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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hepatitis B virus (HBV) subgenotypes C2 and B2 differ in lamivudine- and adefovir-resistance-associated mutational patterns in HBV-infected Chinese patients.

We aimed to study the prevalence and clinical implications of hepatitis B virus (HBV) subgenotypes in Chinese patients. A total of 4,300 patients, mainly from northern China, were enrolled, including 182 patients with acute hepatitis B and 4,118 patients with chronic HBV infection who had been exposed to nucleoside or nucleotide analogs. HBV genotypes/subgenotypes were determined by direct sequencing of the HBV S/Pol region. The prevalence rates were 0.40% for HBV/B1, 14.30% for HBV/B2, 0.25% for HBV/B3, 0.35% for HBV/B4, 1.05% for HBV/C1, 81.72% for HBV/C2, 0.93% for HBV/C3, 0.16% for HBV/C4, and 0.84% for HBV/D. In chronic HBV infection, patients with HBV/B2 were younger and had lower ΗBeAg positive rates than patients with HBV/C2. The incidence of lamivudine-resistant mutations was significantly higher in HBV/C2 compared to HBV/B2 (27.9% versus 19.8%; P<0.01), and the significant difference was observed only for rtM204I and not rtM204V. In addition, compensatory mutations were more frequently detected in HBV/C2. The incidence of adefovir-resistant mutations was similar between the two subsets, but HBV/C2 inclined to show rtA181V (3.6% for C2 versus 0.9% for B2; P<0.01), while HBV/B2 inclined to show rtN236T (4.5% for versus 2.5% for C2; P<0.01). The ratios of HBV/B2 to HBV/C2 infection were 1.7 (110/65), 5.7 (2,653/463), 7.5 (520/69), 8.0 (48/6), and 15.3 (183/12) for acute hepatitis B, chronic hepatitis B, liver cirrhosis, acute-on-chronic liver failure, and hepatocellular carcinoma, respectively. In conclusion, HBV/C2 and HBV/B2, two prevalent subgenotypes, differ in lamivudine- and adefovir-resistance-associated mutational patterns. HBV/C2-infected patients are more likely to have disease progression than HBV/B2-infected ones.

A novel trifunctional IgG-like bispecific antibody to inhibit HIV-1 infection and enhance lysis of HIV by targeting activation of complement.

BACKGROUND: The complement system is not only a key component of innate immunity but also provides a first line of defense against invading pathogens, especially for viral pathogens. Human immunodeficiency virus (HIV), however, possesses several mechanisms to evade complement-mediated lysis (CoML) and exploit the complement system to enhance viral infectivity. Responsible for this intrinsic resistance against complement-mediated virolysis are complement regulatory membrane proteins derived from the host cell that inherently downregulates complement activation at several stages of the cascade. In addition, HIV is protected from complement-mediated lysis by binding soluble factor H (fH) through the viral envelope proteins, gp120 and gp41. Whereas inhibition of complement activity is the desired outcome in the vast majority of therapeutic approaches, there is a broader potential for complement-mediated inhibition of HIV by complement local stimulation. PRESENTATION OF THE HYPOTHESIS: Our previous studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that another new activator of complement, consisting of two dsFv (against gp120 and against C3d respectively) linked to a complement-activating human IgG1 Fc domain ((anti-gp120 x anti-C3d)-Fc), can not only target and amplify complement activation on HIV virions for enhancing the efficiency of HIV lysis, but also reduce the infectivity of HIV through blocking the gp120 and C3d on the surface of HIV. TESTING THE HYPOTHESIS: Our hypothesis was tested using cell-free HIV-1 virions cultivated in vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified (anti-gp120 x anti-C3d)-Fc proteins. As a control group, viruses were incubated with normal human serum under the same conditions. Virus neutralization assays were used to estimate the degree of (anti-gp120 x anti-C3d)-Fc lysis of HIV compared to untreated virus. IMPLICATIONS OF THE HYPOTHESIS: The targeted complement activator, (anti-gp120 x anti-C3d)-Fc, can be used as a novel approach to HIV therapy by abrogating the complement-enhanced HIV infection of cells.

[Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA].

OBJECTIVE: To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). METHODS: HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. RESULTS: The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. CONCLUSION: The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

[Multiple-site analysis of HBV drug-resistant mutations in 340 patients with chronic hepatitis B].

OBJECTIVES: To analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B. METHODS: Serum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients. RESULTS: Drug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues. CONCLUSION: Detection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis.

BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully. rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.

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.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system.

AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS: We constructed p7 protein bait plasmid by cloning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (alpha type) containing liver cDNA library plasmid, pACT2 in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics. RESULTS: Fifty colonies were selected and sequenced. Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens. CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

[Screening of binding proteins to interferon-alpha promoter DNA by phage display technique].

OBJECTIVE: To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display. METHODS: PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis. RESULTS: Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP). CONCLUSION: Many proteins with different functions could bind with interferon alpha promoter.