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ABSTRACT: Chronic hepatitis B virus (HBV) infection due to vertical transmission remains a critical concern with regards to eliminating HBV infection. Implementation of hepatitis B vaccine, the foundation to prevent perinatal and horizontal transmission, has reduced the prevalence of HBV by >80%. In countries where the hepatitis B immune globulin (HBIG) is available, such as China and the United States, the administration of HBIG and hepatitis B vaccine to the infants of mothers who are positive for hepatitis B surface antigen has become a standard practice and is effective in preventing vertical transmission. Accumulating evidence on the efficacy and safety of antiviral prophylaxis during pregnancy indicates the probability of attaining the goal of the World Health Organization to eliminate hepatitis by 2030. In this review, we discuss the transmission routes, diagnostic criteria, and preventive strategies for vertical transmission. A preventive program that includes screening before pregnancy, antiviral prophylaxis during pregnancy, and postpartum immunoprophylaxis provides "perfect strategies" to eliminate vertical transmission. However, there is still a notable gap between "perfect strategies" and real-world application, including insufficient coverage of timely birth dose vaccine and the efficacy and necessity of HBIG, especially in mothers who are negative for hepatitis B envelope antigen. In particular, there is a clear need for a comprehensive long-term safety profile of antiviral prophylaxis. Therefore, feasible and cost-effective preventive strategies need to be determined across regions. Access also needs to be scaled up to meet the demands for prophylaxis and prevalence targets.
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Virus de la Hepatitis B , Hepatitis B Crónica , Femenino , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Humanos , Lactante , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , EmbarazoRESUMEN
Background and Aims: Acute-on-chronic liver failure (ACLF) is a rare, but dramatic clinical syndrome. There is substantial evidence suggesting that immunity-mediated inflammation plays an important role in HBV-ACLF. Our aim was to characterize the proportion and cell counts of peripheral blood lymphocyte subsets in acute-on-chronic liver failure patients caused by HBV infection. Methods: One hundred and seventeen patients were enrolled in this study, including those with HBV-related ACLF (HBV-ACLF; n = 70), and HBV related non-ACLF patients (HBV non-ACLF; n = 47). Demographics, clinical and laboratory data at hospital admission were retrospectively analyzed. The percentage and cell count of peripheral lymphocyte subsets were evaluated by flow cytometry. Comparison analysis was performed by t-test or non-parametric Mann-Whitney U-test. Actuarial probabilities of death were calculated by the Kaplan-Meier method. Results: Both circulating lymphocyte count and lymphocyte percentage were significantly reduced in patients with HBV-ACLF (P < 0.001). The CD8+ T cell, CD4+ T cell, and CD16+CD56+ NK cell counts were significantly decreased in HBV-ACLF. Consistently, flow cytometric analysis showed that CD8+ T cell counts were significantly decreased in non-survivors, while no significant differences were found in CD4+ T cell, CD19+ B cell, or CD56+CD16+ NK cell counts. Furthermore, the group with the lower CD8+ T cell count displayed a significantly higher mortality rate compared with the group with the higher CD8+ T cell count. Conclusions: The abnormal prevalence of lymphocyte subsets may be important in the pathogenesis of HBV-ACLF. The decrease in CD8+ T cell counts may be related to poor survival in HBV-ACLF patients.
