Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay.

BACKGROUND: Adefovir dipivoxil (ADV) is effective for treatment of chronic hepatitis B virus (HBV) infection, but long-time ADV therapy leads to drug resistance because of HBV reverse transcriptase mutations. We developed a sensitive and specific method for detecting the rtA181V/T and rtN236T mutations associated with ADV resistance in chronic hepatitis B patients, based on a ligase detection reaction (LDR). METHODS: HBV templates were amplified by polymerase chain reaction (PCR), followed by LDR and electrophoresis on a sequencer. The assay was evaluated using 165 serum samples, and plasmid controls. RESULTS: In a mixture of wild-type and mutant plasmids, the assay could detect mutant plasmid at 1%. Complete concordance between the PCR-LDR assay and sequencing analysis was observed for 141 of 148 samples (95.3%) at codon 181, and 143 of 148 samples (96.6%) at codon 236. Discordant results were confirmed to be consistent with the PCR-LDR assay by subclone sequencing. Seventeen samples could not be detected by both of the methods due to low HBV DNA levels. CONCLUSIONS: The PCR-LDR assay can sensitively and specifically detect the rtA181V/T and rtN236T mutations, and may be used for monitoring ADV resistance in patients infected with HBV.

[Clinical significance of AFP-L3 variants determined by micro centrifugal column].

OBJECTIVE: To evaluate the clinical significance of AFP-L3 in patients with hepatocellular carcinoma. METHODS: Serum AFP-L3 variants were separated by micro centrifugal column, and detected by chemiluminescence. RESULTS: AFP and AFP-L3 levels were higher in patients with hepatocellular carcinoma than those in patients with chronic hepatitis (P<0.001); as a diagnostic target, the sensitivity and specificity of AFP-L3 were 72.3 percent and 97.2 percent, respectively. Eight patients with hepatitis have higher AFP-L3, but none of them were found with carcinoma by CT three months later. CONCLUSION: AFP-L3 is very useful in the diagnosis of patients with hepatocellular carcinoma.

[Immune response and immunopathology in inducible costimulatory molecule (ICOS) transgenic mice infected with Schistosoma japonicum].

OBJECTIVE: To establish the ICOS transgenic mice schistosomiasis japonica model and to observe the immune response and immunopathology of the model. METHODS: The transgenic mice were infected with Schistosoma japonicum. Spleen cells and sera of mice were harvested at week 4, 6, and 8 after infection. The cytokines IFN-gamma and IL-4 were measured in culture supernatans by ELISA. The serum IgG, IgGI and IgG2a were measured by ELISA at different period of infection. Liver tissue sections were prepared with HE staining. Liver granuloma formation was observed under microscope. RESULTS: The expression level of IFN-y showed no significant difference between ICOS transgenic mice and control, while that of IL-4 in ICOS transgenic mice was significantly up-regulated to 20.81+/-1.95 and 25.31+/-3.37 pg/ml at week 6 and 8 respectively (P<0.01). The serum IgG and IgGl in ICOS transgenic mice were also significantly higher than those in control. Th2 differentiation index and lgC1/IgG2a were used to evaluate the immune regulation balance of Thl/Th2, and results showed that Th2 response in ICOS transgenic mice was significantly stronger than that of the control. The egg granuloma response in ICOS transgenic mice was also significantly stronger than that in control (P<0.01). The rate of egg granuloma enlargement was 24.48% and 26.37% at week 6 and 8 respectively. CONCLUSION: The findings suggest that there is stronger Th2 type response in ICOS transgenic mice infected with Schistosoma japonicum and ICOS may play an important role in the egg granuloma formation of Schistosoma japonicum.

Cloning, structural organization and chromosomal mapping of rat costimulatory molecule 4-1BBL.

4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL (GenBank accession No. AY259541). The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type II transmembrane glycoprotein, the same as human and murine 4-1BBL. "SDAA" in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I ("SXXS" motif), which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned (GenBank accession No. AY332409). Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues: brain, lung, colon, liver, thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 (GenBank accession UniGene No. Rn.46783). Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.