OBJECTIVETo test the infeciton efficiency of recombinant adenoviral vector carrying HBsAg-HSP70 chimeric gene and to abserve its biological characteristics.

METHODSPeripheral blood mononuclear cells (PBMC) were separated from healthy blood donor and they were infected by Ad-HSP70-HBsAg on the first day of isolation. DCs were induced in medium with cytokines IL-4, GM-CSF and TNF-alpha in vitro. The biological characteristics of DC induced were analyzed by inverted fluorecent microscope, RT-PCR, flow cytometer (FACS), and mixed lymphocyte reaction (MLR).

RESULTSThe traced gene-GFP were abserved in DCs by inverted fluorecent microscope and HSP70-HBsAg gene mRNA expression was detected by RT-PCR after the Ad-HSP70-HBsAg infection. FACS analysis shown that the expression of CD1a, CD80, CD86 and HLA-DR on surfece of two groups of DCs were similar. MLR showed that there are not a statitic difference of stimulated index (SI) between two groups.

CONCLUSIONResults indicated that Ad-HSP70-HBsAg can effectively infected DCs without affecting its biological characteristics.

Adenoviridae ; genetics ; physiology ; Cells, Cultured ; Cytokines ; genetics ; immunology ; Dendritic Cells ; immunology ; virology ; Genetic Vectors ; genetics ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Humans ; Lymphocyte Culture Test, Mixed ; Recombinant Fusion Proteins ; genetics ; immunology

OBJECTIVETo construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro.

METHODSHeat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro.

RESULTSThe adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 x 10(12) pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection.

CONCLUSIONThe recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cercopithecus aethiops ; Defective Viruses ; genetics ; Enzyme-Linked Immunosorbent Assay ; Green Fluorescent Proteins ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vero Cells ; Virus Replication ; genetics

OBJECTIVETo explore whether hepatitis B virus (HBV) S gene-modified dendritic cells (DCs) might induce a specific cytotoxic T lymphocyte (CTL) response.

METHODSThe recombinant adenoviruses carrying HBsAg genes were prepared and used to transfect DCs generated from cord blood. The efficacy of transfection was observed through the expression of enhanced green fluorescent protein (EGFP) in DCs and the expression of HBsAg was detected by ELISA. HBV S gene-modified DCs were co-cultured with T cells from cord blood and T cells stimulating activities were detected using mixed lymphocyte reaction (MLR). The CTL assay was carried out to assess the ability of CTL lines to lyse target cells of HepG(2)22.1.5 by measuring lactate dehydrogenase (LDH) release.

RESULTSThe results showed that HBV S genes were expressed in DCs with high efficacy by recombinant adenoviral vector. DCs had a normal shape after transfection. The result of MLR showed that HBV S gene-modified DCs could effectively stimulate naive T cells to proliferate. The induced specific CTL lines could lyse target cells of HepG(2)22.1.5.

CONCLUSIONSHBV S gene-modified DCs enhanced the function to induce a specific CTL effect, showing its promise for developing anti-viral vaccine in future.

Cell Line ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; virology ; Hepatitis B ; immunology ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo study HBV preS2/S gene expression effects in mammalian cells transferred with recombinant adenoviral vector.

METHODSThe replication-deficient recombinant adenoviral vector (Ad-HBs) carrying HBV preS2/S gene were constructed by homologous recombination in bacteria. The 293 cells, Vero cells, HepG2 cells and mesenchymal stem cells (MSCs) were infected with adenoviruses. The expressions of enhanced green fluorescent protein (EGFP) were observed with fluorescence microscope and the expressions of HBsAg were detected by RT-PCR and ELISA in vitro.

RESULTSMore than 90% of 293 cells, Vero cells, HepG2 cells or MSCs expressed EGFP after transfection at the MOI of 20 and the titers of HBsAg were more than 3.229 (A value) in culture supernatant.

CONCLUSIONThe HBV preS2/S gene was not only expressed efficiently in immortalized cells, but also expressed efficiently in stem cells with the recombinant adenoviruses vector.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cercopithecus aethiops ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Humans ; Microscopy, Fluorescence ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vero Cells

OBJECTIVETo evaluate the efficacy and safety of entecavir (ETV) treatment for chronic severe hepatitis B.

METHODS78 patients with chronic severe hepatitis B and positive HBV DNA were divided into ETV group and control group, each group had 39 patients. ETV group was given the same conventional therapy as control group, and was treated with ETV. The change of liver function, PTA, HBV DNA level were observed, and adverse events were recorded. The effective rate of treatment between ETV group and control group, the baseline characteristics between the effective cases and non-responsive cases after ETV treatment were compared at week 12.

