prevents shearing. This property allows large DNA molecules to remain intact after passage through microcapillaries used in the generation of transgenic mice by microinjection of fertilized eggs.


Asunto(s)
Cromosomas Artificiales de Levadura/genética , Cromosomas Fúngicos/genética , ADN/química , ADN/ultraestructura , Poliaminas/química , ADN de Hongos/química , ADN de Hongos/ultraestructura , Electroforesis en Gel de Campo Pulsado , Microscopía Electrónica , Saccharomyces cerevisiae/genética , Cloruro de Sodio/química
13.
J Mol Biol ; 307(1): 183-96, 2001 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-11243813

RESUMEN

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.


Asunto(s)
ADN Circular/química , ADN Viral/química , Virus de la Hepatitis B/genética , Cromatina/química , ADN Circular/ultraestructura , ADN Viral/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas/metabolismo , Virus de la Hepatitis B/química , Peso Molecular , Conformación de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Nucleosomas/química , Staphylococcus aureus/enzimología , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismo
14.
Eur J Cell Biol ; 78(11): 832-41, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10604660

RESUMEN

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.


Asunto(s)
Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Mucinas/genética , Neoplasias Gástricas/genética , Empalme Alternativo , Secuencia de Bases , Biopsia , Western Blotting , Células Epiteliales/metabolismo , Mucosa Gástrica/patología , Humanos , Inmunohistoquímica , Mucinas/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/metabolismo , Células Tumorales Cultivadas
15.
Artículo en Inglés | MEDLINE | ID: mdl-16316397

RESUMEN

The human parvovirus B19 (PVB19), an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes such as hydrops fetalis, is the only known human pathogenic parvovirus so far. Although enteroviruses have long been considered the most common cause of inflammatory cardiomyopathy, PVB19 is emerging as a important candidate. Recent studies have indicated an association of PVB19 with paediatric and adult inflammatory cardiac disease. However, whether or not PVB19 has an impact on inflammatory cardiomyopathy in adult patients is still unclear. The first hints for a possible aetiopathogenetic role of the PVB19-infection and the development of cardiac dysfunction were demonstrated by molecular biology utilizing in situ hybridization (ISH) and polymerase chain reaction (PCR). According to available evidence, PVB19-associated inflammatory cardiomyopathy is characterized by infection of endothelial cells of small intracardiac arterioles and venules, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, and penetration of inflammatory cells into the myocardium.


Asunto(s)
Cardiomiopatía Dilatada/virología , Infecciones por Parvoviridae/complicaciones , Parvovirus B19 Humano/patogenicidad , Cardiomiopatía Dilatada/etiología , Humanos
16.
J Viral Hepat ; 12(2): 160-7, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15720531

RESUMEN

Numerous mutations in the hepatitis B virus (HBV) genome have been described, but in most cases their role in the pathogenesis of HBV infection is still unclear. Therefore, we analysed specific mutations in HBV-infected Vietnamese patients and assessed their potential relationship with their clinical outcome. A total of 153 HBV-infected Vietnamese patients with well-characterised clinical profiles were enrolled. None of the study participants had a history of alcohol or drug use and none received any antiviral or immunosuppressive therapy before or during the course of this study. The HBx- and core promoter regions were analysed by sequencing. The majority of isolates corresponded to genotype A. The presence of hepatitis B e antigen (HBeAg) was associated with significantly higher viral loads in the chronic HBV-infection group (P = 0.026). Double mutations in the core promoter (1762/1764) were more frequent in those with cancer than in noncancer patients (P < 0.01). Mutations at nucleotide (nt) 1766/1773 were found at low prevalence but with no obvious association to clinical presentation. Cytosine at nt 1858 was predominant but the stop codon mutation in the precore region was not detected. In the study, 4/48 hepatocellular carcinoma (HCC) patients revealed truncated HBx, whilst the serine to alanine mutation (codon 31) of HBx was more prevalent in cancer patients than in asymptomatic HBV carriers (P < 0.01). Thus, the low frequency of mutations indicates the relation of the absence of antiviral pressure in this population. The exclusively found prevalence of certain mutations detected in those with HBV-related carcinoma nevertheless indicates a degree of association with disease progression.


Asunto(s)
Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B/etnología , Hepatitis B/genética , Mutación , Adulto , Secuencia de Bases , Estudios de Casos y Controles , Estudios de Cohortes , ADN Viral/análisis , Progresión de la Enfermedad , Femenino , Marcadores Genéticos/genética , Hepatitis B/fisiopatología , Heterocigoto , Humanos , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Probabilidad , Regiones Promotoras Genéticas , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Vietnam
17.
Chromosoma ; 102(4): 249-52, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8486076

RESUMEN

Electron microscopic examination of nucleic acids requires the use of special spreading techniques. The classical method was developed by Kleinschmidt and Zahn in 1959. Modifications of this method increased sensitivity to allow detection of a total amount of about 1 x 10(-3) micrograms of single-stranded DNA and 2 x 10(-5) micrograms of double-stranded DNA. Here we describe two rapid and simple procedures increasing sensitivity by 1-2 orders of magnitude to visualize at least 1 x 10(-5) micrograms of single- and/or double-stranded DNA.


