Characteristics analysis of hepatitis B core-related antigen in children with hepatitis B e antigen-positive chronic viral hepatitis B infection.

BACKGROUND: The objective of antiviral therapy for chronic viral hepatitis B infection (CHB) is to achieve a functional cure. An important viral marker in the serum of patients with CHB is the serum hepatitis B core-related antigen (HBcrAg). However, there is limited research on HBcrAg in juvenile patients with CHB. In this study, we aimed to investigate the correlation between serum HBcrAg and other hepatitis B virus (HBV) markers in children with CHB and its predictive significance for prognosis during antiviral therapy. METHODS: A single-center retrospective study was conducted involving 79 children with CHB, aged between 0 and 16 years. All the children were treated with interferon [or combined nucleos(t)ide analogs] for 48 weeks. HBcrAg, hepatitis B surface antigen (HBsAg), and HBV DNA were measured before treatment, and at 12 and 48 weeks after treatment. The enrolled children were classified into the seroclearance group and the nonseroclearance group based on the therapeutic outcome. RESULTS: HBsAg seroclearance was observed in 28 out of 79 patients and hepatitis B e antigen seroconversion without HBsAg seroclearance was observed in 14 out of 79 patients following the conclusion of the treatment, with baseline HBcrAg titer levels showing no statistical significance in both the seroclearance and nonseroclearance groups (P = 0.277). HBsAg and HBV DNA were positively correlated with HBcrAg in children with CHB (R2 = 0.3289, 0.4388). The area under the receiver operating characteristic curve of the decrease in HBcrAg at 12 weeks of treatment as a predictor of seroclearance at 48 weeks of treatment, exhibited a value of 0.77. CONCLUSION: A decrease in serum HBcrAg levels in children with hepatitis B serves as a prognostic indicator.

Functional cure is associated with younger age in children undergoing antiviral treatment for active chronic hepatitis B.

BACKGROUND AND AIMS: Functional cure is difficult to achieve using current antiviral therapies; moreover, limited data are available regarding treatment outcomes in children. This retrospective study aimed to assess the frequency of functional cure among children undergoing antiviral treatment for active chronic hepatitis B (CHB). METHODS: A total of 372 children aged 1-16 years, with active CHB were enrolled and underwent either nucleos(t)ide analog monotherapy or combination therapy with interferon-α (IFN-α) for 24-36 months. All children attended follow-up visits every 3 months. Functional cure was defined as evidence of hepatitis B virus (HBV) DNA loss, circulating hepatitis B e antigen (HBeAg) loss/seroconversion, and hepatitis B surface antigen (HBsAg) loss. RESULTS: After 36 months of antiviral treatment and/or follow-up visits, children with CHB aged 1- < 7 years exhibited higher rates of HBV DNA clearance, HBeAg seroconversion, and HBsAg loss than CHB children ≥ 7-16 years of age (93.75% versus [vs.] 86.21% [p < 0.0001]; 79.30% vs. 51.72% [p < 0.0001]; and 50.78% vs. 12.93% [p < 0.0001], respectively). Longitudinal investigation revealed more rapid dynamic reduction in HBV DNA, HBeAg, and HBsAg levels in children aged 1-7 years than in those aged ≥ 7-16 years with CHB. According to further age-stratified analysis, HBsAg loss rates were successively decreased in children with CHB who were 1- < 3, 3- < 7, 7- < 12, and 12-16 years of age (62.61% vs. 41.13% vs. 25.45% vs. 1.64%, respectively; p < 0.0001) at 36 months. In addition, baseline HBsAg level < 1,500 IU/mL was found to favor disease cure among these pediatric patients. No serious adverse events were observed throughout the study period. CONCLUSION: Results of the present study demonstrated that children aged 1- < 7 years, with active CHB can achieve a high functional cure rate by undergoing antiviral therapy compared to those aged ≥ 7 years, who undergo antiviral therapy. These data support the use of antiviral treatment at an early age in children with CHB. However, future prospectively randomized controlled trials are necessary to validate the findings of this study.

The utility of hierarchical genetic testing in paediatric liver disease.

