The relationship between HBV lamivudine resistance and HBV genotypes or basic core promoter mutations.

OBJECTIVE: To investigate the relationship between HBV lamivudine resistance and HBV genotypes or basic core promoter (BCP) mutations. METHODS: The common coated probes were synthesized according to the conserved regions of the preC gene of hepatitis B virus (HBV). Different colorized probes were chosen from the sequences of different genotypes of HBV (A to F), BCP and YMDD wild types and mutants, respectively. HBV DNA levels, HBV genotypes, BCP and YMDD resistants were analyzed by PCR microplate hybridization ELISA at the zero and 6th month after the patients were treated with lamivudine. RESULTS: HBV genotyping results showed that HBV types B, C, D accounted for about 30%, 36% and 23% patients respectively. Thirteen BCP mutations (type B in 1 patient, type C in 8 and type D in 4) were found before treatment with lamivudine. HBV DNA levels were lower than 100 pg/ml in 2 patients and higher than 100 pg/ml in 11. 9.4% of the HBV patients (5/43; type C in 3 and type D in 2) showed YMDD resistants and 4 BCP mutations at the same time. CONCLUSION: Oral treatment of lamivudine decreases the level of serum HBV DNA. The appearance of HBV YMDD resistants is related to certain HBV genotypes, and most of them are BCP mutations.

[Bacteriostasis of iodoform in vivo and in vitro].

OBJECTIVE: To investigate the bacteriostasis of iodoform in vivo and in vitro. METHODS: Bacteriostasis of iodoform and economycin against different species of bacteria was observed in vitro and the curves of bacteriostasis were obtained for each agent. In rabbit models traumatic liver injury complicated by infection of the wounds with Staphylococcus aureus, Escherichia coli and Pseudomonas aerudomonas, common bandage supplemented respectively with 6% iodoform, 10% iodoform, economycin ointment, and saline were applied for wound addressing and the changes in the number of bacterium in the wounds was measured 1, 3, and 7 days later, respectively. RESULTS: Iodoform showed better bacteriostatic effect than economycin in inhibiting the growth of the majority of the aerobic bacteria in vitro, and its action on anaerobic bacteria appeared similar to the effect of economycin. In rabbit models, iodoform at the concentration of 10% showed significantly better bacteriostatic effect against the 3 species of bacteria than economycin (P<0.01). CONCLUSION: Iodoform possesses more potent bacteriostatic effect than economycin against most bacteria.

[Establishment and growth regulation of rat hepatocyte colony in vitro].

OBJECTIVE: To investigate the conditions essential for culturing hepatocytes colony in vitro, and to study the growth regulation mechanisms of hepatocyte colony. METHODS: Rat hepatocytes were isolated by two-step in situ preperfusion and collagenase circulatory perfusion. The effects of hepatopoietin (HPN), nicotinamide (NA) and dimethyl sulfoxide (DMSO) on DNA synthesis, mitotic activity, morphology (under inverted microscope) and utrastructure (under TEM) of the hepatocytes were investigated in chemically defined culture medium. RESULTS: The time course of DNA synthesis in cultured hepatocytes showed that 3H-TdR incorporation was dramatically enhanced by 10 mmol/L NA, presenting 2 peaks at 60 and 84 h respectively. The plateau of DNA synthesis was diminished in the presence of DMSO, but a peak occurred again at 132 h upon NA treatment. After cell culture in the presence of HPN, NA, and DMSO for 72 h, the hepatocytes presented sustained regular bipolar mitosis, with considerable mitotic activity at 168 h. The growth characteristics of hepatocyte colony, in addition to its potential for expansion, were captured by both light and electron microscopy on day 28 of cell culture. CONCLUSION: The reciprocal actions of NA and DMSO can control the proliferation of HPN-stimulated hepatocytes, which can be used for studying human hepatocyte metabolism, cytotoxicity, biotransformation and mutagenesis, and may provide experimental evidences for the treatment of liver failure and genetic liver diseases with in vitro hepatocyte clones.

[Effect of inducible nitric oxide on intracellular homeostasis of hepatocytes].

OBJECTIVE: To investigate the effects of inducible nitric oxide (NO) and exogenous NO on the intracellular homeostasis of the hepatocytes. METHODS: Endogenous NO was induced by combined action of lipopolysaccharide (LPS) and cytokines in cultured rat hepatocytes, and exogenous NO was supplied by sodium nitroprusside (SNP) to stimulate the hepatocytes. The changes in intracellular malondialdehyde (MDA), reduced glutathione(GSH) and free calcium ([Ca2+]i) were observed. RESULTS: substantial increase by 7.97 times in intracellular MDA level and a decrease by 57.9% in GSH occurred in the hepatocytes after the cells had been incubated with LPS and cytokines for 24 h, which were reversed by 43.5% and 98.4% respectively by treatment with N(G)-monomethyl-L-arginine (NMMA), a competitive nitric oxide synthase (NOS) inhibitor. Verapamil significantly reduced both endogenous NO production and oxidative stress, while the effect of A23187 was not conspicuous. Incubation with chlorpromazine and Vitamine E (VitE), however, did not result in decreased release of NO by LPS- and cytokines-induced hepatocytes. After SNP exposure of the hepatocytes, the oxidative status was reversibly enhanced in a time-dependent manner. Short exposure to SNP led to a concentration-dependent inhibition of the rapid and transient increase in free calcium induced by K(+) depolarization and hepatopoietin-coupled calcium mobilization. CONCLUSIONS: Inducible NO may initiate and play a key role in the latter stages of metabolic and functional stress responses of hepatocytes against endotoxin and cytokines, when the reduction occurs in the capacity of NO to independently mediate lipid peroxidation and counteract oxidation. The inhibitory effect of NO on [Ca2+]i mobilization may be an important autoregulatory mechanism by means of negative feedback on protein kinase C-associated NOS induction.

HBV resistant to lamivudine: experimental and clinical studies.

OBJECTIVE: To identify the impact of lamivudine on HBV e antigen (HBeAg) seroconversion and HBV DNA level, and the appearance of Tyr-Met-Asn-Asp (YMDD) resistants. METHODS: Forty-seven hepatitis B patients were treated with oral lamivudine. ALT level and HBeAg were detected in the treatment on the zero, 3rd, 6th and 9th month respectively. The levels of HBV DNA and YMDD resistants were analyzed with PCR microplate hybridization-ELISA. RESULTS: After 9 months of treatment, HBV DNA became negative and ALT level was normal in 74% patients. Among these patients, 17% patients had HBeAg converted to negative and anti-HBe antibody positive, whereas another 15% patients showed HBeAg negative. YMDD resistants appeared in 19% patients (9/47). One, three and five resistants were detected in the treatment on the 3rd, 6th and 9th month respectively. CONCLUSIONS: Most HBV DNA in serum became negative after 9 months of treatment, and the rate of HBeAg seroconversion was 17% (HBV DNA level was lower than 100 pg/ml before treatment). YMDD resistants appeared in 19% patients.