[Long-term prognosis and quality of life of survivors with hepatitis B virus-related acute-on-chronic liver failure].

OBJECTIVE: To explore the long-term prognosis and health-related quality of life of patients surviving hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). METHODS: The clinical data were collected from patients with HBV-ACLF, who were hospitalized in our department between November, 2011 and October, 2016 and survived for more than 90 days. The patients were followed for occurrence of newly diagnosed cirrhosis, decompensation events, hepatocellular carcinoma and death. The quality of life of the patients was evaluated using SF-36 score, and the patients with chronic hepatitis B (CHB) and cirrhosis treated during the same period served as controls. RESULTS: A total of 223 ACLF survivors were included in this study. According to the presence of cirrhosis on admission, the enrolled patients were divided into chronic hepatitis B-related ACLF (CHB-ACLF) group (n=130) and liver cirrhosis ACLF (CIR-ACLF) group (n=93). The 12-, 24- and 50-month survival rates in CHB-ACLF group were 97%, 95.7% and 93.9%, respectively, significantly higher than the rates in CIR-ACLF group (91%, 86% and 74%, respectively; P=0.007). In patients with CHB-ACLF, the 12-, 24- and 36-month progression rates of cirrhosis were 37.9%, 58.4% and 68.7% respectively. Multivariate Cox regression identified the peak value of serum creatinine (HR=1.015, P=0.026) and INR (HR=2.032, P=0.006) within 28 days as independent risk factors and serum sodium at baseline (HR=0.84, P=0.035) as an independent protective factor of occurrence of cirrhosis. The score of mental health on SF-36 in ACLF group was significantly lower than the national norms, and the scores for general health and body pain of ACLF patients were significantly higher than those in patients with CHB or cirrhosis. CONCLUSION: The long-term prognosis of ACLF survivors with and without cirrhosis can be different. Acute attacks are associated with an increased rate of cirrhosis progression in CHB patients who recovered from ACLF, possibly in relation with the severity of extra-hepatic organ injuries. The physical and social functions of long-term survivors of ACLF do not significantly decline, but their psychological status can be affected.

LC-MS/MS quantification of sulfotransferases is better than conventional immunogenic methods in determining human liver SULT activities: implication in precision medicine.

This study aims to determine whether enzyme activities are correlated with protein amounts and mRNA expression levels of five major human sulfotransferase (SULT) enzymes in 10 matched pericarcinomatous and hepatocellular carcinoma liver samples. The MRM UHPLC-MS/MS method, Western blot and RT-PCR were used along with SULT activity measurement using probe substrates. The LC-MS/MS method was specific for all five tested SULTs, whereas Western blot was specific for only two isoforms. The activities of SULT1A1, SULT1B1, SULT1E1 and SULT2A1 in 9 of 10 samples showed a significant decrease in tumor tissues relative to matched pericarcinomatous tissues, whereas the activities of SULT1A3 in 7 of 10 samples increased. The turnover numbers of SULTs did not change, except for SULT1A1. A generally high degree of correlations was observed between SULT activities and protein amounts (r2 ≥ 0.59 except one), whereas a low degree of correlations was observed between SULT activities and mRNA expression levels (r2 ≤ 0.48 except one). HCC reduced the SULT activities via impaired protein amounts. LC-MS/MS quantification of SULTs is highly reliable measurement of SULT activities, and may be adopted for implementing precision medicine with respect to drugs mainly metabolized by SULTs in healthy and HCC patients.

[Entecavir combined with Fufang Biejia Ruangan tablet in treatment of chronic hepatitis B patients with liver fibrosis: 96-week efficacy analyses].