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BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) seem common after liver transplantation. AIM: To investigate incidence and predictors of NAFLD and NASH by employing noninvasive testing in liver transplant recipients, namely controlled attenuation parameter (CAP) and the serum biomarker cytokeratin 18 (CK-18). We also evaluated the diagnostic accuracy of CK-18 and CAP compared to liver histology. METHODS: We prospectively recruited consecutive adult patients who received liver transplant at the McGill University Health Centre between 2015-2018. Serial measurements of CK-18 and CAP were recorded. NAFLD and NASH were diagnosed by CAP ≥ 270 dB/m, and a combination of CAP ≥ 270 dB/m with CK-18 > 130.5 U/L, respectively. Incidences and predictors of NAFLD and NASH were investigated using survival analysis and Cox proportional hazards. RESULTS: Overall, 40 liver transplant recipients (mean age 57 years; 70% males) were included. During a median follow-up of 16.8 mo (interquartile range 15.6-18.0), 63.0% and 48.5% of patients developed NAFLD and NASH, respectively. On multivariable analysis, after adjusting for sex and alanine aminotransferase, body mass index was an independent predictor of development of NAFLD [adjusted hazard ratio (aHR): 1.21, 95% confidence interval (CI): 1.04-1.41; P = 0.01] and NASH (aHR: 1.26, 95%CI: 1.06-1.49; P < 0.01). Compared to liver histology, CAP had a 76% accuracy to diagnose NAFLD, while the accuracy of CAP plus CK-18 to diagnose NASH was 82%. CONCLUSION: NAFLD and NASH diagnosed non-invasively are frequent in liver transplant recipients within the first 18 mo. Close follow-up and nutritional counselling should be planned in overweight patients.
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To explore the role of surface receptors natural killer group 2A (NKG2A) and natural killer group 2D (NKG2D) on CD3+CD8+T cells and CD3-CD56+NK cells in the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF), we measured the expression of NKG2A and NKG2D on the surface of these 2 types of circulating cells by flow cytometry in 3 groups. One group consists of 36 patients with chronic hepatitis B (CHB), another one consists of 22 patients with HBV-related ACLF, and the last one has 12 normal controls (NC). The experimental result indicated that there was no significant difference in the proportion of CD3+CD8+T cells in total lymphocytes between the 3 groups. However, the percentage of CD3-CD56+NK cells in ACLF group was evidently higher than that in the CHB group (P < 0.05). In addition, the expression of NKG2D on CD3+CD8+T cells in the ACLF group was significantly lower than that in the CHB group (P < 0.05), but there were no statistically significant differences in its percentages on CD3-CD56+NK cells between the 3 groups. The expression of NKG2A on CD3+CD8+T cells in the ACLF group was significantly higher than that in the NC group (P < 0.05), and on NK cells was significantly higher than that in the CHB group (P < 0.05) and NC group (P < 0.01). The increase in ratios of NKG2A to NKG2D on CD3+CD8+T cells and CD3-CD56+NK cells in the ACLF group was significantly more than that in the CHB group and NC group. The results indicate that the imbalance between NKG2A and NKG2D may contribute to the progression of HBV-related ACLF mediated by CD3-CD56+NK cells and CD3+CD8+T cells. Compared with NKG2D, NKG2A expressed on both peripheral CD3-CD56+NK cells and CD3+CD8+T cells plays a more pivotal negative regulatory role in the progression of HBV-related ACLF.
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Insuficiencia Hepática Crónica Agudizada/etiología , Insuficiencia Hepática Crónica Agudizada/metabolismo , Linfocitos T CD8-positivos/metabolismo , Hepatitis B Crónica/complicaciones , Células Asesinas Naturales/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Subgrupos de Linfocitos T/metabolismo , Insuficiencia Hepática Crónica Agudizada/patología , Adulto , Antígenos CD/metabolismo , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Expresión Génica , Virus de la Hepatitis B , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Humanos , Células Asesinas Naturales/inmunología , Pruebas de Función Hepática , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Subfamília C de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Subgrupos de Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To conduct a prospective randomized controlled trial of infants born to hepatitis B virus (HBV) surface antigen (HBsAg)-positive mothers in order to investigate the dynamic changes in the titer of anti-HBV surface protein (HBS) induced by treatment with combined immunoprophylaxis (200 IU hepatitis B immunoglobulin (HBIG) and 5 or 10 mug yeast recombinant hepatitis B vaccine), to compare the protective effect of 5 and 10 mug hepatitis B vaccine, and to provide an immunization strategy, monitoring mode and booster immunization schedule for the high-risk group. METHODS: Two-hundred-and-sixty-nine infants born to HBsAg positive mothers were given combined immunoprophylaxis at birth, and the venous blood samples (at birth, and 1, 7 and 12 months) were tested for HBV DNA load, and HBsAg and anti-HBS titers. RESULTS: The overall 1-year protective rate of combined immunoprophylaxis was 95.9%. There was no significant difference between the infectious rates of infants given the 5 mug or the 10 mug hepatitis B vaccine (x2 = 0.876, P = 0.377). The geometric mean titers (GMTs) of anti-HBS were 144.1 mIU/ml at 1-month old and 564.9 mIU/ml at the age of 7 months old (the highest point), but declined to 397.6 mIU/ml at the age of 12 months old. The rate of infants with anti-HBS titer less than 100 mIU/ml was 20.9%, and that of less than 10 mIU/ml was 7.4% at 7-month-old; the rate of infants with anti-HBS titer less than 100 mIU/ml increased to 30.2% and that of less than 10 mIU/ml increased to 15.9% at 12-month-old. At 7-month-old, the GMT of the 10 mug vaccine group was higher than that of the 5 mug vaccine group (675.3 mIU/ml vs. 25.0 mIU/ml, P = 0.001) and the rate of infants with anti-HBS titer less than 10 mIU/ml was significantly lower in the 10 mug vaccine group (2.3% vs. 12.6%, P = 0.002); at 12-month-old, the rate of infants with anti-HBS titer less than 100 mIU/ml was also significantly lower in the 10 mug group (20.6% vs. 40.2%, P = 0.001). CONCLUSION: Combined immunoprophylaxis is therapeutically efficacious for treating infants born to HBsAg positive mothers. Monitoring these infants' anti-HBs titer will help to identify non- or low-responders in a timely manner. The high-dose hepatitis B vaccine is preferable to the low-dose, and should be considered for use in immunization strategies for these infants.
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Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B/uso terapéutico , Hepatitis B/inmunología , Hepatitis B/prevención & control , Femenino , Hepatitis B/sangre , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Lactante , Madres , Estudios Prospectivos , Carga ViralRESUMEN
Hepatitis B virus (HBV) infection and its associated liver diseases have characteristics of familial clustering in China. However, the reasons for this are not understood fully. To address this issue, the prevalence HBV infection and the characteristics of unfavorable prognoses in clustering of infection in families in northwest China were investigated. Families with clustering of infection and unfavorable prognoses were enrolled, and general information and serum samples were collected. The clinical features and sequelae of HBV infection were compared among the blood relatives (including the first-, second-, and third-degree blood relatives) and spouses using the chi-square test or Fisher's exact test. A total of 102 clusterings of infection families with unfavorable prognoses were interviewed. In the first-, second-, and third-degree blood relatives and spouses, the prevalences of cirrhosis of the liver were 29.2%, 11.9%, and 8.7%, respectively, while those of hepatocellular carcinoma (HCC) were 21.8%, 1.4%, and 4.3%, respectively (P<0.05). The mean ages of the onset of cirrhosis of the liver in the first-, second-, and third-degree blood relatives and spouses were 57 ± 9.91, 47 ± 9.96, 38 ± 10.35, and 57 ± 8.49 years, respectively, while the mean ages of the onset of HCC were 60 ± 7.92, 49 ± 8.57, 41 ± 3.54, and 50 ± 0 years, respectively, (P<0.05). The first-, second-, and third-degree blood relatives from clustering of infection in families with unfavorable prognoses had prevalences of cirrhosis or HCC in descending order of relationship. The findings suggest that genetic factors may be associated with a familial tendency for cirrhosis of the liver and HCC.