RESULTSThe baseline characteristics were well balanced between ETV group and control group. The effective rate of ETV group was 56.41% versus 33.33% of control group at week 12 (P = 0.0405). The effective rate of ETV group was higher than that of control group, in the early stage of chronic severe hepatitis B (P = 0.0275), but there was no statistically significant in the middle or late stage (P = 0.4687). The comparison result of baseline characteristics between the effective and non-responsive cases after ETV treatment showed: there were statistically different in age, bilirubin level, HBV DNA level and stage of the severe hepatitis, proportion of cirrhosis, but no statistically different in cholinesterase level, alpha-fetoprotein level and sex ratio, the proportion of ascites, positive HBeAg (P > 0.05). No serious adverse events occurred.

CONCLUSIONSETV improves the curative effect when used in the early stage of chronic severe hepatitis B, and may not in the middle and late stage. The curative effect of ETV may be affected by age, bilirubin level, HBV DNA level and stage of the severe hepatitis, cirrhosis. ETV has good security in the treatment for chronic severe hepatitis B.

Adult ; Aged ; Aged, 80 and over ; Antiviral Agents ; therapeutic use ; Female ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B, Chronic ; drug therapy ; pathology ; Humans ; Male ; Middle Aged ; Severity of Illness Index ; Treatment Outcome ; Young Adult

OBJECTIVETo establish a sequential antiviral regime and evaluate its efficacy in patients with chronic hepatitis B using a controlled trial.

METHODSSeventy-four patients with chronic hepatitis B were divided into 3 groups: 30 cases were enrolled in the sequential antiviral group in which patients received eight-week treatment with thymosin alpha1 (1.6 mg/time, subcutaneous injection, 2 times/week), six-month treatment with interferon (500 MU/ times, muscle inject, every other day) begun in the fifth week of the therapeutic course, and lamivudine treatment (100 mg/days) begun 2 months later after HBeAg seroconversion or just after the withdrawal of interferon to more than eighteen months. Fourteen cases were enrolled in combination group in which patients received six-month treatment with interferon and thymosin alpha1 simultaneously in the same manner as in sequential antiviral group. Thirty cases were enrolled in lamivudine group in which patients received more than eighteen-month treatment with lamivudine.

RESULTSThe temporary rates of HBeAg seroconversion and normalization of alanine aminotransferase (effective rate) in sequential antiviral group, combination group and lamivudine group were 76.7%, 78.6% and 13.3%, respectively. The effective rates of sequential group and combination group were very similar, and significantly higher than that of lamivudine group (P less than 0.01). Long-term efficacy rates were 76.7%, 57.1% and 16.7% among the three groups, respectively. The long-term effective rate of sequential group was relatively higher. The rate of liver damage sensitive period in sequential antiviral group and combination group was 47.7%. The time of onset was from 2 to 8 weeks after the treatment begun, earlier than that from 6 to 8 weeks after the beginning of interferon alone in the literature.

CONCLUSIONSequential antiviral therapy had much higher rates of long-term HBeAg seroconversion, undetectable HBV DNA and normalization of alanine aminotransferase with good cost-effectiveness. Its mechanism to promote the antiviral effect might be dependent on the immunoregulatory action of thymosin alpha1 in the earlier period and the specific inhibition of HBV DNA replication by lamivudine in the later period of the therapeutic course.

Adjuvants, Immunologic ; administration & dosage ; Antiviral Agents ; administration & dosage ; China ; Drug Therapy, Combination ; Hepatitis B, Chronic ; drug therapy ; Humans ; Interferon-alpha ; administration & dosage ; Lamivudine ; administration & dosage ; Thymosin ; administration & dosage ; analogs & derivatives ; Treatment Outcome

Objective To investigate the diagnostic accuracy of 64-slice spiral computed tomography(MSCT)in detecting coronary artery lesions and to analyze the impacts of coronary artery calcium on its diagnostic accuracy.Methods Sixty patients underwent 64-MSCT coronary angiography and conventional coronary angiography(CCA).Calcium scoring was estimated on plain scans.The diagnostic accuracy of MSCT to detect significant lesions(≥50%)was evaluated referring to quantitative coronary angiography(QCA).The impacts of coronary artery calcium on the diagnostic accuracy was analyzed.Results A total of 797 segments were diagnositc.The overall sensitivity,specificity,positive predictive value and negative predictive value of 64-MSCT were 96%(174/182),98%(601/615),93% (174/188),and 99%(601/609),respectively.When calcium score ≥100(Agatston score),the specificity and positive predictive value of 64-MSCT was 63%(12/19)and 81%(30/37), respectively.Conclusion In patients with no or mild coronary calcification,the 64-MSCT coronary angiography had a reliable detection of coronary artery stenoses.But severe calcification in coronary artery may degrade diagnostic specificity and positive predictive value of MSCT coronary angiography.

OBJECTIVETo study the relationship between pulmonary surfactant-associated protein B (SP-B) gene polymorphisms and their susceptibility to neonatal respiratory distress syndrome (RDS).

METHODSEighty-eight preterm infants with RDS (RDS group) and 103 infants without RDS (control group) were enrolled. The genomic DNA was isolated using DNA kits. Polymerase chain reaction with restriction fragment length polymorphism technique was used to detect the genotype and allele frequency of the SP-B -18A/C and SP-B 1580C/T single nucleotide polymorphisms. The association between the polymorphisms and RDS was analyzed.