BACKGROUND & AIMS: Genetic factors underlie a substantial proportion of paediatric liver diseases. Hereditary liver diseases have considerable genetic heterogeneity and variable clinical manifestations, which bring great challenges to clinical and molecular diagnoses. In this study, we investigated a group of paediatric patients with varying degrees of liver dysfunction using a hierarchical genetic testing strategy. METHODS: We first applied a panel encompassing 166 known causal genes of liver disease. We then used exome sequencing (ES) in those patients whose cases remained undiagnosed to identify the genetic aetiology of their symptoms. RESULTS: In total, we enrolled 131 unrelated paediatric patients with liver disease of Chinese Han ethnicity. We first applied targeted gene sequencing of 166 genes to all patients and yielded a diagnostic rate of 35.9% (47 of 131). Eighty-four patients who remained undiagnosed after target gene sequencing were subjected to ES. As a result, eight (8/84, 9.5%) of them obtained molecular diagnoses, including four patients suspected of abnormal bilirubin metabolism and four idiopathic cases. Non-typical genetic findings, including digenic inheritance and dual molecular diagnosis, were also identified. Through a comprehensive assessment of novel candidate variants of uncertain disease association, 11 patients of the remaining undiagnosed patients were able to obtain likely molecular diagnoses. CONCLUSIONS: Our study presents evidence for the diagnostic utility of sequential genetic testing in a cohort of patients with paediatric liver disease. Our findings expand the understanding of the phenotypic and mutational spectrum underlying this heterogeneous group of diseases.

Novel and Specific MRI Features Indicate the Clinical Features of Patients With Rare Hepatic Tumor Epithelioid Hemangioendothelioma.

OBJECTIVE: To investigate the MRI features and clinical significance of hepatic epithelioid hemangioendothelioma (HEHE). METHODS: Clinical records and MRI findings were retrospectively evaluated in nine HEHE patients from May 2010 to January 2020. RESULT: There were 121 lesions in nine patients with a predominantly peripheral distribution. Five lesions (4.13%) in two patients (22.22%) had evidence of capsular retraction, and three patients had lung metastasis (33.33%). Dynamic contrast-enhanced MRI showed progressive enhancement, mainly in two ways: ring enhancement with hypovascularity in four patients (44.44%) and ring enhancement with hypervascularity in five patients (55.56%). Imaging demonstrated a multilayer ring appearance, which was typically observed on T2-weighted imaging (T2WI). The most common appearance consisted of two layers of varying signal, with some images displaying up to four layers. There were significant differences in the size of lesions between different layers of multilayer ring appearance (p < 0.001). All lesions exhibited a two-layer appearance on diffusion-weighted imaging (DWI), with hyperintensity at the periphery and a slightly high signal at the center (except for those with a single layer on T2WI). The "vascular penetration sign" was observed in most lesions, and the blood vessels of 112 lesions (92.56%) were portal vein branches, and five (4.13%) were hepatic vein branches. Pulmonary metastasis was found in three patients with the "vascular penetration sign" of hepatic vein branches. CONCLUSION: The multilayer ring appearance on T2WI, the "vascular penetration sign", and the two enhancement patterns may be of great significance in the diagnosis and treatment of HEHE. The "vascular penetration sign" of hepatic vein branches may indicate extrahepatic metastasis.

Prediction for HBsAg seroconversion in children with chronic hepatitis B.