Asunto(s)
ADN/análisis , ADN/ultraestructura , Microscopía Electrónica/métodos , Animales , ADN de Cadena Simple/análisis , ADN de Cadena Simple/ultraestructura , Estudios de Evaluación como Asunto , Microquímica/métodos , Microquímica/estadística & datos numéricos , Microscopía Electrónica/estadística & datos numéricos , Ácidos Nucleicos Heterodúplex/análisis , Ácidos Nucleicos Heterodúplex/ultraestructura , Sensibilidad y Especificidad
18.
Chromosoma ; 103(9): 653-7, 1995 May.
Artículo en Inglés | MEDLINE | ID: mdl-7587588

RESUMEN

Heteroduplex analysis is commonly used to map homologous sequences in DNA:DNA or DNA:RNA hybrids in spread preparations by electron microscopy. However, the standard procedures are not suitable to detect the orientation of a fragment with a defined sequence in a hybrid molecule. Here, we describe an alternative protocol for the visualization of DNA:DNA "diheteroduplex" structures based on digoxigenin/anti-digoxigenin gold labeling that allows determination of the position and orientation of a fragment. Single-stranded polymerase chain reaction (PCR) generated fragments labeled at their 3' ends are hybridized to double-stranded plasmid DNA. Electron microscopy of spread preparations visualizes the gold label and, in combination with morphometric measurements, it is possible to determine the position and orientation of the fragment with the diheteroduplex molecule.


Asunto(s)
ADN de Cadena Simple/biosíntesis , Microscopía Electrónica/métodos , Ácidos Nucleicos Heterodúplex , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Mapeo Cromosómico , Genoma Viral , Virus de la Hepatitis B/genética , Inmunohistoquímica , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/genética
19.
J Virol ; 67(12): 7482-92, 1993 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8230469

RESUMEN

The B-lymphotropic papovavirus (LPV) productively infects only a subset of human B-lymphoma-derived cell lines while transfection of the viral genome yields infectious viral particles in a much wider variety of human hematopoietic cell lines. We have analyzed the contribution of a putative LPV receptor on the cell surface of B-cell lines in restricting the virus host range. In order to establish a quantitative virus binding assay for LPV, infectious virus particles were highly purified by metrizamide equilibrium density centrifugation and used as immunogens to raise seven mouse monoclonal antibodies specific for LPV VP1. Virus particle binding was quantitated in an indirect, nonradioactive assay with an LPV VP1-specific enzyme-linked immunosorbent assay. Binding of LPV particles to permissive human B-lymphoma cell line BJA-B occurred within minutes. Kinetics and capacity of binding were similar at 4 and 37 degrees C. A BJA-B cell was estimated to bind approximately 600 virus particles at conditions under which 50% of the administered virus was bound. The sialidase and trypsin sensitivities of the cellular virus binding moiety show that sialylated and proteinaceous components are necessary components of the LPV receptor on BJA-B cells. Despite a high binding capacity of BJA-B cells for simian virus 40, LPV binding was not significantly affected by a 20-fold excess of simian virus 40 particles, indicating that these related polyomaviruses do not bind to the same receptor on BJA-B cells. Reduction of LPV binding to sialidase-pretreated BJA-B cells was accompanied by a similar reduction of infection, indicating that virus binding may be a limiting factor in the LPV replicative cycle. The two highly LPV-permissive human B-lymphoma cell lines BJA-B and Namalwa displayed high virus binding whereas low and nonpermissive hematopoietic cell lines showed reduced or undetectable virus binding. We conclude that the inability of LPV particles to productively infect the nonpermissive human hematopoietic cell lines analyzed is probably due to the absence or insufficient expression of a functional cell surface receptor.


Asunto(s)
Linfocitos B/microbiología , Cápside/inmunología , Poliomavirus/patogenicidad , Receptores Virales/metabolismo , Anticuerpos Monoclonales , Anticuerpos Antivirales , Unión Competitiva , Proteínas de la Cápside , Centrifugación Isopicnica , Ensayo de Inmunoadsorción Enzimática/métodos , Células Madre Hematopoyéticas/microbiología , Humanos , Neuraminidasa/farmacología , Poliomavirus/inmunología , Poliomavirus/aislamiento & purificación , Poliomavirus/ultraestructura , Receptores Virales/efectos de los fármacos , Virus 40 de los Simios/metabolismo , Especificidad de la Especie , Tripsina/farmacología , Células Tumorales Cultivadas , Virulencia
20.
Virus Genes ; 8(3): 215-29, 1994 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-7975268

RESUMEN

Hepatitis B virus (HBV) nucleoprotein complexes were isolated from nuclei of the human hepatoblastoma cell line HepG2.2.15. Under conditions of physiological ionic strength, the complexes sedimented at a rate corresponding to about 82 S. They contained viral DNA, histone, and nonhistone proteins. For DNA a circular, covalently closed structure was shown both by CsCl gradient centrifugation and electron microscopy. Spread preparations revealed the typical "beads-on-a-string" appearance of nucleosomally organized DNA. The average number of nucleosomes was 18, resulting in a biochemical repeat unit of HBV chromatin of approximately 180 base pairs of DNA. This value was confirmed by experiments analyzing the structure of the HBV chromatin by the use of micrococcal nuclease. Electron microscopy demonstrated that exposure to high ionic strength conditions resulted in removal of nucleosomes from the complexes, but also revealed proteinaceous structures remaining bound to viral DNA molecules. The nature of these residual proteins is discussed. Since native nucleoprotein complexes could be precipitated with HBV-core antibodies, core protein appeared to be one of the nonhistone proteins.


Asunto(s)
Virus de la Hepatitis B/genética , Hepatoblastoma/virología , Neoplasias Hepáticas/virología , Nucleosomas/virología , Integración Viral , Centrifugación por Gradiente de Densidad , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Electroforesis en Gel de Poliacrilamida , Genoma Viral , Humanos , Immunoblotting , Nucleoproteínas/química , Nucleoproteínas/ultraestructura , Nucleosomas/química , Pruebas de Precipitina , Células Tumorales Cultivadas/virología , Proteínas del Núcleo Viral/metabolismo
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