Nov. 2 Abstracts
Disease Progression
– Role of HBeAg Status
861. Hepatitis B Viral Factors Influencing
the Histological Changes Of HBeAg-negative Chronic HBV Infected Patients With Persistently
Y. Yang; Q. Xie; H. Zhou; H. Wang; H. Gui; W. Cai; L.
Lin; G. Zhao; C. Shi; H. Yu; Q. Guo
Background and Aims:
Several viral factors have been identified to
be associated with a higher risk of
Methods:
HBV
Results:
Of 52 subjects, 67.3% and 59.6% had
Conclusions:
Patients of HBeAg negative chronic HBV
infection with PNAL have a higher prevalence of
862. Hepatitis B Virus Basal Core Promoter Mutation and
C. Liu; P. Chen; Y. Jeng; J. Kao; D. Chen
Background & Aims:
Expression of intrahepatic hepatitis B core
antigen (HBcAg) and mutations of hepatitis B virus (HBV) genome are closely
related to the immunopathogenesis and disease activity of chronic HBV
infection. The role of HBV genotype and basal core promoter (
Results:
Fifty-two (74.3%) patients were infected with
HBV genotype B and 18 (25.7%) with genotype C. Virus nucleotide sequences
displayed
Conclusions:
HBV
838. Histological
Findings Of HBeAg-negative
Chronic HBV -Infected Patients With Persistently
Q. Xie; Y. Yang; H. Zhou; H. Wang; H. Gui; L. Lin; W. Cai; W. Zhu; X. Xiang; B. Gong; Q. Guo; H. Yu
Background and Aims:
As the increasing prevalence, poor clinical
prognosis and lower response of anti-viral therapy, HBeAg-negative chronic
hepatitis has attracted the attention of experts in liver disease. However,
research on HBeAg-negative carriers with persistently normal
Methods:
154 HBeAg-negative subjects underwent
percutaneous liver biopsy were enrolled, 56 with abnormal
Results:
Compared with abnormal
Significant histological findings may be seen
in various HBV
Conclusions:
HBeAg-negative chronic HBV infected patients
with PNAL are not a homogenous group. Those with age>45yrs,
815. Age-specific
Prognosis Following HBeAg Seroconversion in Chronic Hepatitis B
Y. Chen; C. Chu; C. Yeh; Y. Liaw
Background and Objectives:
HBeAg seroconversion confers favorable prognosis but untoward outcomes may develop thereafter. Since age is an important factor in the events of chronic HBV infection, we conducted a cohort study to elucidate the prognosis following spontaneous HBeAg seroconversion in different age groups in chronic hepatitis B infection.
Patients and Methods:
Five hundred and 51 patients with biopsy-proved
chronic hepatitis B virus (HBV) monoinfection had been followed up in our liver
unit for at least 1 year since 1977. Eight-three patients with persistent HBeAg
> 3 years and 441 patients with HBeAg seroconversion and then followed >
1 year were included in our analysis. Follow-up studies included liver
biochemistry, alfa-fetoprotein and ultrasonography every 3-6 months or more
frequently if clinically indicated. Occurrence of hepatitis relapse,
development of cirrhosis and hepatocellular carcinoma (
Results:
Of 441patients with spontaneous HBeAg
seroconversion, 207 were seroconverted before age 30 (group A), 181 between age
31 and 40 (group B) and 53 after age 40 (group C). The calculated annual
incidence of hepatitis relapse, cirrhosis,
Conclusion:
Untoward outcomes can develop after spontaneous HBeAg seroconversion, especially in patients HBeAg seroconverted after age 40.
829. Evolution of
HBV Quasipecies Generates New CpG Dinucleotides Prior to HBeAg Seroconversion
Y. Cheng; M. Aung; S. Lim
We recently showed that HBV quasispecies diversity was significantly higher in HBeAg seroconverters compared to non-seroconverters (Lim et al, Gastroenterology 2007; 133:951-8). We postulate that this increase in viral diversity may result in formation of new CpG motifs that may stimulate toll receptor 9 receptors. In this study, we analyzed sequences of HBV quasispecies in patients before seroconversion compared to non-seroconverters to determine if significant new CpG motifs had developed.
Methods:
Eight chronic hepatitis B HBeAg
seroconverters, with long term stored serum and clinical follow-up of at least
6-15 years were studied. Two chronic hepatitis B patients without HBeAg
seroconversion with matching length of follow-up were included as controls.