RESULTSSP-B -18A/C and SP-B 1580C/T were found to be polymorphic in both RDS and control groups. The frequencies of CC genotype (X2=12.26, P<0.01) and C allele (X2=11.97, P<0.01) of SP-B 1580C/T were significantly higher in the RDS group than in the control group. The C allele significantly increased the risk of RDS (OR=2.26, 95%CI: 1.42-3.60). The frequencies of genotype and allele of SP-B -18A/C showed no significant difference between the two groups.

CONCLUSIONSSP-B 1580C/T polymorphism contributes to the etiology of RDS and may serve as the susceptibility gene for RDS. The C allele increases the risk of RDS. SP-B -18A/C shows no association with the etiology of RDS.

Genetic Predisposition to Disease ; Genotype ; Humans ; Infant, Newborn ; Polymorphism, Single Nucleotide ; Pulmonary Surfactant-Associated Protein B ; genetics ; Respiratory Distress Syndrome, Newborn ; etiology ; genetics

BACKGROUNDLaparoscopic Heller cardiomyotomy and Dor fundoplication is the surgical procedure of choice for esophageal achalasia. The aim of this study was to investigate the clinical outcome of laparoscopic Heller-Dor procedure in our initial series of 25 patients with achalasia.

METHODSBetween October 2003 and January 2006, a total of 25 patients with achalasia underwent laparoscopic Heller-Dor operation. Among them, 9 were male and 16 were female with an average age of (41.5 +/- 5.1) years (21-66). All the patients received upper gastrointestinal series (barium swallow), esophagogastroscopy, esophageal manometry to exclude esophageal carcinoma and to confirm the diagnosis, and 21 patients also had 24-hour ambulatory pH studies. All the patients were operated by laparoscopic modified Heller's myotomy with Dor fundoplication. In addition, 2 of them had combined laparoscopic cholecystectomy + excision of hepatic hemangioma and laparoscopic cholecystectomy, respectively.

RESULTSThe average operating time was (110.6 +/- 12.9) minutes (range, 60-180), operative blood loss averaged (18.6 +/- 7.1) ml (5-50), the median time to oral feeding was (1.6 +/- 0.4) days (1-4) and the median hospital stay was (12.6 +/- 1.2) days (10-20). There was no conversion to open surgery. Intraoperative mucosal perforation was encountered in six patients and was repaired in all of them by laparoscopic suture. All the patients had an uneventful recovery without postoperative complication. After a median follow-up of (10.6 +/- 7.2) months (1-27), 24 patients were asymptomatic and 1 had mild postoperative dysphagia.

CONCLUSIONSLaparoscopic Heller-Dor operation had the advantages of reduced compromise of the cardiopulmonary function, with less disruption of the supporting structures (phrenoesophageal membrane) of the antireflux mechanism, requiring simpler general anesthesia and providing excellent exposure permitting an easy fundoplication, less pain and reduced morbidity, shorter hospitalization and faster convalescence.

Adult ; Aged ; Digestive System Surgical Procedures ; methods ; Esophageal Achalasia ; surgery ; Esophagus ; surgery ; Female ; Follow-Up Studies ; Fundoplication ; methods ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods

OBJECTIVETo generate hepatitis C virus pseudo-particles (HCVpp) containing the complete E1-E2 envelope glycoprotein, in order to establish a HCVpp database covering the six major genotypes of HCV (1b, 2a, 3b, 4, 5, and 6) and to develop a simple and effective method for detection of neutralizing antibodies in HCV patients.

METHODSHCVpp were generated for the six genotypes by co-transfecting 293T cells with a plasmid expressing the respective E1-E2 (p HR, CMVA 8.2 construct) and a MLV-GFP plasmid. Titration of each HCVpp was carried out by p24 ELISA. Infectivity of each HCVpp was assessed by mixing the harvested supernatant of producer cells with sera from HCV patients, adding the mixture to Huh-7 cells, and detecting the subsequent titers of neutralizing antibodies against HCVpp.

RESULTSAll six types of HCVpp were able to infect Huh-7 cells in vitro. For healthy HCV carriers, only two genotypes of HCVpp (1b and 2a) produced neutralizing antibody titers more than 1:40. For cured HCV patients, only the 1b genotype produced neutralizing antibody titers more than 1:40. One patient showed titer of 1:200 for genotype 4. A healthy spouse of a chronic hepatitis C patient showed titers more than 1:40 for four genotypes of HCVpp (3a, 4, 5, 6).

CONCLUSIONWe generated six different genotypes of HCVpp successfully, established the in vitro neutralizing antibody detection method, and provided an effective model for screening antiviral drugs.

Adolescent ; Adult ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Female ; Genotype ; Hepacivirus ; classification ; Hepatitis C ; blood ; immunology ; Humans ; Male ; Middle Aged ; RNA, Viral ; blood ; Viral Envelope Proteins ; immunology ; Young Adult