Serum N-glycan markers for diagnosing liver fibrosis induced by hepatitis B virus.

BACKGROUND: Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection, which may develop into liver fibrosis, cirrhosis and hepatocellular carcinoma. Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion. Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis; however, this method is invasive and prone to clinical sampling error. In order to address these issues, we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis. AIM: To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes. METHODS: N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed. Significant changed N-glycan levels (peaks) (P < 0.05) in different fibrosis stages were selected in the modeling group, and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis. The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models. These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI) , fibrosis index based on the four factors (FIB-4), glutamyltranspeptidase platelet albumin index (S index), GlycoCirrho-test, and GlycoFibro-test. Furthermore, we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power. In addition, the diagnostic accuracy of N-glycan models was also verified in the validation group of patients. RESULTS: Multiparameter diagnostic models constructed based on N-glycan peak 1, 3, 4 and 8 could distinguish between different stages of liver fibrosis. The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752, respectively differentiating fibrosis F0-F1 from F2-F4, and F0-F2 from F3-F4, and surpassing other serum panels. However, AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4. In combination with ALT and PLT, the multiparameter models showed better diagnostic power (AUROC = 0.912, 0.829, 0.885, respectively) when compared with other models. In the validation group, the AUROCs of the three combined models (0.929, 0.858, and 0.867, respectively) were still satisfactory. We also applied the combined models to distinguish adjacent fibrosis stages of 432 patients (F0-F1/F2/F3/F4), and the AUROCs were 0.917, 0.720 and 0.785. CONCLUSION: Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV, especially in combination with ALT and PLT.

Comparison of immunogenicity between hepatitis B vaccines with different dosages and schedules among healthy young adults in China: A 2-year follow-up study.

Immunogenicity of hepatitis B vaccine between 20 µg with 3-dose schedule and 60 µg with 2-dose regimens was compared 2 years after primary immunization. A total of 353 healthy adults aged 18-25 years were enrolled in the study and randomly assigned (1: 1: 1) into 3 vaccine groups: A (20 µg, 0-1-6 month), B (60 µg, 0-1 month) and C (60 µg, 0-2 month). Serum samples were collected at 1 month after a series vaccination and 12 months, 24 months after the first-dose. The GMC level of anti-HBs antibody was measured using Chemiluminescent Microparticle ImmunoAssay (CMIA). There were 59, 45 and 55 vaccinees available to follow-up with 2 year later in vaccine groups A, B and C, respectively. No significant differences existed in sex ratio, age and body mass index (BMI) among vaccinees at month 24 and the corresponding participants at baseline in each group (P > 0.05). The seroprotection rates in group A, B and C were 98.31%, 88.37% and 85.19%, respectively (P = 0.014), reflecting the fact that the rate of group A was significantly higher than that in group C (P = 0.026). Also, the GMC level of anti-HBs antibody in group A was significantly higher than those of other two groups (427.46 mIU/ml vs. 89.74 mIU/ml, 89.80 mIU/ml, respectively; all P < 0.01). This data suggested that the standard 20 µg (0-1-6 month) regimen of hepatitis B vaccine should be recommended as a priority on the premise of complete compliance in adults.

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.

BACKGROUND & AIMS: As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. METHODS: Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. RESULTS: Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. CONCLUSION: The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. LAY SUMMARY: The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.

Long-term persistence in protection and response to a hepatitis B vaccine booster among adolescents immunized in infancy in the western region of China.

OBJECTIVES: To evaluate the persistence of protection from hepatitis B (HB) vaccination among adolescents immunized with a primary series of HB vaccine as infants, and the immune response to booster doses. METHODS: Healthy adolescents aged 15-17 y vaccinated with HB vaccine only at birth were enrolled. Baseline serum hepatitis B surface antigen (HBsAg), antibody against hepatitis B surface antigen (anti-HBs) and antibody against hepatitis B core antigen (anti-HBc) were detected by Enzyme-Linked Immunosorbent Assay (ELISA) and anti-HBs level was measured using Chemiluminescent Microparticle Immunoassay (CMIA). The rate of HBV infection was calculated. The seroprotection rate of anti-HBs (≥ 10 mIU/ml) and GMC level were used to evaluate the persistence of immunity from HB vaccination. Those with anti-HBs < 10 mIU/ml were immunized with booster doses of HB vaccine and the anamnestic response was assessed. RESULTS: Of 180 adolescents who received a primary series of HB vaccinations as infants, 3 (1.7%) had HBV infection and 74 (41.1%) had anti-HBs ≥ 10 mIU/ml with a GMC of 145.11 mIU/ml. The remaining 103 (57.2%) with anti-HBs < 10 mIU/ml received a booster dose of 20 µg HB vaccine and achieved the seroprotection rate of 84% (84/100) and a GMC of 875.19 mIU/ml at one month post-booster. An additional dose of 60 µg HB vaccine was administered to the 16 adolescents with anti-HBs < 10 mIU/ml after the first booster. All of them obtained anti-HBs seroprotection with a GMC of 271.02 mIU/ml at 1.5 months after an additional dose. CONCLUSIONS: Vaccine-induced immunity persisted for up to 15-17 y in 89.3% (158/177) of participants after a primary HB vaccination in infancy. Administering a booster dose of 20µg HB vaccine elicited an anamnestic immune responses in the majority of individuals with baseline anti-HBs <10 mIU/ml.