BACKGROUND: To establish a prediction of HBsAg seroconversion in children with chronic hepatitis B (CHB), so as to help clinicians to choose therapeutic strategy. METHODS: A total of 63 children with HBeAg-positive CHB aged 1 to 17 years, who admitted to the fifth medical center of Chinese PLA general hospital and treated with interferon α (IFNα) 48 weeks were enrolled, the clinical data were measured. Based on the results of HBsAg seroconversion (HBsAg < 0.05 IU/mL and anti-HBsAg > 10 IU/L) at week 48, the patients were divided into HBsAg seroconversion (S) group and non-HBsAg seroconversion (NS) group. Multivariate COX regression was used to identify the impact factors associated with HBsAg seroconversion. A novel prediction index was established and the area under the receiver operating characteristic curve (AUROC) was used to assess the prediction for HBsAg seroconversion. RESULTS: The 63 patients were divided into S group (20.6%, 13/63) and NS group (79.4%, 50/63). Univariate and multivariate analysis identified age, baseline intrahepatic cccDNA and serum HBsAg levels were independent impact factors for HBsAg seroconversion. Intrahepatic cccDNA was positively correlated with serum HBsAg (r = 0.464, p = 0.000). AUROC of HBV cccDNA was 0.83 (95% CI 0.71 to 0.95) and AUROC of baseline HBsAg was 0.77 (95% CI 0.61 to 0.92). Intrahepatic cccDNA ≤ 0.08 log10 copies/106 cell is regarded as cutoff value, the positive predictive value(PPV) and negative predictive value(NPV) for HBsAg seroconversion were 86.8% and 60.0%, respectively, with a sensitivity of 92.0% and specificity of 56.2%. HBsAg ≤ 3.68 log10 IU/mL is used as cut off value, the PPV and NPV for HBsAg seroconversion were 91.2% and 56.3%, respectively; the sensitivity and specificity was 86.0% of 69.2%, respectively. There was no statistical difference between them for predicting HBsAg seroconversion (p = 0.146). CONCLUSIONS: HBsAg seroconversion can be predicted by the baseline serum HBsAg or intrahepatic cccDNA in children with CHB. Using the index, clinicians can choose more reasonable therapeutic strategy and reduce the waste of medical resources.

Assessment of liver fibrosis by transient elastography in young children with chronic hepatitis B virus infection.

BACKGROUND: This study aimed to compare the diagnostic accuracy of transient elastography (TE) and biopsy for the detection of liver fibrosis in children with chronic hepatitis B (CHB). METHODS: This single-center prospective study included 157 CHB children aged 0-6 years. All patients underwent liver stiffness measurement (LSM) by TE and liver biopsy, separated by an interval of less than 1 week. RESULTS: The LSM, aspartate aminotransferase-platelet ratio index (APRI), and fibrosis-4 index (FIB-4) were positively correlated with activity grade and fibrosis stage in CHB children. The areas under the receiver operating characteristic curves (AUCs) of LSM for identifying significant (F ≥ 2) and advanced (F ≥ 3) fibrosis were 0.732 and 0.941, respectively. The cut-off values, specificity, and sensitivity for significant fibrosis were 5.6 kPa, 75.7%, and 67.4%, respectively; the corresponding values for advanced fibrosis were 6.9 kPa, 91.5%, and 81.3%, respectively. Compared to LSM, the overall diagnostic performances of APRI and FIB-4 for significant and advanced fibrosis were suboptimal, with low AUCs and sensitivity. Since LSM, platelet, and Log10 (hepatitis B surface antigen) were independent factors associated with the fibrosis stage (F < 2 and F ≥ 2), they were used to formulate the "LPS" index for the prediction of F ≥ 2. The AUC of LPS (for F ≥ 2) was higher than that of LSM (0.792 vs. 0.732, p < 0.05), and had an improved sensitivity (76.6% vs. 67.4%). CONCLUSIONS: TE is a promising technology for the diagnosis of advanced fibrosis in CHB children aged 0-6 years.

Nucleoside analogs treatment delay the onset of hepatocellular carcinoma in patients with HBV-related cirrhosis.