Serum samples from 6-10 time points per patients were involved in this study.
Conclusion:
New CpG dinucleotides occurred along during course of HBeAg seroconversion and may play an important role in HBeAg seroconversion. Functional studies of these new CpG dinucleotides are needed.
Disease
Progression: Viral Replication and
Integration
858. High HBV
A. Mamun; R. Salimur; S. Akbar; K. Mobin;
K. Md. Fazal; K. Md.
Introduction:
Inactive hepatitis B virus (HBV) carriers
constitute one of the major reservoirs of the HBV, especially in the developing
countries of the world. But, present management guidelines provide inadequate
treatment modalities for these patients. Generally, these patients are
recommended for regular check up, and treatment is only recommended when
patients exhibit evidences of liver damages. The condition can mainly be
attributable to lack of information about extent of liver damages of inactive
HBV carriers in developing countries. The purpose of this study was to assess
the extent of liver damages in patients who were completely unaware of their
HBV infection at
Methods:
Three hundred ten HBV-infected subjects were
enrolled in this study. HBV infection was detected in these subjects
incidentally either during (1) health check up, or (2) pre-vaccination
screening, or (3) assessment before blood donation or (4) screening due to
presence of HBsAg-positive patients among family members. They did not know
that they were infected with the HBV, and none of them had any symptoms related
to liver damages. HBV infection was detected from hepatitis B surface antigen
(HBsAg) positivity in these subjects. Subsequently, all patients were tested
for serum alanine aminotransferase (
Conclusion:
This study indicates that machinery should be developed to characterize undetected HBV carriers in developing countries by conducting multicenter clinical studies. We have shown that considerable numbers of patients those are unaware of their HBV infection suffer from progressive liver damages. The overall strategy of management of chronic HBV infection in developing countries should be revisited.
859. Lack of Correlation Between Viral Load and Extent of Liver Damage in Patients with
Chronic HBV Infection in
A.
Mamun; R. Salimur; S. Akbar; K. Mobin; K. Md. Fazal; K. Md.
Introduction:
In general, it is assumed that patients with chronic hepatitis B virus (HBV) infection with high viral load exhibit increased liver damages. Accordingly, the treatment guidelines emphasize on reducing viral load in chronic HBV carriers. The ethical and scientific basis of these observations was mainly accumulated from investigations from developed countries of the world. However, more than 80% chronic HBV carriers live in the developing nations of the world, but little is known about relationship between HBV viral load and extent of liver damages in these countries. In this study, we addressed this issue to provide insights about this so that better management guidelines can be developed for chronic HBV carriers of developing nations.
Methods:
A total of 402 Bangladeshi patients with
chronic hepatitis B (
Results:
High HBV
Conclusion:
This study shows that a correlation could not
be established between viral load and liver damage in patients with
860. Modulation of ERK, AKT, apoptosis and cell cycle
in primary mouse hepatocytes and Huh7 cells by wild-type and rtM204I HBV.
R. Chin; U. Nachbur; C. Bock; J. Silke; J.
Torresi
Introduction:
Lamivudine resistant hepatitis B viruses (HBV)
are not infrequent in patients on antiviral therapy and who develop
hepatocellular carcinoma (
Methods:
Recombinant adenoviruses expressing a
full-length infectious genome of wt and rtM204I HBV together with an internal
GFP reporter or a control adeno-GFP virus were used to infect primary mouse
hepatocytes (PMH) and Huh7 cells. Intracellular HBV cccDNA was analysed by real
time
Results:
Infection of PMH and Huh7 cells with rAd-HBV-wt and rAd-HBV-M204I resulted in up-regulation of ERK phosphorylation when compared to mock infected or control adeno-GFP-infected cells. In contrast to mock-infected cells infection of cells with rAd-HBV-M204I resulted in a 150% increase in pERK, a 160% increase pAkt, and a 30% increase in p-cMyc. In both rAd-HBV-wt and rAd-HBV-M204I infected cells ERK phosphorylation was also strongly increased following PDGF stimulation. The level of the inactivated p-Ser9-GSK3-beta was increased by 140%. The p53 and p21cip1 proteins were both increased by 40% and 35% respectively. Relative to mock-infected cells cyclin A was increased by 95% in rAd-HBV-wt and 140% in rAd-HBV-M204I infected cells. Cyclin B1 was increased by 225% in rAd-HBV-wt and 210% in rAd-HBV-M204I infected cells. Both rAd-HBV-wt and rAd-HBV-M204I infected cells demonstrated increased levels of p-cdc2 (158% wild type and 128% rtM204I). Cell cycle analysis showed a greater proportion of the rAd-M204I HBV infected cells (16% ±SD 0.6) were in G2 phase compared to mock infected cells (7% ±SD 3.3). Cell viability of HBV infected hepatocytes was significantly reduced after treatment with TNF-alpha, FasL and TRAIL. cIAP was increased in the first 24 hours post-infection but this was followed by a reduction in cIAP and a rise in cleaved caspase 3, consistent with increased apoptosis in HBV infected hepatocytes.