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Análisis por Conglomerados , Salud de la Familia , Hepatitis B Crónica/epidemiología , Adolescente , Anciano , Carcinoma Hepatocelular/epidemiología , Carcinoma Hepatocelular/genética , Niño , China/epidemiología , Femenino , Predisposición Genética a la Enfermedad , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/genética , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/epidemiología , Cirrosis Hepática/genética , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Prevalencia , Pronóstico , Adulto JovenRESUMEN
AIM: To investigate the role of T helper 17 cells (Th17) and regulatory T cells (Treg) in hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). METHODS: We enrolled 79 patients with HBV infection into the study, 50 patients with HBV-related ACLF and 29 patients with chronic hepatitis B (CHB), from the First Affiliated Hospital of Medical College from January 2009 to June 2012. The ACLF patients were diagnosed according to the criteria recommended by The 19(th) Conference of the Asian Pacific Association for the Study of the Liver in 2009. Twenty healthy individuals with a similar gender and age structures to the two patient groups were also included as the normal controls (NC). Of the 50 ACLF patients, 28 were subsequently classified as non-survivors: 19 patients died from multi-organ failure, 3 underwent liver transplantation, and 6 discontinued therapy during follow-up because of financial reasons. The remaining 22 ACLF patients whose liver and anticoagulation function recovered to nearly normal levels within the next 6 mo were classified as survivors. The number of circulating Treg and Th17 cells was determined upon diagnosis and during the 8th week of follow-up through flow cytometry. RESULTS: The percentage of circulating Treg cells in the ACLF group was significantly higher than that in the CHB group (5.50% ± 1.15% vs 3.30% ± 1.13%, P < 0.01). The percentages of circulating Th17 cells in the ACLF and the CHB groups were significantly higher than that in the NC group (6.32% ± 2.22% vs 1.56% ± 0.44%, P < 0.01; 3.53% ± 1.65% vs 1.56% ± 0.44%, P < 0.01). No significant difference in Treg cell to Th17 cell ratio was observed between the ACLF group and the CHB group (0.98 ± 0.44 vs 1.12 ± 0.64, P = 0.991), whereas those in the two HBV infection groups were significantly lower than that in the NC group (1.85 ± 1.22; both P < 0.01). The percentage of Treg cells in the survivors during the 8(th) week of follow-up was significantly lower than that during peak ACLF severity [total bilirubin (TBIL) peak] (3.45% ± 0.97% vs 5.18% ± 1.02%, P < 0.01). The percentage of Th17 cells in survivors during the 8(th) week of follow-up was significantly lower than that during the peak TBIL (2.89% ± 0.60% vs 5.24% ± 1.46%; P < 0.01). The Treg cell to Th17 cell ratio during the 8(th) week of follow-up was significantly higher than that during the TBIL peak (1.22 ± 0.36 vs 1.10 ± 0.54; P < 0.05). CONCLUSION: Restoring the Treg cell to Th17 cell ratio during the follow-up phase of ACLF could maintain the immune system at a steady state, which favours good prognosis.
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Enfermedad Hepática en Estado Terminal/inmunología , Hepatitis B Crónica/inmunología , Fallo Hepático Agudo/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Adulto , Antivirales/uso terapéutico , Estudios de Casos y Controles , Células Cultivadas , Enfermedad Hepática en Estado Terminal/diagnóstico , Enfermedad Hepática en Estado Terminal/mortalidad , Enfermedad Hepática en Estado Terminal/terapia , Enfermedad Hepática en Estado Terminal/virología , Femenino , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/mortalidad , Hepatitis B Crónica/terapia , Humanos , Fallo Hepático Agudo/diagnóstico , Fallo Hepático Agudo/mortalidad , Fallo Hepático Agudo/terapia , Fallo Hepático Agudo/virología , Trasplante de Hígado , Masculino , Persona de Mediana Edad , Insuficiencia Multiorgánica/inmunología , Insuficiencia Multiorgánica/mortalidad , Insuficiencia Multiorgánica/virología , Linfocitos T Reguladores/virología , Células Th17/virología , Factores de Tiempo , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate father-to-infant transmission of hepatitis B virus (HBV) by detecting HBV mRNA in the IVF embryos with paternal HBV infection. METHODS: We collected 18 discarded IVF embryos (9 cases) with paternal chronic HBV infection, and detected HBV mRNA in the embryos by single-cell RT-PCR. RESULTS: HBV mRNA positive signals were found in 1 of the 18 embryos with paternal serum HBV positive markers (5.6%), but no specific HBV mRNA signals were observed in the 84 embryos of the negative control group. Follow-up visits revealed no significant difference between the experimental and negative control groups either in the rate of clinical pregnancy (P > 0.05) or in that of early abortion (P > 0.05). The IVF embryo with paternal HBV mRNA positive signals was successfully implanted, but early abortion occurred. HBV infection was not transmitted to progeny in either of the two groups. CONCLUSION: The positive results of HBV mRNA indicate that HBV can get into early-cleavage embryos through sperm and replicate there, which may be the main channel of father-to-infant transmission. HBV may interfere with the development of embryos, and even result in abortion and other adverse outcomes.
