The significance of Toll-like receptor 4 (TLR4) expression in patients with chronic hepatitis B.

PURPOSE: To investigate the importance of Toll-like receptor 4 (TLR4) expression on hepatocytes obtained from Chronic Hepatitis B patients as well as on hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines. METHODS: Expression of TLR4 in liver tissues was determined by immunohistochemistry in 75 patients with CHB and in10 healthy controls. The protein and mRNA 1eve1s of TLR4 in hepatocellular carcinoma HepG2 and HepG2.2.15 cells were measured by flow cytometry (FCM) and real-time quantitative PCR (RQ-PCR), and endotoxin triggered TNF-alpha secretion in HepG2 and HepG2.2.15 cells was evaluated by ELISA. RESULTS: TLR4 expressed mainly in the cytoplasm and some on cell membrane in hepatocytes. The staining scores of TLR4 expression in the liver tissues of patients with CHB were significantly higher than that of healthy controls. The liver tissues from patients with severe CHB expressed higher level of TLR4 than those from patients with mild CHB. Furthermore, the staining scores of TLR4 expression in the liver tissues of patients with CHB were positively correlated with the grading scores. Our results also showed that the mean fluorescence intensity and TNF-alpha secretion induced by endotoxin as well as the protein and mRNA 1eve1s of TLR4 in HepG2.2.15 cells were all significantly higher than those in HepG2 cells. CONCLUSION: TLR4 was up-regulated in the hepatocytes in patients with CHB. This indicates a potentially important interaction between TLR4 expression and the pathogenesis of CHB.

[A close relationship between viral hepatitis B and Toll-like receptor 2].

OBJECTIVE: To investigate the relationship of Toll-like receptor 2 (TLR2) and viral hepatitis B through testing the expression of TLR2 in liver tissues of patients infected with HBV and in HepG2 and HepG2.2.15 cell lines. METHODS: Expression of TLR2 was determined by Elivision immunohistochemistry in liver tissues from patients with chronic viral hepatitis B (CHB), chronic severe hepatitis (CSH), from healthy controls and from cells of HepG2 and HepG2.2.15 hepatocellular carcinoma cell lines. Direct immunofluorescence flow cytometry was used to detect TLR2+ cell percentage and mean fluorescence intensity (MFI). RESULTS: The intensity of TLR2 expression in liver tissues of CHB and CSH was significantly higher than that of the healthy controls (probability value less than 0.01). The positive staining was mainly located in the cytoplasm and on some cell membranes. In CHB, the intensity of TLR2 expression was positively correlated with the grade of necroinflammatory activity (r=0.597, P less than 0.01). There were significant differences of serum total bilirubin levels with different grades of positive staining of TLR2 (P less than 0.05). In liver tissues of the CHB patients, the positive staining of TLR2 was shown in small foci. MFI of TLR2 (10.7+/-2.8) and TLR2+ cell percentage (16.3%+/-7.0%) of HepG2.2.15 cells were both significantly higher than those of HepG2 cells (1.0+/-0.3, 0.4%+/-0.1%, P less than 0.01). CONCLUSIONS: Expression of TLR2 is closely correlated with hepatitis B, especially to the grades of its necroinflammatory activity.

Effects of c-myb antisense RNA on TGF-beta1 and beta1-I collagen expression in cultured hepatic stellate cells.

AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-beta1 and beta1-I collagen in cultured hepatic stellate cells (HSC) from rats. METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP. The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4, 5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide(MTT) method. The expression of c-myb, alpha (1)-I collagen and TGF-beta1 mRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively. RESULTS: HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, the c-myb protein expression, cell proliferation,and alpha (1)-I collagen and TGF-beta1 mRNA expression were repressed significantly compared with their corresponding control groups (P<0.01). CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, alpha (1)-I collagen and TGF-beta1 mRNA expression, suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.

[Clinical characteristics of and the related factors to the relapse of chronic hepatitis B after lamivudine withdrawal].

OBJECTIVES: To investigate the cases of chronic hepatitis B relapse after lamivudine withdrawal, and to find clinical characteristics and related factors to them. METHODS: 46 cases of chronic hepatitis B relapse after lamivudine withdrawal were investigated and followed up. The diagnosis and the course of the diseases before therapy with lamivudine, the dosage, effects, period of treatment, the reasons for lamivudine withdrawal, the biochemistry, immunological and virulogical data in each period, YMDD mutation, pre-C mutation and prognosis after relapse were recorded. RESULTS: The periods of treatment of 32.6% of patients in this group were shorter than 52 weeks. The HBV DNA of 93.6% of patients turned negative at the time of lamivudine withdrawal and returned to positive in all of those patients when relapsed; of the 6.4% of patients who did not turn negative at the time of lamivudine withdrawal, their HBV DNA was elevated more than 2 log when relapsed. A majority of patients (71.7%) did not ask their physicians when they decided to withdraw from lamivudine. There were 61.5% of the other patients who withdrew from lamivudine on the advice of physicians but they were not followed up. The state of their illness in 71.7% (33/46) patients was more severe than before their lamivudine therapy. In these cases, the YMDD mutation was detected in 78.8% of patients (chi2 = 23.23, P<0.01), and pre-C mutation was detected in 84.8% of patients (chi2 = 21.04, P<0.01), higher than that in the cases with aggrevation of their illnesses before the lamivudine therapy. The median time between lamivudine withdrawal and relapse was 12 weeks; it was negatively correlated with ALT (r = 0.32, P<0.05) detected at the time of lamivudine withdrawal. The severity of the illness at the time of relapse was related with age (r = 0.40, P<0.01) and YMDD mutation (r = 0.31, P<0.05). The prognosis was related with the age of the patient (r = 0.49, P<0.01), the diagnosis (r = 0.39, P<0.01), TBil (r = 0.46, P<0.05) and ALT (r = 0.32, P<0.05) detected before lamivudine therapy, HBeAg became negative when lamivudine was withdrawn (r = 0.31, P<0.05), TBil (r = 0.55, P<0.01) and PTA (r = 0.57, P<0.01) detected when relapsed. CONCLUSION: A majority of patients did not follow the lamivudine treatment recommendation of the experts group on lamivudine clinical practice in China. The major reason for relapse perhaps was revived HBV DNA multiplication, which induces damage of hepatocytes after lamivudine withdrawal. The YMDD mutation and/or pre-C mutation may be one of the factors that aggravate the damage of hepatocytes during the relapse. The factors including age of the patient, diagnosis before the treatment, TBil and ALT detected before lamivudine therapy, HBeAg turning to negative when lamivudine is withdrawn, and TBil and PTA detected during relapse may all impact the prognosis.