Conclusion:
In both PMH and Huh7 cells
wild-type and rtM204I HBV strongly upregulate the pERK, pAkt and cMyc. These changes were accompanied by increased levels of p53, p21,
cyclin B1 and pcdc2 and in Huh7 cells and resulted in G2/M cell cycle arrest.
Apoptosis was increased in both HBV infected PMH and Huh7 cells. The
combination of these effects may contribute to
854. Plasmacytoid Dendritic
Cell Function Is Impaired in Chronic Hepatitis B Infection
D. Ratnam; K. Visvanathan; P. U. Cameron;
W. Sievert
Background:
Dendritic cells (DC) are potent antigen
presenting cells involved in the induction of adaptive immune responses and may
also play a role in immunological tolerance. The two major subtypes found in
human peripheral blood are myeloid (mDC) and plasmacytoid (pDC) and much of
their function is mediated through pattern recognition receptors known as Toll
like receptors (TLR). pDCs play an important role in
host defences against viruses, with the ability to secrete large amounts of
type 1 interferons. Previous studies of DC frequency and function in chronic
HBV have yielded conflicting results. The aim of this study was do determine
whether a TLR mediated impairment of DC function contributes to the suboptimal
immune responses seen in chronic HBV(
Methods:
Peripheral blood was obtained from patients in immunoactive (both HBeAg+ and HBeAg-), immunotolerant (IT) and inactive stages of chronic HBV infection and from healthy volunteers. All patients in the immunoactive and IT phase had viral loads above four log IU/ml compared to undetectable viral loads in patients in the inactive stage. PBMCs were isolated using Ficoll Density Centrifugation and then cultured with purified lipopolysaccharide (pLPS), or Influenza virus (H1 strain) in order to stimulate TLR4 on mDCs, and TLR9 on pDCs respectively. Cells were then stained with combinations of flurochrome conjugated antibodies to identify the individual DC subsets as well as intracellular TNF-α and IFN-α. Flow cytometry was then used to determine the frequency of the DC subsets and their levels of intracellular cytokine production (expressed as the percentage of the DC subset expressing either cytokine).
Results:
pDCs from individuals in the immunoactive and immunotolerant
phases of infection demonstrated an impaired capacity to secrete IFN-α in
response to Influenza virus compared to healthy controls (p<0.02). There was
no difference in pDC IFN-α production from patients in the inactive phase
compared to controls. mDCs from chronic HBV
individuals did not demonstrate a difference in capacity to secrete TNF-α
in response to pLPS stimulation compared to controls. No difference was
detected in the frequency of circulating mDCs or pDCs in
Conclusion:
An impaired capacity of pDCs to produce
IFN-α in
855. Assessment of Delta and Hepatitis B Viremia in Patients with
Chronic Hepatitis Delta. Influence of HIV Infection
and Antiviral Therapy.
A. Madejón; G. Gaeta; M. Bottecchia; J.
García-Samaniego; M. Romero; V. Soriano
Background:
Complex interactions between hepatitis B (HBV) and delta (HDV) viruses, occur in chronic HDV carriers.
Methods:
A total of 226 sera from 42 patients with
chronic HDV infection were examined (mean follow-up: 26.1+16.4 months).