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Embrión de Mamíferos/virología , Padre , Virus de la Hepatitis B/genética , Hepatitis B/transmisión , Transmisión Vertical de Enfermedad Infecciosa , ARN Viral/genética , Adulto , Femenino , Fertilización In Vitro , Hepatitis B/virología , Humanos , Masculino , Embarazo , ARN Mensajero/genética , Adulto JovenRESUMEN
OBJECTIVE: To study the expressions of cyclooxygenase-2 (COX-2) and Peroxisome proliferator-activated receptor gamma (PPARg) in liver of patients with hepatitis B virus (HBV) related acute-on-chronic liver failure (ACLF) and their correlation with clinical parameters. METHODS: 35 patients with ACLF, 35 patients with HBV related chronic liver failure (CLF), 27 patients with chronic hepatitis B(CHB) and 15 normal control were enrolled to study the expressions of COX-2 and PPARg in the liver tissues by immunohistochemical staining, and to analyze the correlation of the COX-2 and PPARg levels in liver tissues with clinical parameters. RESULTS: COX-2 was distinctly expressed in the cytoplasm of the hepatocytes, but PPARg was mostly expressed in the nuclei of the hepatocytes and also could be seen in the cytoplasm. The expressions of COX-2 in the liver of ACLF, CLF and CHB groups increased significantly as compared with NC group (z = -5.18, -4.50, -5.32, P is less than 0.01). The levels of COX-2 in ACLF livers also increased evidently as compared with CLF groups (z = -1.98, P is less than 0.05). The expression levels of PPARg in ACLF liver tissues were much higher than the other three groups, and statistical significances existed between ACLF group and the other two groups (CLF, NC groups) (z = -2.62, -4.28, P is less than 0.01). In ACLF group, the expression of COX-2 correlated with MELD score (r = 0.337, P is less than 0.05) and the expression of PPARg correlated with HBV DNA load (r = 0.348, P is less than 0.05). Clinical data showed that the levels of AST, TBil, CHOL, PT, INR, FIB and MELD score in ACLF group were significantly different from that in CLF, CHB and NC groups. CONCLUSIONS: COX-2 expressed in liver may be a marker to reflect the degree of inflammation and injury of liver tissue. The PPARg expression of liver could be increased during chronic HBV infection and may be a protective mechanism against liver injury.
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Ciclooxigenasa 2/metabolismo , Enfermedad Hepática en Estado Terminal/metabolismo , Fallo Hepático Agudo/metabolismo , PPAR gamma/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Enfermedad Hepática en Estado Terminal/virología , Femenino , Virus de la Hepatitis B , Humanos , Hígado/metabolismo , Fallo Hepático Agudo/virología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: The preS1 domain of the large envelope protein has been identified as an essential viral structure involved in hepatitis B virus (HBV) attachment. However, the cellular receptor(s) for HBV has not yet been identified. AIMS: To identify a cell-surface receptor for HBV, which could elucidate the molecular mechanism of HBV infection. METHODS: A novel yeast two-hybrid system was used to screen proteins interacting with the preS1 region of HBV. Their interaction was verified by yeast cotransformation, coimmunoprecipitation and mammalian two-hybrid assay, while their intracellular and tissue localization was analyzed by confocal microscopy and immunohistochemistry, respectively. RESULTS: Asialoglycoprotein receptor (ASGPR) interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro. The levels of expression of preS1 and ASGPR in the liver were similar and correlated with each other. CONCLUSIONS: ASGPR is a candidate receptor for HBV that mediates further steps of HBV entry.
