Search

Objective: To detect the levels of miR-146a and miR-155 in different samples from chronic hepatitis B (CHB), reveal whether there is a correlation between the 2 miRNAs in different samples, and to provide a theoretical basis for sample choice of miRNA research in liver.
 Methods: Real-time PCR was conducted to examine the expression of miR-146a and miR-155 in the plasma, peripheral blood mononuclear cell (PBMC), and liver tissues from 41 CHB patients who underwent nucleoside analogues antiviral therapy for 104 weeks. Correlations between the levels of miR-146a and miR-155 among the 3 samples were analyzed.
 Results: The expressions of miR-146a and miR-155 in the plasma, PBMC and liver tissues were significantly down-regulated at the 104th week than those at the baseline (all P<0.05). There was a correlation in the expression of miR-146a between plasma and liver tissues (r=0.560, P=0.007), PBMC and liver tissues (r=0.428, P=0.047) at baseline. There was a correlation in the expression of miR-155 between plasma and liver tissue (r=0.587, P=0.004), PBMC and liver tissue (r=0.483, P=0.023) at baseline. The expressions of miR-146a and miR-155 between the plasma and PBMC were not correlated (P>0.05).
 Conclusion: Compared with PBMC, miR-146a and miR-155 from plasma can better reflect the expression in the liver tissues, suggesting that plasma can be applied in the mechanism research on miR-146a and miR-155 in the liver diseases instead of liver tissues.

Objective: To investigate expression profiles of the plasma exosomal miRNAs of the chronic hepatitis B (CHB) patients with persistently normal alamine aminotransferase (PNALT) for the first time and try to find exosomal miRNAs which could reflect liver inflammation better. 
 Methods: Five CHB patients with liver tissue inflammation grade ≥A2 of PNALT and 5 CHB patients with liver tissue inflammation grade

Objective: To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).
 Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis.
 Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance.
 Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.

Objective: To observe the stability and sensitivity of transcription mediated amplification (TMA) system, and to compare it with real-time reverse transcription polymerase chain reaction (RT-PCR) in amplifying serum HCV RNA in HCV infected patients., Methods: TMA system was established by moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase and 2 specific primers firstly, and then its stability and repeatability were compared at different storage temperatures by the correlation change of HCV RNA amplification curve. The sensitivity difference between TMA and RT-PCR was evaluated by amplifying a group of 10-fold diluted HCV RNA samples which were transcribed in vitro. A total of 101 serums of chronic HCV infected patients were measured by TMA system and RT-PCR to observe the positive rate and their correlation. Linear correlation and linear regression were used to observe the correlation of the two methods., Results: TMA system was successfully established. TMA system was not stable when stored at 20 °C (placed for 24 hours only), but it was stable for 6 days when stored at 4°C or within 6 months when stored at -20 °C . Compared with RT-PCR whose reagent was made by Hunan Sansure Biotechology Corporation, TMA system showed 20 positive samples and 11 negative samples in a total of 31 samples. So was the RT-PCR kit of the Sansure Biotechology Corporation, and the concordance rate of the two methods was 100%. Advanced quantitative study of the 20 positive samples found that the two methods had good correlation and consistency (r=0.91, P<0.01). Compared with the results of RT-PCR whose reagent was made by Shanghai Kehua Bio-engineering Corporation, TMA system had 34 positive samples and 36 negative samples, while the RT-PCR technology had 32 positive samples and 38 negative samples out of 70 samples. The concordance rate of the two methods was 97.1%, with no statistical difference in the positive rate of the two methods (P>0.05). Advanced quantitative study of 29 positive samples found that the two methods had good correlation and consistency (r=0.96, P<0.01)., Conclusion: The stability and repeatability of TMA system are good within 6 months when stored at -20 °C storage temperature. Both TMA and RT-PCR HCV RNA can detect serum HCV RNA well, and the two methods have good correlation and consistency.

