Hepatitis B, Chronic ; therapy ; Humans ; Immunotherapy

Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P

Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.

Objective To detect the sperm apoptosis rate and level of reactive oxygen species (ROS) in seminal plasma and explore their correlation among infertile males. Methods Ninety-two inferitile males were divided into varicocele (VC) group (n=32), leukocytospermia group(n=30) and the other cause group (n=30), and another 24 in vitro fertilization sperm samples were sereved as controls. The routine sperm parameters including seminal pH, sperm viability and sperm density were examined by computer assisted sperm analysis, the sperm apoptosis rate was asseseed using Annexin V/PI staining, and the ROS level in seminal plasma was detected by TBA method. The differences in seminal parameters between three infertile groups and control group were compared, and the correlation of sperm apoptosis rate with level of ROS in seminal plasma was explored in each group. Results The sperm viability of three infertile groups was significantly lower than that of control group (P<0.01). The sperm apoptosis rates and levels of ROS in seminal plasma in VC group and leukocytospermia group were significantly higher than those in control group (P < 0.05 or P < 0.01). The sperm apoptosis rate was positively correlated with the level of ROS in seminal plasma in leukocytospermia group(r=0. 573, P < 0.05). Conclusion The increased sperm apoptosis rate and level of seminal plasma ROS may be related to the infertility of patients with VC and leukocytospermia. The increased level of seminal plasma ROS may be one of the causes of increased sperm apoptosis rate in patients with leukocytospermia.

Objective To study the molecular mechanisms of ampicillin- resistant haemophilius influenzae (Hi)in Nanjing. Methods One hundred and fifty- eight strains of Hi isolated from children were collected to detect bata-lactamase. TEM and ROB bata- lacta-mase genes were detected by polymerase chain reaction (PCR) ,and cloned into T vector for sequencing. Results The rate of ampicillin resistance was 41. 77% in Hi isolated from children in Nanjing,40.51 % was found to be bata-lactamase production. Eighty-nine strain were TEM positives, 1 strain was ROB positive,63 strains bata - lactamase positive ampicillin- resistant Hi were identified. The resistance mechanism of ampicillin resistant Hi was production of bata - lactamase , mainly TEM - type enzyme. Two bata - lactamase negative ampicillin - resistant Hi were identified , predicts the other mechanisms of ampicillin - resistant Hi was occuered yet . One strain of non -TEM - type,and non - ROB - type bata - lactamase - producing Hi was identified. Conclusions Ampicillin - resisitant in Hi isolated from children in this region is challenging. TEM bata - lactamase is the principal mechanism of ampicillin - resistant of Hi.

DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the relationship between the drug resistance of Pseudomonas aeruginosa (PA) isolated from burn patients wounds and its mobile genetic elements, including plasmid, transposon, and integron.

METHODSThirty-two strains of PA were isolated from wounds exudate of hospitalized burn patients in Ningbo No. 2 Hospital. PA drug sensitivity was determined using GNS-448 drug sensitivity card and K-B tests. The genetic markers of plasmid, transposon and integron including traA, traF, tnpA, tnpU, merA, int I 1 were amplified by PCR and verified by gene sequencing.

RESULTSDrug resistant rate of 32 PA strains to gentamicin, amikacin, cefoperazone/sulbactam, ciprofloxacin was 43.7%, 32.0%, 46.8%, 49.9%, respectively. PA drug resistant rates to piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, levofloxacin, imipenem and meropenem were all above 56.0%. Seventeen out of 32 PA strains were found to carry transposon and (or) integron genetic markers. One strain was positive for both tnpA and merA, 8 strains were positive for both merA and int I 1, 1 strain was only positive for tnpA, 2 strains were only positive for merA, and 5 strains were positive for int I 1 only.

CONCLUSIONSPA isolated from burn wounds of hospitalized patients in Ningbo No. 2 Hospital is seriously drug resistant, which may relate with its high positive rate of mobile genetic elements of transposon and (or) integron.

Anti-Bacterial Agents ; pharmacology ; Burns ; microbiology ; DNA Transposable Elements ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Integrons ; Microbial Sensitivity Tests ; Plasmids ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification

OBJECTIVESTo observe immunogenicity of new DNA vaccine encoding for hepatitis B virus core antigen (HBcAg).

METHODSA new DNA vaccine (pSW3891/HBc) encoding for hepatitis B virus core antigen was constructed using plasmid pSW3891 which can be used in human. Control and experiment groups of Balb/c mice were immunized with pSW3891 or pSW3891/HBc by gene gun. Anti-HBc in sera of mice was tested by ELISA (enzyme linked immune sorbent assay). Specific cytotoxicity of cytotoxic T lymphocyte (CTL) of mice was detected by LDH release assay.

RESULTSpSW3891/HBc can express in 293T cell line effectively. Mice immunized with pSW3891/HBc showed strong anti-HBc response and specific high cytotoxicity of CTL.

CONCLUSIONpSW3891/HBc induced significantly humoral and cellular immune responses in Balb/c mice.

Animals ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Immunity, Cellular ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA ; immunology

OBJECTIVETo determine the expression level changes of survivin, a inhibitor of apoptosis protein, followed by activation of insulin receptors in human hepatocellular carcinoma HepG2 cell line, and to investigate the signalling pathway involved in the regulation.

METHODSHuman hepatocellular carcinoma HepG2 cells were treated with insulin alone or pre-treated with LY294002, a specific inhibitor of PI3K signalling pathway, to determine whether blocking PI3K signaling can attenuate the up-regulation of survivin expression. Real time RT-PCR and Western blot analysis were used to measure survivin mRNA and protein changes before and after treatment, respectively.

RESULTSWithout serum supplement, HepG2 cells expressed a small amount of survivin. Insulin induced survivin expression in a dose- and time-dependent fashion. Survivin expression was blocked if cells were pre-treated with LY294002 prior to insulin stimulation.

CONCLUSIONInsulin induces survivin expression via PI3K signalling pathway, suggesting that to interfere the key gene in this signalling pathway may block survivin expression, therefore, promoting apoptosis in hepatocellular carcinoma cells.

Apoptosis ; Chromones ; pharmacology ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Insulin ; administration & dosage ; pharmacology ; Microtubule-Associated Proteins ; genetics ; metabolism ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Signal Transduction ; Up-Regulation

OBJECTIVEThe aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen.

METHODSSurvivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry.

RESULTSTamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells.

CONCLUSIONSOur results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Synergism ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Tamoxifen ; pharmacology