Twenty-four (57%) were anti-HIV-neg, 36(85%) anti-HBe+ and 39 (93%)
anti-HCV-neg. Six HIV-neg patients (25%) were treated with interferon (IFN),
and 18 HIV+ patients received HAART with anti-HBV drugs (tenofovir and/or
lamivudine/emtricitabine). Serum HDV-RNA was quantified with a real time
Results:
Baseline mean serum HDV-RNA tended to be
higher in HIV-pos than in HIV-neg patients (4x107 vs 6.8x106
copies/ml; p=0.073). Different HDV replication patterns were observed in
HIV-neg untreated patients; serum HDV-RNA was detected during the entire
follow-up in 11/18 (61%) patients, intermittently in 3 (17%), and was always
negative in the remaining 3 (17%) patients. In contrast, almost all HIV
patients were delta viremic. Serum HBV-
Conclusions:
Most patients with chronic HDV infection show
an inhibition of HBV replication. Serum HDV-RNA titers are generally greater
than HBV-
Y.
Rodriguez-Muñoz; S. Martin-Vilchez; P. Sanz-Cameno; M. Lopez-Cabrera; R.
Moreno-Otero; E. Lara-Pezzi
VHB chronic infection in liver fibrosis may
lead to cirrhosis and higher risk of hepatocellular carcinoma, due to the host
immune response and to the expression of the viral transactivator HBx, which
exhibits pleiotropic biological effects, being able to modify different cell
functions.
Aim:
Our aim is to analyze HBx contribution to liver fibrosis through its effects over ECM homeostasis, MMP-2 expression and HSCs activation.
Methods:
Primary human HSCs were incubated with
conditioned media from hepatic cell lines:
Results:
HSCs activation was demonstrated by the “de novo” expression of α-SMA as showed in immunofluorescence. Zymography from HSCs exposed to conditioned media from HBx expressing cells displayed an increased expression of MMP-2 as compared to HSCs incubated with control media. In the same cases HSCs showed an enhanced proliferation rate too in comparison with those exposed to control media. This increased proliferation was aborted when we blockade MMP-2 activity with TIMP2.
Conclusion:
HBx contribute to the fibrotic process inducing HSCs activation and proliferation. Moreover, HBx protein also mediates in ECM integrity, through its capacity to induce MMP-2 in HSCs. Interestingly, MMP-2 neutralization with TIMP-2 prevented HSCs proliferation, suggesting the role of this metalloproteinase in the enhacement of HSCs proliferation by HBx. Taken together our results demonstrate that HBx could be a promoting agent of hepatic fibrosis through its ability to modulate key features, like HSCs activation and ECM integrity modulated at least by MMP-2 expression.
S. Martin-Vilchez; Y. Rodriguez-Muñoz; P.
Sanz-Cameno; M. Lopez-Cabrera; R. Moreno-Otero; E. Lara-Pezzi
Introduction:
Hepatic fibrosis is a scarring process that
leads to extracellular matrix (ECM) accumulation and whose central mediators
are HSCs. HSCs activation, is characterized by increased expression of matrix
components and growth factors such collagen-I (COL-I), connective tissue growth
factor (CTGF) and TGF-beta, as well as their enhanced proliferation. HBx
protein is a promoting agent of
Aim:
Our aimis to study HBx contribution to the fibrotic process through the analysis of its effects on human HSCs behaviour.
Methods:
Primary human HSCs were incubated with
conditioned media from the following hepatic cell lines;
Results:
α-SMA expression was detected in HSCs exposed to
849. Control of HBV Replication by HepaRG
cells Innate Response
J.
Lucifora; D. Durantel; B. Testoni; O. Hantz; M. Levrero; F. Zoulim
Background
and Aims:
To gain more insight on the
HBV/host-cell interactions, one of the major problems is to improve the
existing cellular models for the study of HBV replication. As human primary hepatocytes
are difficult to obtain, we have improved the system based on the transduction
of HepG2 cells with a recombinant baculovirus thanks to the construction of a
new HBV recombinant baculovirus (Bac-HBV-1.1-WT).Then, we asked whether a
persistent HBV infection may be obtained after transduction of the
differentiated HepaRG cells which are susceptible to viral infection.
Methods:
HepaRG cells were transduced by Bac-HBV-1.1-WT
and kinetics of synthesis of HBV replication intermediates were
analyzed. Cellular response to infection via the IFN pathway was studied by
qRT-
Results:
Following transduction by Bac-HBV-1.1-WT,
HepaRG cells initiate an HBV replication cycle with viral
Conclusions:
Our results suggest that the innate response may play a critical role in the control of HBV infection. The magnitude of the innate response was dependent on the cell type and may therefore play a role in the clearance or control of HBV infection. The modulation of the IFN-β pathway may be a prerequisite to establish persistent hepatocyte infection by HBV.