Objective: To evaluate the mid-term prognostic value of procalcitonin (PCT), endotoxin and common inflammatory markers combining the model for end-stage liver disease (MELD) score in patients with chronic severe hepatitis., Methods: A total of 124 chronic severe hepatitis patients were enrolled, who were hospitalized in the Department of Infectious Diseases, Xiangya Hospital, Central South University from May 2011 to December 2011. Indexes of inflammation, liver and kidney function tests and MELD were determined within 24 h after the admission, and blood samples were collected for measurement of endotoxin , procalcitonin (PCT), and C-reactin protein (CRP). The outcome was confirmed after discharge follow-up at the end of the 3rd month. According to the outcome, the 124 patients were divided into a survival group (n=58) and a death group(n=66)., Results: 1) Of the 124 patients, 66 died and 58 survived, with statistical difference in age, MELD score, white blood cell (WBC), polymorphonuclear (PMN), CRP and PCT by single factor analysis between the 2 groups(P<0.05). Binary logistic regression analysis indicated that age, MELD scores and PCT were highly correlated with the outcome (OR=1.07, 1.42 and 1.02 respectively, P<0.05), which could be used to predict the 3 month mid-term mortality of chronic severe hepatitis. 2)There was significant correlation between the MELD scores and the mid-term mortality. Age was positively correlated with the MELD score, and Pearson's correlation coefficient was 0.21 (P<0.05). PCT was also positively correlated with the MELD, and Spearman's correlation coefficient was 0.54 (P<0.01). 3)According to the receiver operation characteristic (ROC) curve analysis , the area under the curve (AUC) of MELD score and PCT were 0.91 and 0.77 respectively, higher than those of other indexes (P<0.01). When the MELD score was up to 30.09 or higher, the predicted mortality risk among these tested patients was the highest(82.26%). The mortality risk predicted by PCT combining MELD score and PCT alone was lower than by MELD score alone (75.00%), but the specificity of MELD score combining PCT was 100%, and the positive prediction value was 1.00., Conclusion: Endotoxin and common inflammatory markers (WBC, PMN, and CRP) are not reliable indicators to predict the prognosis in patients with chronic-severe hepatitis. MELD score is significantly correlated with the outcome of mid-term chronic severe hepatitis, PCT and age are both positively correlated with the MELD score. PCT and age combining MELD score can be used to predict the 3 month mid-term mortality of chronic severe hepatitis. MELD score has better prognostic value than PCT. MELD score combining PCT can improve the specificity of prediction.

Objective: To determine CD4(+) CD25(+) T regulatory cells (Tregs), forkhead box P3 (FoxP3) mRNA expression and levels of cytokines secreted by peripheral blood mononuclear cells (PBMCs) in individuals responsive or unresponsive to hepatitis B (HB) vaccination, and to explore the relationships between immune response and immune regulatory cells or cytokines., Methods: Based on the antibody against hepatitis B surface antigen (HBsAg) after HB vaccination, the CD4(+) CD25(+) Tregs frequencies in PBMCs from 18 responders, 22 nonresponders and 10 non-immunized healthy controls were analyzed by flow cytometry. The expression of FoxP3 mRNA in PBMCs with or without stimulation of phytohemagglutinin (PHA)and HBsAg was analyzed by real-time quantitative PCR. Levels of IL-4, IL-12, IL-18, and IFN-γ secreted by PBMCs after PHA and HBsAg stimulation were analyzed by enzyme-linked immunosorbent assay., Results: The ratio of CD4(+) CD25(+) Tregs to CD4(+) T cells in the nonresponders was markedly higher than that in the responders (P<0.05), but lower than that in the controls (P<0.01). FoxP3 was differentially expressed among the responders, nonresponders, and controls in PBMCs before and after PHA and HBsAg stimulation, and nonresponders had the highest FoxP3 mRNA expression (P<0.05 or P<0.01). The content of IFN-γ by PBMCs after PHA and HBsAg stimulation was markedly lower in the nonresponders as compared with the controls and responders (P<0.05). However, there were no significant differences in the levels of IL-18, IL-4, and IL-12 from PBMCs after PHA and HBsAg stimulation between the responders and controls as well as the nonresponders (P>0.05)., Conclusion: CD4(+) CD25(+) FoxP3(+) Tregs may be involved in the negative regulation of responses to hepatitis B vaccination. Immunologic non-responses to hepatitis B vaccination may be related to IFN-γ hyposecretion in PBMCs.

Objective: To investigate whether there is mutation in DC-SIGN promoter region in patients with chronic hepatitis B (CHB) and healthy persons previously infected with hepatitis B virus (HBV) and to explore the relationship between the mutation in dendritic cell-specific intercellular adhension molecule-3-grabbing nonintegrin (DC-SIGN) promoter region and HBV., Methods: The studied population was composed of two cohorts: 47 CHB patients and 20 healthy persons previously infected with HBV. The mutation in DC-SIGN promoter region was detected with PCR, single-stranded conformational polymorphism and heteroduplex analysis, cloning, sequencing and aligning the published DC-SIGN promoter sequence., Results: The characteristic mutation within DC-SIGN promoter region in HBV infected individuals was observed. In the DC-SIGN promoter region, 4 hot spot mutations located in positions -139, -142, -222, and -336 were observed in the CHB patients, but only 1 spot mutation located in position -139 was observed in the healthy persons previously infected with HBV. The -336C which was absent in the healthy persons previously infected with HBV was shown in 11 CHB patients (23.40%). The -139T was far more frequent in the healthy persons previously infected with HBV (100%) than in the CHB patients (34.04%)., Conclusion: In the DC-SIGN promoter region, -336C may be a genetic risk factor for developing CHB, but -139T may be associated with protection against HBV.