OBJECTIVE: To evaluate the effect of long-term therapy with entecavir and Fufang Biejia Ruangan tablet in patients with chronic hepatitis B (CHB)-associated fibrosis and explore the synergistic therapy that accelerates the reversion of liver fibrosis. METHODS: A total of 197 patients with CHB-associated fibrosis were recruited from Nanfang Hospital between June, 2010 and June, 2015. The patients were divided into two groups after matching for age, gender and liver stiffness measurement (LSM), namely group A (n=98) treated with Fufang Biejia Ruangan Tablet plus entecavir, and group B (n=99) to receive entecavir only. HBV DNA quantification, HBV serological indicators, blood biochemical indexes, and results of abdominal ultrasound and FibroScan were recorded every 12 weeks. FibroScan values were converted to Metavir staging. RESULTS: Both groups showed significant decreases in serum levels of HBV DNA, alanine aminotransferase (ALT), and LSM value from baseline (all P<0.05). The median time to achieve Metavir fibrosis staging improvement were 72 weeks in group A and 96 weeks in group B (P<0.05), and the median time to achieve ALT and AST normalization were 12 and 24 weeks in Group A, respectively, significantly shorter than the time in group B (P<0.05). No significant difference was found between the two groups in HBV DNA undetectable rate and HBeAg seroconversion rate. CONCLUSION: The combination therapy with Fufang Biejia Ruangan tablet and entecavir produces a stronger efficacy than entecavir alone in the treatment of chronic hepatitis B patients with liver fibrosis, and Fufang Biejia Ruangan tablet shows an obvious hepatoprotective effect in these patients.

Drug-Metabolizing Activity, Protein and Gene Expression of UDP-Glucuronosyltransferases Are Significantly Altered in Hepatocellular Carcinoma Patients.

UDP-glucuronosyltransferases (UGTs), the most important enzymes in body detoxification and homeostasis maintaining, govern the glucuronidation reaction of various endogenous and environmental carcinogens. The metabolic function of UGTs can be severely influenced by hepatocellular carcinoma (HCC), the fifth prevalent and third malignant cancer worldwide. Particularly in China, HBV-positive HCC account for approximately 80% of HCC patients. But rare papers addressed the alteration on the metabolism of UGTs specific substrates, translational and transcriptional activity of UGTs in HBV-positive HCC patients. In present study, we choose the main UGT isoforms, UGT1As, UGT1A1, UGT1A9, UGT1A4 and UGT2B7, to determine the alterations of metabolic activity, protein and gene expression of UGTs in HBV-positive HCC. The corresponding specific substrates such as genistein, SN-38, tamoxifen, propofol and zidovudine were utilized respectively in UGTs metabolic activity determination. Furthermore, the plausible mechanism responsible for UGTs alterations was addressed by analyzing the protein and gene expressions in tumor and the adjacent normal tissues in HBV-positive HCC. The results revealed that in the tumor human liver microsomes (HLMs), either V(max) (maximum reaction rate, R(max) for UGT1A1) or the clearance rates (V(max)/K(m), Clint) of UGT1A, UGT1A1, UGT1A4, UGT1A9 and UGT2B7 were significant lower than those of in the adjacent normal HLMs. Subsequently, the relative protein and gene expressions of these isoforms were notably decreased in most of tumor tissues comparing with the adjacent normal tissues. More interestingly, in tumor tissues, the metabolic activity reduction ratio of each UGT isoform was closely related to its protein reduction ratio, indicating that decreasing protein level would contribute to the reduced metabolic function of UGTs in HBV-positive HCC. In summary, our study firstly determined the alteration of UGT function in HBV-positive HCC patients, which would provide an important insight for toxicity or efficacy determination of chemotherapeutic drugs, and even bring a new strategy for clinical regimen in the health cares for the relative patients.

Pharmacokinetic characterization of oxymatrine and matrine in rats after oral administration of radix Sophorae tonkinensis extract and oxymatrine by sensitive and robust UPLC-MS/MS method.

The purpose of this study is to systematically investigate the pharmacokinetic (PK) behaviors of radix Sophorae tonkinensis (S. tonkinensis) using oxymatrine (OMT) and matrine (MT) as the target markers (2 mg/kg OMT and 1.3 mg/kg MT, oral administration). The PK characteristics in radix S. tonkinensis extracts were also compared with those of pure OMT. A fast ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed. OMT absorption was very fast, and no significant differences were observed (p>0.05) in tmax, CL, and t1/2 for both pure OMT and extracts. Cmax and AUC0→∞ of pure OMT were significantly higher than those of S. tonkinensis extracts (Cmax, 61.64±6.65 vs. 43.24±10.14 ng/mL; AUC, 9894.48±2234.99 vs. 4730.30±3503.8 min ng/mL) (p<0.05). However, the absolute OMT bioavailability of pure OMT was higher than that of the compound in radix S. tonkinensis extracts (6.79±2.52% vs. 1.87±2.66%). By contrast, the bioavailability of total alkaloids (OMT+MT) after pure OMT administration was 81.14±8.83%, similar to that of radix S. tonkinensis extracts (69.36±17.37%) (p>0.05). It was presumed that OMT absorption has no effect on the bioavailability of the two alkaloids. Other constituents in radix S. tonkinensis extracts can influence the transformation of OMT to MT, which directly leads to variations in the PK behavior of OMT. In addition, the protein binding of OMT and MT in plasma was very low (4.80%-8.95% for OMT, 5.10-10.55% for MT). In conclusion, OMT in radix S. tonkinensis extracts exhibits different PK behaviors with pure OMT through the transformation of OMT to MT due to other complex ingredients.