Whether Nucleos(t)ide analogs(NA) treatment can delay the onset of HCC remains unclear. We retrospectively analyzed the clinical data of patients with HBV-related cirrhosis and HCC from 2000 to 2012. Cox proportional hazards model was used to explore the association between NA treatment and postponement of HCC development, the dependent variable was time interval from cirrhosis treatment towards the onset of HCC, and the covariates included age, sex, family history, compensation status at baseline. A total of 1155 HCC patients treated with NAs (n = 528, lamivudine, adefovir, entecavir) and non NA (n = 627) for more than 24 months before the occurrence of HCC were incorporated into the cohort. Compared with the non-NA group, NAs therapy was associated with delaying the onset of HCC in patients with cirrhosis. Significant factors were: adefovir treatment (n = 181; p = 0.0072; HR: 0.792; 90% CI: 0.687-0.914), entecavir treatment (n = 83; p = 0.0068; HR: 0.716; 90% CI: 0.585-0.877), lamivudine switched to adefovir treatment (n = 95, p = 0.0808; HR: 0.822; 90% CI: 0.684 to 0.989). But Lamivudine monotherapy was not a significant factor (n = 102; p = 0.6877; HR: 1.045; 90% CI: 0.873-1.250). Long-term NA treatment (> 6 months, except for lamivudine monotherapy) can delay the onset of HCC in patients with HBV-related cirrhosis, and applying high barrier NA to resistance is important in these patients.

IL28B SNP rs12979860 is the Critical Predictor for Sustained Viral Response in Chinese Children Aged 1 to 6 Years with Chronic Hepatitis C.

Clinical data on children with chronic hepatitis C (CHC) remain extremely limited. This study investigated sustained virologic response (SVR) to alfa-interferon 2b plus RBV treatment in children aged 1-6 years with unsafe injection-acquired CHC. 154 children with CHC aged 1 to 6 years were enrolled, 101 of them were male (65.6%) and 53 were female (34.4%), and they were treated with alfa-interferon at a dose of 1-5 MIU/m2 3 times weekly plus oral RBV (15 mg/kg/day) for 48 weeks. 57(39.3 %) of them were genotype 1b, 73(50.3%) were genotypes 2a, 15(10.3%) were undecided type. SVR was achieved in 53 of 57(93.0%) patients with genotype 1b, in 72 (98.6%) of 73 with genotype 2a, 15(100.0%) of 15 with undecided type. There was no significant statistical difference in SVR between male and female (98.0% vs 94.3%, p=0.340), genotype 2a and those with genotype 1b(98.6% vs 93.0%, p=0.160), ALT>40U/L group and ALT≤40U/L group(96.7% vs 96.8%, p=1.000), AST>40U/L group and AST≤40U/L group(95.9% vs 98.2%, p=0.654) as well as lower baseline viral load group (<6×105 IU/ml) and higher baseline viral load group(≥6×105 IU/ml)(97.3% vs 95.3%, p=0.916). Leucopenia, neutropenia, hemoglobin concentration decrease, fever, platelet count decrease and rash were 8.4%, 69.5%, 24.0%, 50.6%, 1.9% and 4.5%, respectively. And only 12(7.8%) individuals developed thyroid autoantibodies. The SVR was higher in patients with IL-28B genotype C/C than C/T (99.0% vs 80%, p=0.002). Compared with HCV viral genotype, ALT level and baseline viral load, IL-28B rs12979860 is more suitable for predicting antiviral efficacy in children with CHC. It is inappropriate to take the increase of ALT level as an essential indicator for antiviral treatment in children aged 1-6 years.

Clinical Features of Patients With Ebola Virus Disease in Sierra Leone.

BACKGROUND: Clinical and laboratory data available on patients with Ebola virus disease (EVD) remain extremely limited. We summarized the clinical characteristics of patients with EVD and analyzed the factors related to their death. METHODS: Patients admitted for care at the Freetown China-Sierra Leone Friendship Hospital during 1 October-14 November 2014 were enrolled in this study. The clinical data of these patients were retrospectively analyzed. RESULTS: Sixty-one patients were confirmed to have EVD; 28 of them (45.9%) were male and 33 (54.1%) were female. Their median age was 28 years (range, 1.17-67 years). The median duration from symptom onset to clinic visit time was 5 days (range, 1-16 days). Among these patients, 42 of them (68.9%) died. Of the confirmed cases, 18.0% did not present with fever. Patients aged >30 years had a higher fatality rate than those <30 years (87.0% vs 57.9%; P = .0175). The mean duration from symptom onset to clinic presentation of the survivors (4.57 ± 2.79 days) was shorter than that of the nonsurvivors (6.34 ± 3.33 days). Clinical factors associated with a fatal outcome included weakness, extreme fatigue, vomiting, diarrhea, mental symptoms, bleeding, and loss of appetite. No statistical difference in the case fatality rate between males and females was found (P = .2061). CONCLUSIONS: The mortality of patients with EVD was closely associated with age and duration from symptom onset to presentation for care. Patients with EVD identified in the current outbreak did not necessarily have fever. Early diagnosis of the disease and timely symptomatic treatment may greatly contribute to the reduction of fatality rate of patients with EVD.