On-treatment quantitative hepatitis B e antigen predicted response to nucleos(t)ide analogues in chronic hepatitis B.

AIM: To investigate potential predictors for treatment response to nucleos(t)ide analogues (NAs) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. METHODS: Seventy-six HBeAg-positive CHB patients received 96-wk NAs optimized therapy (lamivudine and adefovir dipivoxil) were studied retrospectively. Serum hepatitis B surface antigen, HBeAg, hepatitis B core antibody, hepatitis B virus (HBV) DNA and alanine aminotransferase levels were quantitatively measured before and during the treatment at 12 and 24 wk. Stepwise logistic regression analyses were performed to identify predictors for treatment response, and areas under the receiver operating characteristic curves (AUROC) of the independent predictors were calculated. RESULTS: Forty-three CHB patients (56.6%) achieved virological response (VR: HBV DNA ≤ 300 copies/mL) and 15 patients (19.7%) developed HBeAg seroconversion (SC) after the 96-wk NAs treatment. The HBeAg level (OR = 0.45, P = 0.003) as well as its declined value (OR = 2.03, P = 0.024) at 24-wk independently predicted VR, with the AUROC of 0.788 and 0.736, respectively. The combination of HBeAg titer < 1.3 lg PEIU/mL and its decreased value > 1.6 lg PEIU/mL at 24-wk predicted VR with a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of 85%, 100%, 100% and 83%, respectively, and the AUROC increased to 0.923. The HBeAg level (OR = 0.37, P = 0.013) as well as its declined value (OR = 2.02, P = 0.012) at 24-wk also independently predicted HBeAg SC, with the AUROC of 0.828 and 0.814, respectively. The HBeAg titer < -0.5 lg PEIU/mL combined with its declined value > 2.2 lg PEIU/mL at 24-wk predicted HBeAg SC with a sensitivity, specificity, PPV, NPV of 88%, 98%, 88% and 98%, respectively, and the AUROC reached 0.928. CONCLUSION: The combination of HBeAg level and its declined value at 24-wk may be used as a reference parameter to optimize NAs therapy.

Comparative immunogenicity of hepatitis B vaccine with different dosages and schedules in healthy young adults in China.

OBJECTIVES: To compare immunogenicity of hepatitis B vaccine between the standard 3-dose (20 µg) and 2-dose with higher-dosage (60 µg) regimens in healthy young adults and evaluate the safety profile. METHODS: A randomized, parallel-group clinical trial was conducted among healthy young adults aged 18-25 years. Subjects were randomly assigned to three groups. One group was administered hepatitis B vaccine with the standard regimen of 0-1-6 month (20 µg) and other groups were immunized with regimens of 0-1 or 0-2 month (60 µg) respectively. Serum samples were collected at 1 month after a series vaccination and 12 months after the first-dose inoculation for anti-HBs antibody measurement with a Chemiluminescent Microparticle ImmunoAssay (CMIA). RESULTS: The seroprotection rates in 20 µg (0-1-6 month), 60 µg (0-1 month) and 60 µg (0-2 month) groups were 100, 93.64 and 99.19% at month 7/2/3, and 100, 96.04 and 95.90% at month 12, respectively. There were no significant differences among three vaccine groups (p>0.05). The geometric mean concentration (GMC) of anti-HBs was significantly higher in 20 µg (0-1-6 month) group than that in 60 µg (0-1 month) group at month 7/2 (1847.99 vs. 839.27 mIU/ml, p=0.004), but was similar to that in 60µg (0-2 month) group at month 7/3 (1847.99 vs. 1244.80 mIU/ml, p=0.138). At month 12, the GMC in 20 µg (0-1-6 month) group was significantly higher than those of other groups (1456.63 vs. 256.30, 235.15 mIU/ml, respectively, p<0.001). The total incidence of injection-site or systemic adverse reactions was <3%. CONCLUSIONS: A 2-dose with higher-dosage hepatitis B vaccine regimens are comparable to the standard 3-dose regimen in terms of immunogenicity except a relatively rapid decline in GMC levels which are associated with the longevity of protection. All formulations of hepatitis B vaccine were well tolerated. CLINICALTRIALS.GOV IDENTIfiER: NCT02203357.