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Receptor de Asialoglicoproteína/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Mapeo de Interacción de Proteínas , Precursores de Proteínas/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Animales , Línea Celular , Hepatocitos/virología , Humanos , Inmunoprecipitación , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: To construct a yeast expression vector of hepatitis B virus (HBV) PreS1 gene using the Sos-recruitment system (SRS), and evaluate the effect of the expression product on the growth of the yeast cells and activation of the reporter gene. METHODS: The coding sequence of HBV preS1 was amplified by PCR and cloned into the yeast expression plasmid pSos. The recombinant bait plasmid pSos- PreS1 was verified by sequencing before transformation into competent yeast cells. The effects of the expression product on the yeast cell growth and activation of the reporter gene were evaluated. RESULTS: The yeast expression vector of HBV PreS1 gene was constructed successfully. The recombinant bait plasmid showed no toxic effect on yeast cdc25H cells without a self-activation of the reporter gene. CONCLUSION: The SRS can be used to study the proteins interacting with HBV PreS1 protein and provides a means for obtaining insight into the pathogenic mechanism of HBV.
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Vectores Genéticos/genética , Antígenos de Superficie de la Hepatitis B/biosíntesis , Precursores de Proteínas/biosíntesis , Receptores Virales/metabolismo , Técnicas del Sistema de Dos Híbridos , Clonación Molecular , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Plásmidos/genética , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
BACKGROUND: Hepatitis C virus (HCV) core protein is a multi-functional viral protein that interacts with several target proteins of both viral and cellular origin. AIM AND METHODS: To gain insight into the mechanism of action of HCV core protein, we used a yeast two-hybrid system to identify the core protein-interacting cellular targets. RESULTS: A cDNA clone encoding an aspartoacylase was obtained, termed aspartoacylase 3 (ACY3). Interaction between ACY3 and HCV core protein was verified using a co-immunoprecipitation assay in vitro, and a mammalian two-hybrid system in vivo. Fluorescence microscopy showed green fluorescence protein-fused ACY3 localized in the cytoplasm. CONCLUSION: Our data suggest that ACY3 is an HCV core binding protein, which may play a role in the development of HCV-associated diseases.
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Amidohidrolasas/metabolismo , Hepacivirus/metabolismo , Hígado/enzimología , Proteínas del Núcleo Viral/metabolismo , Amidohidrolasas/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Citosol/enzimología , Hepacivirus/genética , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas del Núcleo Viral/genéticaRESUMEN
OBJECTIVE: To analyze the interaction of hepatitis C virus (HCV) core protein with HCBP1 and observe the expression and cellular localization of HCBP1. METHODS: The cDNA fragments encoding HCV core protein and HCBP1 were amplified by PCR and subsequently cloned into pGEM T vector, respectively. After sequence verification, the two recombined vectors were respectively subcloned into two hybrid plasmids, pM and pVP16. pM-core, pVP16- HCBP1 and the reporter vector pG5CAT were co-transfected into COS-7 cells, and the interaction between HCV core protein and HCBP1 was assayed by detecting CAT gene expression after 48 h. The expression and subcellular localization of the fusion protein in the transfected COS-7 cells were analyzed by Western blotting and fluorescence microscopy, respectively. RESULTS: CAT-ELISA showed that the absorbance of the co-transfection group was significantly higher than that o f the negative control groups but lower than that of the positive control group. Western blotting confirmed the expression of fusion protein in the transfected COS-7 cells. Fluorescence microscopy showed that the fusion protein was distributed mainly in the cytoplasm, and in contrast, diffuse EGFP expression was detected in COS-7 cells transfected with the empty vector. CONCLUSION: Mammalian two-hybrid assay confirms the capacity of HCBP1 to bind HCV core protein, and the expression vector for HCBP1-EGFP fusion gene has been constructed successfully and expressed in COS-7 cells.