Peripheral blood mononuclear cell traffic plays a crucial role in mother-to-infant transmission of hepatitis B virus.

The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.

A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR.

BACKGROUND: Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR. METHODS: Biopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR. RESULTS: HBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control. CONCLUSIONS: RCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics.

[A retrospective study on pathological and clinical characteristics of 3 932 children with liver diseases].

OBJECTIVE: To explore the pathological and clinical characteristics of children with liver diseases by retrospective study on clinical and liver biopsy pathological data of children with liver diseases. METHOD: This retrospective analysis was performed at Beijing No. 302 Hospital among 3 932 children with liver diseases who visited the hospital from January 2001 to December 2012. The kinds of diseases were compared with the results of 1983-2000. RESULT: (1) Liver biopsy was successful in 99.72% (3 932/3 943) of cases of 2001-2012 group, complications occurred in 31 children only. (2) Of the 3 932 cases, 2 647 (67.32%) had hepatitis , non-hepatotropic viral hepatitis and non viral liver disease were seen in 365 cases (9.28%), and 920 cases (23.4%), respectively. Among 2 647 cases with viral hepatitis, 2 115 were hepatitis B (79.90%), 521 hepatitis C (19.69%), 7 were hepatitis A (0.26%) and 4 hepatitis E (0.15%), respectively. (3) In 2001-2012 group, the degrees of inflammatory activity (>G2) of liver were seen in 9.57% (202/2 111) patients with hepatitis B, while 23.57% (132/560) in 1983-2000 group. There was significant difference between the two groups (χ(2)=80.36, P=0.00 ). (4) Significant difference was observed in the rate of non viral liver disease between 2001-2012 group (23.40%, 920/3 932) and 1983-2000 group (9.61%, 98/1 020) (χ(2)=93.46, P=0.00). In 2001-2012 group, including 46 kinds of diseases, which were significantly higher than those of 1983-2000 group (18 kinds). In 2000-2012, the main causes of diseases were liver degeneration (18.26%, 168/920), drug-induced liver injury (13.59%, 125/920), fatty liver (8.80%, 81/920) and liver glycogen accumulation disease (8.70%, 80/920). While in 1983-2000 group, the main causes were liver degeneration (20.41%, 20/98), fatty liver (16.33%, 16/98), glycogen storage disease (10.20%, 10/98) and myopathy (9.18%, 9/98). CONCLUSION: Liver biopsy in children is safe and feasible. Hepatitis B virus was ranked first in children with liver diseases in 2001-2012 group. The kinds of non viral hepatic disorders had changed and extended.

Combining Oxymatrine or Matrine with Lamivudine Increased Its Antireplication Effect against the Hepatitis B Virus In Vitro.

Some recent clinical reports have shown that the combination of oxymatrine, a phyto-derived drug, with lamivudine (3TC) could improve its curative effect against hepatitis B virus (HBV) infection. However, the experimental data in support of this combination strategy are lacking. In this study, we investigated the anti-HBV activity of the combination of 3TC and either oxymatrine or matrine on HepG2 2.2.15 in vitro. The activities of the combination and the solo compound, each in different concentrations, were compared on the 3rd, 6th, and 9th experimental days. The cytotoxicity results showed that the nontoxic concentrations of both oxymatrine and matrine to HepG2 2.2.15 cells were 800 µg/mL. We found that the single use of oxymatrine below 100 µg/ml, matrine below 200 µg/ml, and 3TC below 30 µg/ml showed weak inhibitory effects on the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV-DNA in culture media; the combination of 3TC (30 µg/ml) with oxymatrine (100 µg/ml) or matrine (100 µg/ml) showed significant inhibitory effects that were higher than or equivalent to the single use of 3TC at 100 µg/ml. The results provide a new impetus to develop novel, multicomponent anti-HBV drugs through the combination of natural products with nucleoside analogs to enhance their activity.

Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B.

Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype.

Evaluation of the liver protection and toxicity of Da-Huang-Zhe-Chong pill in rats.

CONTEXT: Da-Huang-Zhe-Chong pill (DHZCP), a classical traditional Chinese formula, consists of 12 crude drugs which have been widely used with significant therapeutic effects. Some drugs in this formula have toxicities that might result in some adverse effects of DHZCP. OBJECTIVE: The liver protection and toxicity of DHZCP were first evaluated against chronic carbon tetrachloride (CCl(4))-induced liver injury in rats. MATERIALS AND METHODS: The rats were treated by intraperitoneal injection of 10% CCl(4) for 12 weeks. At the end of week 4, DHZCP at doses of 44 g/kg (high-dose group) and 22 g/kg (low-dose group) was intragastrically administered to CCl(4)-treated rats, once a day for four weeks. At the end of weeks 8 and 12, the general status of the rats, histopathology of liver, serum alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) levels were observed or determined and recorded. By correspondence analysis (CA) on biochemical markers and liver histopathological score (HS), the "dose-time-response" relationship of DHZCP on the hepatic injury rats was evaluated. RESULTS: The results showed that DHZCP exhibited a significant protective effect on liver injury by reversing the biochemical parameters and histopathological changes. However, this hepatoprotective effect may be weakened, or even be transferred to toxicity with the increase of the administration dose (44 g·kg(-1)·d(-1)) and time (more than 2 months) of this formula. These results were consistent with the histopathological observation and the serum levels. DISCUSSION AND CONCLUSION: Administration of proper dose and time of DHZCP could well play its hepatoprotective effect and even treat hepatitis, but the safety on liver should be considered when large-dose (44 g·kg(-1)·d(-1)) DHZCP is used for long time (more than 2 months). We suggest that the administration dose and time of DHZCP in clinical use should not be increased and prolonged, and simultaneously liver function should be regularly monitored.

Mimotopes selected with neutralizing antibodies against multiple subtypes of influenza A.

BACKGROUND: The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice. METHODS: The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE). RESULTS: Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups. CONCLUSIONS: Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.

Virologic and clinical characteristics of HBV genotypes/subgenotypes in 487 Chinese pediatric patients with CHB.

BACKGROUND: The association of hepatitis B virus (HBV) genotypes/subgenotypes with clinical characteristics is increasingly recognized. However, the virologic and clinical features of HBV genotypes/subgenotypes in pediatric patients remain largely unknown. METHODS: Four hundred and eighty-seven pediatric inpatients with CHB were investigated, including 217 nucleos(t)ide analog-experienced patients. HBV genotypes/subgenotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. The stage of fibrosis and degree of inflammatory activity were evaluated by the Metavir score system. RESULTS: Among 487 enrolled pediatric patients, HBV genotype C2 and B2 were the most two prevalent (73.7% and 21.1%). Comparing with HBV/B2 infected patients, no significant difference was observed in the incidence rate and mutant patterns of lamivudine- or adefovir-resistant mutations in HBV/C2 infected patients (P > 0.05). Importantly, we found that the degree of hepatic inflammation degree, fibrosis stage and ALT level were significantly higher in HBV/C2-infected HBeAg positive patients than it was in HBV/B2-infected ones. CONCLUSIONS: The pediatric patients with HBV/C2 infection might be more susceptible to develop severe liver pathogenesis.

Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR.

BACKGROUND: The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy. METHODS: Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control. RESULTS: Compared to the classical method, i.e., PSAD digestion+real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25copies/cell, P<0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels. CONCLUSIONS: The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.

Cloning, expression and identification by immunohistochemistry of humanized single-chain variable fragment antibody against hepatitis C virus core protein.

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.