The detection of (total and ccc) HBV DNA in liver transplant recipients with hepatitis B vaccine against HBV reinfection.

To investigate the levels of hepatitis B virus total DNA (HBV DNA) and covalently closed circular (ccc) DNA in liver transplant recipients who received hepatitis B vaccination, including responders and non-responders, following liver transplantation due to hepatitis B-related diseases and to investigate the efficacy of hepatitis B immune reconstitution against HBV reinfection. Twenty responders and 34 non-responders were enrolled in the present study. The levels of HBV total DNA and ccc DNA in peripheral blood mononuclear cells (PBMCs) and the liver and plasma were detected by real-time polymerase chain reaction (PCR). Fifty-three blood samples and 38 liver allograft tissues were acquired. For the responders, the mean serum titer for anti-HBs (antibodies against hepatitis B surface antigen) was 289 (46.64-1000) IU/ml. Also for the responders, HBV total DNA was detected in PBMCs for one recipient and in the liver for another recipient, but ccc DNA was not detected in either of those 2 recipients. For the non-responders, HBV total DNA was detected in PBMCS for 2 recipients, neither of whom had ccc DNA. Also for the non-responders, HBV total DNA was detected in the livers of 3 recipients, 2 of whom also had ccc DNA. All responders had discontinued hepatitis B immunoglobulin (HBIG), and 13 responders had discontinued antiviral agents. One responder experienced HBV recurrence during the follow-up period. For the majority of liver transplant recipients, no HBV total DNA or ccc DNA was detected in the blood or liver. The lack of HBV total DNA and ccc DNA both in PBMCs and the liver in liver transplant recipients who received hepatitis B vaccination to prevent HBV reinfection should be a prerequisite for the withdrawal of HBIG and/or antiviral agents.

Comparison of Immunogenicity Between Inactivated and Live Attenuated Hepatitis A Vaccines Among Young Adults: A 3-Year Follow-up Study.

UNLABELLED: A randomized clinical trial of hepatitis A vaccines (1 or 2 doses of inactivated vaccine [Healive] or 1 dose of live attenuated vaccine [Biovac]) was conducted among adults to evaluate seroprotection rates and geometric mean concentrations of antibody against hepatitis A virus for 36 months. High rates of seroprotection persisted for at least 36 months among adults who received 1 or 2 doses of inactivated hepatitis A vaccine but not among adults who received 1 dose of live attenuated hepatitis A vaccine. The long-term serial monitoring of immunogenicity induced by 1 dose of inactivated hepatitis A vaccine is needed to determine an effective alternative to a 2-dose schedule. CLINICAL TRIALS REGISTRATION: NCT01865968.

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA.

AIM: To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay. METHODS: HBV DNA standards as well as a total of 180 clinical serum samples from patients with chronic hepatitis B were measured using the Abbott and Da-an real-time polymerase chain reaction (PCR) assays. Correlation and Bland-Altman plot analysis was used to compare the performance of the Abbott and Da-an assays. The HBV DNA levels were logarithmically transformed for analysis. All statistical analyses were performed using SPSS for Windows version 18.0. The correlation between the two assays was analyzed by Pearson's correlation and linear regression. The Bland-Altman plots were used for the analysis of agreement between the two assays. A P value of < 0.05 was considered statistically significant. RESULTS: The HBV DNA values measured by the Abbott or Da-an assay were significantly correlated with the expected values of HBV DNA standards (r = 0.999, for Abbott; r = 0.987, for Da-an, P < 0.001). A Bland-Altman plot showed good agreement between these two assays in detecting HBV DNA standards. Among the 180 clinical serum samples, 126 were quantifiable by both assays. Fifty-two samples were detectable by the Abbott assay but below the detection limit of the Da-an assay. Moreover, HBV DNA levels measured by the Abbott assay were significantly higher than those of the Da-an assay (6.23 ± 1.76 log IU/mL vs 5.46 ± 1.55 log IU/mL, P < 0.001). A positive correlation was observed between HBV DNA concentrations determined by the two assays in 126 paired samples (r = 0.648, P < 0.001). One hundred and fifteen of 126 (91.3%) specimens tested with both assays were within mean difference ± 1.96 SD of HBV DNA levels. CONCLUSION: The Da-an assay presented lower sensitivity and a narrower linear range as compared to the Abbott assay, suggesting the need to be improved.