843. Genetic
Variability on the cccDNA Regulatory Regions Changes Significantly in the Different
Phases of Chronic HBV Infection
A. Koepke; T. Volz; M. Lutgehetmann; A.
W. Lohse; M. Dandri; J. Petersen
Knowledge of factors regulating the template
of HBV replication, the covalently closed circular
Methods:
The cccDNA of treatment naïve 21
HBeAg-positive and 22 HBeAg-negative, anti-HBe positive individuals was
compared by direct sequencing of cccDNA-specific
Conclusions:
The significantly lower mutation rates
determined in the low replicative phase of HBV chronic infection reveals that
impairment of virion productivity is not due to the emergence of specific
mutation patterns within the regulatory regions, suggesting that other
host-mediated molecular mechanisms control HBV replicative activity, while the
selection of
X. Zhang
Aims:
To investigate the association of the polymorphisms in CIITA coding region with the outcome of chronic HBV infection.
Methods:
The two SNPs at C30799G (Ala500Gly) and
C19170G (Leu45Val) sites of CIITA gene coding region were genotyped using ARMS-
Results:
1. The frequencies of C allele and CC genetype at 30799 site were significantly higher among patients with hepatocellular carcinoma than those among patients with liver cirrhosis(χ2=4.861,4.993;P=0.027,0.025). Logistic regression analysis indicated that there were significantly different with the genotype frequencies at 30799 site in the patients between liver cirrhosis and hepatocellular carcinoma(OR 0.557;95%CI,0.334-0.930;P=0.025).
2. The frequencies of G allele and genetypes carring G allele (GG and GC) at 19170 site were significantly higher among patients with liver cirrhosis than those among nonprogressed liver diseases including asymptomatic carriers and patients with chronic hepatitis B χ2=7.128,6.464;P=0.008,0.011). Logistic regression analysis indicated that there were significantly different with the allele frequencies at 19170 site in the patients between liver cirrhosis and nonprogressed liver diseases (OR 0.742;95%CI,0.552-0.998;P=0.048).3.There were not significantly different with the levels of HLA class II antigen (HLA-DR,DP,DQ) expression among Hela cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA (P>0.05).
Conclusions:
1. The polymorphism at 30799 site of CIITA gene was associated with hepatocellular carcinoma in chronic HBV infection. The polymorphism at 19170 site of CIITA was associated with cirrhosis in chronic HBV infection.
2. The two SNP at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of CIITA gene did not influence capability of CIITA trans-activating HLA class II gene expression.
J. Bockmann; M. Lutgehetmann; T. Volz; A. W. Lohse; M. Dandri; J. Petersen
Accumulating evidence suggests that HBV may prevent activation of the Toll-like receptor signaling, while antiviral noncytolytic mechanisms can inhibit virion productivity.
Aim:
The aim of the study was to investigate whether intrahepatic changes in the expression of innate immunity genes correlate with changes in viral activity in chronic HBV infection (CH-B).
Methods:
Results:
In general, moderate changes in cytokine
levels (i.e. TGF-beta, IFN-alpha/beta) were found in different patient cohorts
and only TNF alpha, IFN gamma and IL10 levels significantly differed in CH-B
livers compared to uninfected controls, but did not vary between HBeAg+ and
HBeAg- patients. Intrahepatic expression of TLR3 and TLR4 did not differ
between groups, but levels of TLR4 and down-stream effector HMGB1 inversely
correlated with
Conclusions:
This analysis indicates that interactions between HBV and components of the innate immunity occur in the clinical setting and that intrahepatic HBV levels may favour suppression of the immune surveillance.
832.
G. Zhao; Q. Xie; H. Wang; L. Lin; H.
Zhou; N. Jia; Y. Yang; C. Shi; B. Gong; X. Xiang; Q. Huang; Q. Guo; H. Yu
Background and Aims:
The retinoic acid-inducible gene I (
Methods:
Peripheral blood was collected from 28
hepatitis B (HB) virus-infected persons, including 21 cases of chronic
hepatitis B (
Results:
The expression of
Conclusions:
Our results showed that the expression of
Y. Kondo; Y. Ueno; S. Kon, J.
Inoue; M. Ninomiya; E. Kakazu; M. Shiina; K. Tamai; Y. Wakui; K. Kobayashi; K.