[Relationship between single nucleotide polymorphisms in IL28B gene and response to interferon treatment in chronic hepatitis B patients].

OBJECTIVE: To explore the relationship between the single nucleotide polymorphisms (SNPs) of rs12979860 and rs8099917 in IL28B gene and the response to interferon treatment in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B patients. METHODS: Peripheral blood samples were collected from 82 HBeAg-positive patients with chronic hepatitis B receiving interferon treatment, including 38 with favorable response to the treatment and 44 without response. IL28B gene was amplified from the chromosomal DNA, and rs8099917 SNP was genotyped based on PCR-RLFP and rs12979860 SNP by sequencing. RESULTS: In the responsive patients, the distribution frequencies of TT and TG+GG genotypes and allele G in SNPrs8099917 were 81.6% (31/38), 18.4% (7/38), and 9.2% (7/76), as compared to the frequencies of 97.7% (43/44), 2.3% (1/44), and 1.1% (1/88) in nonresponsive patients, respectively. The frequencies showed significant differences between the responsive and nonresponsive patients (P=0.014 for genotypes and P=0.025 for allele G). The distribution frequencies of CT genotypes and allele T in SNPrs12979860 showed no differences between the responsive and nonresponsive patients (P<0.05). CONCLUSION: rs8099917 SNP is probably associated with the response to interferon treatment in HBeAg-positive patients with chronic hepatitis B, and Allele G may be predictive of the treatment success.

Simple nucleos(t)ides as HBV prophylaxis regime of post-liver transplantation: Six-year followed up.

A combination of nucleos(t)ides and hepatitis B immunoglobulin (HBIg) has been found to be effective for the prevention of hepatitis B viral (HBV) reinfection after liver transplantation (LT), but its administration is costly, and not always available. We report the case of a male, 33-year-old cirrhotic patient who has tested positive for serum HBsAg, and HBeAg, with 9.04 × 10(7) copies/mL of HBV DNA. He suffered from acute liver failure and was near death before undergoing emergency LT. No HBIg was available at the time, so only lamivudine was used. He routinely received immunosuppression medication. Serum HBV DNA and HBsAg still showed positive post-LT, and the graft re-infected. Hepatitis B flared three months later. Adefovir dipivoxil was added to the treatment, but in the 24(th) mo of treatment, the patient developed lamivudine resistance and a worsening of the hepatitis occurred shortly thereafter. The treatment combination was then changed to a double dosage of entecavir and the disease was gradually resolved. After 60-mo of post-LT nucleos(t)ide analogue therapy, anti-HBs seroconverted, and the antiviral was stopped. By the end of a 12-mo follow-up, the patient had achieved sustained recovery. In conclusion, the case seems to point to evidence that more potent and less resistant analogues like entecavir might fully replace HBIg as an HBV prophylaxis and treatment regimen.

[Preparation of human anti-HBs from volunteers with hepatitis B vaccine boost vaccination by modified B-cell immortalization technique].

OBJECTIVE: To establish immortalized B lymphoblast cell lines (B-LCLs) from healthy anti-HBs antibody (anti-HBs)-positive volunteers and screen for human anti-HBs and the antibody-secreting cells. METHODS: The peripheral blood mononuclear cells (PBMC) isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus (EBV) and incubated in the presence of CpG DNA motifs and cyclosporin A (CyA). The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs. RESULTS: Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138. CONCLUSION: Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.

[Stable expression of HBV C gene mutants in immortalized human B-cell lines].

OBJECTIVE: To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. METHODS: V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. RESULTS: DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant. CONCLUSION: Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.