Comparison of immunogenicity between inactivated and live attenuated hepatitis A vaccines: a single-blind, randomized, parallel-group clinical trial among children in Xinjiang Uighur Autonomous Region, China.

OBJECTIVES: To compare immunogenicity among an inactivated hepatitis A vaccine (Healive(®)) with one-dose and two-dose regimens, and three kinds of live attenuated vaccines in children. METHODS: A single-blind, randomized, parallel-group clinical trial was conducted among healthy children aged 1.5-6 y in Xinjiang Uighur Autonomous Region, China. Subjects were randomly assigned to 5 groups. Two groups were administered one-dose or two-dose inactivated vaccine and the remaining groups were immunized with one of three kinds of attenuated vaccines, respectively. Serum samples were collected at 6- and 12-mo follow-ups. Anti-HAV IgG was measured with a microparticle enzyme immunoassay. RESULTS: No significant differences were observed in seroconversion rates (seroprotection rates) among the five groups at 6 or 12 mo (p>0.05). The geometric mean concentration (GMC) of anti-HAV IgG was significantly higher in the two-dose Healive(®) group than in the one-dose Healive(®) group and the attenuated vaccine groups at 12 mo (932.4 vs. 112.7, 135.8, 203.3, 212.8 mIU/ml, respectively, p<0.05). In the one-dose Healive(®) group, the GMC was significantly lower than that in the attenuated vaccine B and C groups at 6 mo (152.6 vs. 212, 204 mIU/ml, p<0.05) and at 12 mo (112.7 vs. 203.3, 212.8, p<0.05), but was similar to the attenuated vaccine A group at 12 mo (112.7 vs. 135.8 mIU/ml, p>0.05). The GMCs were significantly higher in the 1-2 y of age group than in the 3-6 y of age group for all types of vaccines except the attenuated vaccine C (p<0.05) at 12 mo. CONCLUSIONS: A higher GMC of anti-HAV IgG was induced in the two-dose Healive(®) than in the one-dose and the attenuated vaccines at 12 mo. The attenuated vaccine B or C produced higher GMCs than the one-dose Healive(®) at 6-12 mo after vaccination.

Discrepancy of potential antiviral resistance mutation profiles within the HBV reverse transcriptase between nucleos(t)ide analogue-untreated and -treated patients with chronic hepatitis B in a hospital in China.

Little is known about the discrepancy of the potential antiviral resistance mutation profiles within the hepatitis B virus (HBV) reverse transcriptase (RT) between nucleos(t)ide analogue (NA)-untreated and -treated patients with chronic hepatitis B. Full-length HBV RT sequences from 59 NA-treated and 105 NA-untreated Chinese patients were amplified and sequenced. Forty-two potential NA resistance (NAr) mutation sites were screened within these 164 RT sequences. The NAr mutation prevalence and frequency in the NA-treated group were significantly higher than those in the NA-untreated one (P < 0.001, respectively). The classical primary drug resistance and secondary/compensatory mutations were only detected at seven sites (rtL80, rtI169, rtL180, rtA181, rtT184, rtM204, and rtN236) in NA-treated patients. The non-classical putative NAr and pre-treatment mutations were observed at 22 sites (rtT38, rtN/S53, rtL82, rtL/I91, rtN/Y124, rtH126, rtT128, rtN/D134, rtN139, rtR153, rtV191, rtV207, rtS213, rtV214, rtE218, rtY/F221, rtV/I224, rtL229, rtI233, rtN/H238, rtR242, and rtS/C256) in both groups. Substitutions at seven non-classical mutation sites were of interest due to either detection only in patients with virologic breakthrough (rtL82 and rtV214), or potential ties with HBV genotypes (rtV191 and rtL229), or coexistence with rtM204I/V (rtL229), or increased mutation trends after NA-treatment (rtT128, rtV207, and rtN/H238). In conclusion, NA treatment not only constitutes a major selection factor for the primary and secondary/compensatory NAr mutations but also drives the changes of some of the putative NAr mutation sites, most of which are the genotype-independent RT sites (rtL82, rtT128, rtV191, rtV207, rtV214, rtL229, and rtN/H238). Their antiviral resistance potential calls for further investigations.