Fukushima; T. Kogure; H. Niitsuma; Y. Tanaka; M. Mizokami; T. Shimosegawa
Background:
We previously reported that HBcAg specific
regulatory T cells (Tregs) play an important role in the pathogenesis of
chronic hepatitis B virus. Some groups reported that heat shock protein 60 (
Aim:
The aim of this study is to analyze whether
soluble
Methods:
Chronic Hepatitis B (CH-B) HBeAg+ (n=24), CH-B HBeAb+ (n=24) were studied. As control subjects, CH-C (n=24) and Healthy subjects (n=10) were simultaneously studied.
Ex-vivo study. The amounts of
sHSP60 in serum were quantified by ELISA. PBMCs of selected CH-B subjects who
had received Entercavir (
In-vitro study. Two kinds of plasmids carrying a 1.3-fold of the HBV genome that could replicate in HepG2 cells were used to analyze whether HBV replication may affect the production of sHSP60 in culture medium.
Results:
The serum
levels of sHSP60 in
Conclusion:
sHSP60 produced from HBV infected hepatocytes could enhance
the HBcAg specific function of regulatory T cells. The suppression of HBV
replication by
834. T-1993C
Polymorphism in The Promoter of
TBX21 Gene Is Associated With Susceptibility to Persistent Hepatitis B Virus
Infection
S. Chen; W. Zhao; G. Deng
Transcription factor T-bet is responsible for
the differentiation of naive T lymphocytes, and its expression level is linked
with different responses to some viral infections, including hepatitis B virus
(HBV) infection. In this report we examine whether promoter polymorphisms of TBX21
gene (encoding T-bet) are associated with persistent HBV infection. Three
previously reported promoter polymorphisms, the T-1993C, T-1514C, and G-1499A , were analyzed by
Two common polymorphisms, the T-1993C and T-1514C were selected for genotyping in 436 persistent HBV-infected cases, 310 spontaneously recovered controls, and 302 HBV naïve controls. Haplotypes were constructed for each subject and association with the susceptibility to persistent HBV infection was estimated by logistic regression. Case-control association study showed that the -1993C and -1514C allele were associated with persistent HBV infection (for T-1993C, χ2 = 20.4, P = 0.0000063; for T-1514C, χ2 = 5.6, P = 0.018). Similarly, haplotype -1993T-1514T was associated with decreased susceptibility to the persistence of HBV infection (P = 0.000017, OR = 0.46, 95% CI 0.32-0.65). Our results suggest that it is the common regulatory T-1993C polymorphism, not the uncommon G-1499A polymorphism in TBX21 promoter, influences susceptibility to persistent HBV infection in a Chinese population.
Table 1.TBX21 genotype distributions in naïve controls, persistent HBV infected group and spontaneously recovered controls.
|
|
Naïve
controls (n =
302) |
Spontaneously
recovered individuals (n =
310) |
Persistent
HBV infected
group (n =
436) |
Allele T
versus allele C† χ2
P Value |
|
T-1993C |
|
|
|
|
|
TT, no. (%) |
231 ( 76.5 ) |
262 ( 84.5 ) |
310 ( 71.1 ) |
|
|
TC, no. (%) |
66 ( 21.8 ) |
45 ( 14.5 ) |
111 ( 25.5 |
|
|
CC, no. (%) |
5 ( 1.7 ) |
3 ( 1.0 ) |
15 ( 3.4 ) |
|
|
T allele, no. (%) |
528 ( 87.4 ) |
569 (91.8 ) |
731 (83.8 ) |
20.4 0.0000063 |
|
C allele, no. (%) |
76 (12.6 ) |
51 (8.2 ) |
141 (16.2 ) |
|
|
T-1514C |
|
|
|
|
|
TT, no. (%) |
265 ( 87.8 ) |
281 ( 90.7 ) |
366 ( 84.0 ) |
|
|
TC, no. (%) |
36 ( 11.9 ) |
27 ( 8.7 ) |
69 ( 15.8 ) |
|
|
69 ( 15.8 ) |
1 ( 0.3 ) |
2 ( 0.6 ) |
1 ( 0.2 ) |
|
|
T allele, no. (%) |
566 ( 93.7 ) |
589 (95.0 ) |
801 (91.9 ) |
5.6 0.018 |
|
C allele, no. (%) |
38 (6.3 ) |
31 (5.0 ) |
71 (8.1 ) |
|
†Comparison is between persistent HBV infected group and spontaneously recovered individuals