Objective: To investigate the impact of coagulative parameters on different anticoagulation systems in molecular adsorbent recirculating system (MARS) in subjects with liver failure, and to evaluate the safety of different anticoagulation methods ., Methods: A prospective experimental observation was designed. According to anticoagulation Methods , 174 MARS treatment sessions for 146 patients with liver failure and prothrombin time activity percentage (PTA) ≤ 40% were randomly divided into 2 groups: 92 MARS treatment sessions in the heparin-free group and 82 in the low-dose heparin group. Time points of 0, 0.5, 1, 2, 3, 4, 5 and 6 h were selected to observe the coagulation changes of prothrombin time (PT), PTA, thrombin time (TT), activated partial thromboplastin time (APTT) and international normalized ratio (INR) dynamically. Adverse events such as line / filter coagulation, rupture and bleeding were also investigated and compared due to frequency and severity between the 2 groups., Results: There was no difference in PT, PTA, INR between the 2 groups, but significant differences were observed in APTT and TT and fibrinogen (Fbg). APTT and TT levels in the low-dose heparin group was increased rapidly after the first given dose of anticoagulant heparin and reached the peak within 30 min.The levels at each time point was statistically different between the 2 groups (P<0.05). A significant difference in the Fbg level was obtained between the 2 groups. In the low-dose heparin group it was stabilized and increased slightly at the end of the treatment. While in the heparin-free group it was decreased gradually and reached a ravine at the end of the treatment. A curve was observed after 2.5 h treatment between the 2 groups (P=0.001). There were 2 cases of severe bleeding after MARS was finished in the heparin group, and 1 was terminated because of degree III clotting in the heparin-free group., Conclusion: Fibrinogen should be adsorbed while the blood touches the MARS circuit path and anticoagulants can prevent it. Comprehensive analysis of blood platelet count (BPC), fibrin degradation products (FDP), D-dimer and clinical symptoms is critical and required to determine the coagulation status to select an anticoagulation system before MARS. The use of low dose heparin in MARS improves the disorder of hypercoagulable state during the high coaguation period, while heparin-free during low coagulation period can effectively prevent the occurrence of bleeding and improve the mechanism of blood coagulation by reducing heparin-like substance in the blood.

Objective: To investigate clinical features and antifungal therapeutic effect of chronic severe hepatitis (CSH) patients with invasive fungal infection (IFI), and to improve the diagnosis and treatment., Methods: Clinical manifestation, blood routine, imageology and mycetology characteristic, antifungal treatment perscription and therapeutic effect of 79 CSH patients with IFI were retrospectively analyzed. Antifungal therapeutic effect was compared between fluconazole and voriconazole., Results: Thirteen (16.5%) patients received glucocorticoid or other immunodepressants for a relatively long time, 40 (50.6%) patients had invasive operation, and 61 (77.2 %) patients were administered 1-6 kinds of broad-spectrum antibiotics. Seventy-three patients had fever. Leucocytes and neutrophilic granulocyte increased in 96.2% of the patients. Lung (31.6%), intestinal tract (26.2%) and oral cavity (14%) infections were common. Fungus was found in 70.9% of the patients. Candida albicans (40.9%) and aspergillus (21.1%) were often seen. Halo signs and crescent signs on lung CT were relatively specific in 40% of the patients with fungal pneumonia. Voriconazole was more effective than fluconazole(71.4% vs. 39.0%, P<0.05). Twelve patients with lung aspergillus infection were administered voriconazole, 8 (66.7%) patients of whom was effective, and the other 4 (33.3%) patients died., Conclusion: There are high risk factors in major CSH patients with IFI. The most common clinical manifestations of CSH patients with IFI are fever, leukocytosis, lung and intestinal tract infection. Candida albicans and aspergillus infection are common. Voriconazole is more effective than fluconazole, and can increase the survival rate of CSH patients with IFI.

Objective: To determine whether HBx gene can directly induce hepatocellular carcinoma in vivo, and to explore the mechanism of transplantation tumor in nude mice., Methods: pCMVX/QSG7701 cell lines were vaccinated into subcutaneous tissue of nude mice. pRcCMV2/QSG7701 and QSG7701 cell lines were used as controls. The sections of transplantation tumor were observed microscopically by HE staining. RT-PCR was used to detect the expression of mutant p53 and c-Myc mRNA in transplant tumor and an other 3 cell lines., Results: The transplant tumor occurred within the subcutaneous tissue of the nude mice inoculated with pCMVX/QSG7701 cell lines at 2nd week after the vaccination. No metastatic tumor was found in other organs. Transplant tumor was not formed in all the controls. HE staining confirmed that the transplant tumor was hepatocellular carcinoma. The mutant p53 mRNA and c-Myc mRNA expression level of transplant tumor and pCMVX/QSG7701 cells was significantly higher than that of pRcCMV2/QSG7701 and QSG7701 cells, respectively (P<0.01)., Conclusion: HBx gene can up-regulate the expression of mutant p53 and c-Myc genes, and directly induce hepatocellular carcinoma in vivo.