[A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].

OBJECTIVE: To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue. METHODS: Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and ß-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using ß-globin housekeeping gene as a quantitation standard. RESULTS: The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%. CONCLUSION: A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

Alteration of N-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a non-invasive monitoring tool for liver cancer.

BACKGROUND AND AIMS: There is a demand for serum markers that can routinely assess the progression of liver cancer. DENA (diethylnitrosamine), a hepatocarcinogen, is commonly used in an experimental mouse model to induce liver cancer that closely mimics a subclass of human hepatocellular carcinoma (HCC). However, blood monitoring of the progression of HCC in mouse model has not yet been achieved. In this report, we studied glycomics during the development of mouse HCC induced by DENA. METHODS: Mouse HCC was induced by DENA. Serum N-glycans were profiled using the sequencer assisted-Fluorophore-assisted carbohydrate electrophoresis technique developed in our laboratory. Possible alteration in the transcription of genes relevant to the synthesis of the changed glycans was analysed by real-time polymerase chain reaction. RESULTS: In comparison with the control mice that received the same volume of saline, a tri-antennary glycan (peak 8) and a biantennary glycan (peak 4) in serum total glycans of DENA mice increased gradually but significantly during progression of liver cancer, whereas a core-fucosylated biantennary glycan (peak 6) decreased. Expression of alpha-1,6-fucosyltransferase 8 (Fut8), which is responsible for core fucosylation, decreased in the liver of DENA mice compared with that of age-matched control mice. Likewise, the expression level of Mgat4a, which is responsible for tri-antennary, significantly increased in the liver of DENA mice (P<0.001). CONCLUSIONS: The changes of N-glycan levels in the serum could be used as a biomarker to monitor the progress of HCC and to follow up the treatment of liver tumours in this DENA mouse model.

Altered serum N-glycomics in chronic hepatitis B patients.

BACKGROUND: We previously reported on serum N-glycans as markers for the diagnosis of cirrhosis in patients with chronic hepatitis C infection. Our present study aimed to evaluate the use of serum glycan markers for the diagnosis of liver fibrosis in patients with chronic hepatitis B infection. METHODS: Patients with hepatitis B virus (HBV) infection (n=173) were diagnosed by clinical laboratory analysis and histological examination. Liver fibrosis was staged using Ishak score. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling. RESULTS: We found that in HBV patients, like in hepatitis C virus patients, several serum N-glycans were altered during the development of liver fibrosis. We found higher levels of total agalactosylated biantennary glycans in fibrosis patients with HBV infection than in healthy controls. The biantennary (NA2) and the triantennary (NA3) N-glycans decreased significantly (P<0.001) with increased severity of fibrosis. The diagnostic power of serum glycan marker (GlycoFibroTest) [area under the curve (AUC)=0.735) was similar to that of FibroTest (AUC=0.740) for discriminating between moderate and advanced fibrosis (F3-F6) from non- or mild fibrosis (F0-F2). However, GlycoFibroTest (AUC=0.740) was slightly better than FibroTest (AUC=0.696) for distinguishing fibrotic patients (F1 or more) from non-fibrotic patients (F0). CONCLUSIONS: The assay for serum glycan profiling showed satisfactory reproducibility and is a non-invasive blood test for the diagnosis of liver fibrosis. The changes of N-glycan level in serum can be used to monitor or follow-up the progress of fibrosis using specific N-glycan markers.

[A study on seroprevalence of anti-HAV IgG in adults of 4 cities in China].