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Receptores Virales/metabolismo , Proteínas del Núcleo Viral/metabolismo , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
AIM: To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells (PBMCs). METHODS: HBcAg region was amplified by polymerase chain reaction (PCR) and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains (Western blot analysis), yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (QDO) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (TDO). The second screening was performed with the LacZ report gene ( yeast cells were grown in QDO medium containing X-alpha-gal). The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods. RESULTS: Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 (3 colones), eukaryotic translation elongation factor 2 (2 colones), acetyl-coenzyme A synthetase 3 (1 colone), DNA polymerase gamma (1 colone), putative translation initiation factor (1 colone), chemokine (C-C motif) receptor 5 (1 colone), mitochondrial ribosomal protein L41 (1 colone), kyot binding protein genes (1 colone), RanBPM (1 colone), HBeAg-binding protein 3 (1 colone), programmed cell death 2 (1 colone). Four new genes with unknown function were identified. CONCLUSION: Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.
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Clonación Molecular , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B , Leucocitos/metabolismo , Proteínas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Biología Computacional , Regulación Viral de la Expresión Génica , Genes Virales , Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Operón Lac , Leucocitos/virología , Datos de Secuencia Molecular , Unión Proteica , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos , Proteínas del Núcleo Viral/genética , Replicación Viral , Levaduras/genéticaRESUMEN
OBJECTIVE: To investigate the expression of human annexin-V (HA-V) in relation to HBV infection in different fetal tissues. METHODS: Immunohistochemistry was employed to detect the expression and distribution of HA-V in the liver, kidney, ovary, heart, fallopian tube, spleen, and thymus gland of human fetus. RESULTS: HA-V expression was detected in different tissues including the ovary, liver, intrahepatic bile duct, heart, kidney, lymphocytic cells in the thymus gland, epithelial cells of the fallopian, and cortical and medullary cells of the spleen. HA-V was distributed mainly in the cytoplasm of the cells. The liver tissues exhibited greater gray scale for HA-V expression than in the other tissues (P<0.05) and no significant difference was observed in the other tissues than the liver (P>0.05) in image analysis with Photoshop 7.0. CONCLUSION: HA-V is an inherent protein in fetal tissues with possible relation to HBV infection of different tissues as a HBV receptor. Greater amount of HA-V in the liver may account for the vulnerability of the liver to HBV infection.
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Anexina A5/análisis , Feto/química , Hígado/química , Feto/virología , Hepatitis B/metabolismo , Hepatitis B/virología , Virus de la Hepatitis B/crecimiento & desarrollo , Humanos , Inmunohistoquímica , Hígado/virología , Distribución TisularRESUMEN
AIM: To investigate the expression and distribution of HBV in the ovaries and ova. METHODS: The immunohistochemistry method was used to detect the HBsAg and HBcAg in the ovaries of patients with chronic hepatitis B. RESULTS: Expression of HBsAg in the ova, granular and interstitial cells of the ovaries was located in the cytomembrane and cytoplasm. Expression of HBcAg in the ova, granular, interstitial and endothelial cells of interstitial blood vessels of the ovaries was found in the cytomembrane, cytoplasm, and nuclei. CONCLUSION: HBV can infect the ova at different stages of development and replicate in it.
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Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Hepatitis B Crónica/virología , Ovario/virología , Óvulo/virología , Femenino , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Ovario/citología , Óvulo/citologíaRESUMEN
OBJECTIVE: To screen and identify the protein interacting with HBV antigen in hepatocytes. Then investigate the biological functions of hepatitis B virus antigen in the pathogenesis of hepatitis B and seek effective methods to prevent and treat it. METHODS: The yeast two-hybrid system-3 technique was used to construct HBV PreS2, HBeAg, HBcAg, HBxAg bait plasmids. The bait plasmids transformed the yeast AH109 and expressed themselves in it. After being identified by SDS-PAGE and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium to form diploid yeast and was then plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. To further prove the interaction between HBV antigen and metallothionein, translation was performed by using reticulocyte lysate and coimmunoprecipitation was displayed in vitro. RESULTS: Genes coding for HBV antigen binding protein were successfully cloned and metallothionein was found in that protein. The interaction between HBeAg, HBcAg and HBxAg and metallothionein were further proved by coimmunoprecipitation in vitro. CONCLUSION: The interaction between HBV antigen and metallothionein indicates that metallothionein may participate in the pathogenesis of hepatitis B