OBJECTIVE: To investigate the seroprevalence of anti-HAV IgG in adults of 4 cities in China. METHODS: Serum samples were collected from 2390 local residents aged between 20 to 88 years from Beijing, Shanghai, Wuhan and Guangzhou. The anti-HAV IgG in sera was detected with a microparticle enzyme immunoassay (MEIA). RESULTS: The anti-HAV IgG seroprevalence in female of 30 to 39 years in Beijing (64.58%, 62/96) was higher than that in male (45.57% 36/79)) (x(2) = 6.358, P = 0.012). It increased with age in adults of Beijing and Guangzhou. The rates were 54.22 % (90/166), 56.00% (98/175) and 67.18% (88/131) for the 20-, 30- and 40-49 age groups in Beijing (x(2) = 4.76, P = 0.03); and 52.83% (56/106), 52.50% (63/120), 82.46% (94/114), 89.80% (88/98) and 96.77% (60/62) for the 20-, 30-, 40-, 50- and 60-88 age groups in Guangzhou, respectively (x(2) = 72.58, P less than 0.01). This trend was not found in Shanghai and Wuhan (x2 = 0.96, 2.99; P = 0.33, 0.08 respectively). The seroprevalence rates of anti-HAV IgG in the 20 to 39 age group of Beijing, Shanghai, Guangzhou and Wuhan were 55.13% (188/341), 63.93% (429/671), 52.65% (119/226) and 78.37% (308/393), respectively. CONCLUSION: The seroprevalence rates of anti-HAV IgG in young adults aged 20 to 39 years of the four cities are relatively low, and HAV vaccination should be suggested for the susceptible population of this age group in China.

Serum protein N-glycans profiling for the discovery of potential biomarkers for nonalcoholic steatohepatitis.

The hepatic histology in nonalcoholic fatty liver disease can vary from isolated hepatic steatosis to steatohepatitis can progress to cirrhosis and liver-related death. The aim was to evaluate the use of blood serum N-glycan fingerprinting as a tool for differential diagnosis of nonalcoholic steatohepatitis from steatosis. A group of 47 patients with NAFLD was diagnosed by clinical laboratory analysis and ultrasonography, and was studied histologically using the Brunt's scoring system. The control group included 13 healthy individuals. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling. We have found that the concentrations of two glycans (NGA2F and NA2) and their logarithm ratio of NGA2F versus NA2 (named GlycoNashTest) were associated with the degree of NASH-related fibrosis, but had no correlation with the grade of inflammation nor steatosis severity. When used to screen NAFLD patients, GlycoNashTest could identify advanced NASH-related fibrosis (F3-F4) with the diagnosis sensitivity of 89.5% and specificity of 71.4%. The serum N-glycan profile is a promising noninvasive method for detecting NASH or NASH-related fibrosis in NAFLD patients, which could be a valuable supplement to other markers currently used in diagnosis of NASH.

N-glycan profiles as tools in diagnosis of hepatocellular carcinoma and prediction of healthy human ageing.

Protein glycosylation, the most common form of co-translational modification of proteins, is the enzymatic addition of sugars or oligosaccharides (glycans) to proteins. Protein glycosylation increases the diversity of the functions of proteins encoded in the genome. The result is that different glycomes of the same protein may have different functional, kinetic or physical properties. The glycosylation pathway is largely regulated by the condition of the cells, which means that the sugar chains can be altered by the physiological or pathophysiological condition of the cell. Thus, the type of glycans produced by cells, tissues, or organism could reflect their current physiological state. We determined the N-glycan profiles of serum proteins by using DNA sequencer-based carbohydrate analytical profiling technology. We show that two N-glycan structures (NGA2F and NA2F) present in human blood glycoproteins change with ageing, and that one triantennary glycan (NA3Fb) is correlated with tumor stage in HCC patients. Therefore, examining alterations in serum glycan fingerprint by using our platform could be a suitable tool for monitoring the healthiness of ageing and for the follow-up of pathophysiological conditions such as liver cancer.

Over-expression of heat shock protein 70 in mice is associated with growth retardation, tumor formation, and early death.

Experiments in lower organisms, such as worms and flies, indicate that the molecular chaperone protein heat shock protein 70 (HSP70) is a longevity factor. In contrast, we demonstrate here that mice overexpressing HSP70 display growth retardation and early death. HSP70 transgenic mice displayed increased levels of serum corticosterone and weaker expression and activity of the glucocorticoid receptor in the liver. Serum insulin-like growth factor-1 (IGF-1) concentrations in the transgenic mice were 50% lower than in the control mice, leading to growth retardation. HSP70 transgenic mice showed decreased expression of Casp9, which encodes caspase-9, and increased expression of the anti-apoptotic Bcl-2 gene, indicating that apoptosis is suppressed. Consequently, most of the transgenic animals died before the age of 18 months from tumors in their lungs and lymph nodes. We suggest that the proinflammatory and antiapoptotic effects of HSP70 might be responsible for the growth retardation, tumor formation, and early death observed in the HSP70 transgenic mice.