W. Yan; X. Wang; Q. Xiaoming; J. Zhang; T. Chen; H. Wang; X. Luo; Q. Ning
Objective:
To study the contribution of previously
unidentified subset of double negative T cells (DNT cells) in MHV-3 induced
chronic viral hepatitis in C3H/HeJ mouse and patients with chronic hepatitis B(
Methods:
Murine hepatitis virus stain 3(MHV-3) infected
C3H/HeJ displayed as chronic hepatitis and Balb/cJ mice displayed as fulminant
hepatitis were used in this study. Splenocytes from MHV-3 infected C3H/HeJ mice
were adaptively transferred into Balb/cJ mice, the
surviral rate and pathohistology of Balb/cJ mice were examined. The proportions
of various T cell subsets in CD3+ T cells in C3H/HeJ mice were examined post
infection. Magnetic bead sorting was applied for the purification of T cell
subsets. The cytotoxic effects, Transwell experiment and Perforin/granzyme/Fas-FasL
pathways identification were performed. Cell surface markers and cytokines of
the DNT cells were identified by cytofluorometric analysis. The frequencies of
DNT cells from healthy and patients with
Results:
About 63% of MHV-3 infected C3H/HeJ mice developed a chronic course of virus infection and all of the Balb/cJ mice receiving MHV-3 died within 3 to 5 days. After adaptive transfer of splenocytes from MHV-3 infected C3H/HeJ mice, the survival rate(~30%)of Balb/cJ mice post MHV-3 infection increased significantly to. The DNT cell and CD4+CD25+T cell proportions raised Significantly since 2 days post MHV-3 infection in C3H/HeJ mice. DNT cells showed significant and specific cytotoxic effects on CD8+T cells. More than 14% of infected DNT cells expressing IL-2, and about 8% expressing IFN-γ. None has shown the expression of IL-4,IL-10, TNF-γ or Foxp3. The cell surface markers of the DNT cells is TCRαβ+ CD4-CD8-CD25-CD28-CD30-CD44+. Cytotoxicity of DNT cells is only about 20% when cultured with CD8+T cells in separate chambers of Transwell. Fas-FasL but not Perforin/granzyme pathway was involved in the virus specific cytotoxicity of DNT cells.
Patients with
M. Bes; S. Sauleda; N. Casamitjana; A.
Esplugas; J. I. Esteban; M. Pirón; L. Puig; J. Guardia; V. Vargas
Introduction:
Since implementation of highly sensitive HBV
Aim:
To assess cellular immune response to HBV recombinant proteins in blood donors with occult hepatitis B infection.
Subjects and Methods:
Nineteen blood donors with OBI (HBsAg
negative, HBV
Results:
From January 2006 to April 2008, 647.953 blood
donations were routinely screened for HBV
Conclusions:
Cellular immune response to HBV recombinant
proteins in OBI donors is multispecific (surface, core and HBeAg), shows a Th1
profile (IFN-gamma production) and is significantly higher than in inactive HBV
carriers. In OBI donors, low level HBV
822. Cytopathic
Effect of Hepatitis B Virus Obtained from Fulminant Hepatitis Patients
J. Inoue; Y. Ueno; Y. Wakui; K.
Fukushima; T. Kogure; Y. Kondo; E. Kakazu; H. Niitsuma; T. Shimosegawa
Background and Aims:
It is generally thought that hepatitis B virus (HBV) has no direct cytopathic effect and that the host immune response to infected hepatocytes leads to liver injury. However, the presence of a direct effect on hepatocytes was suggested based on the HBV-induced histological changes of a uPA-SCID mouse model with the liver replaced by human liver. We reported in the 58th annual meeting of AASLD that genotype B2 HBV obtained from patients of epidemic fulminant hepatitis accumulates its replicative intermediates in cells, and mutations in the core promoter region (A1762T/G1764A) and precore region (G1862T and G1896A) are related to its effects. We aimed to analyze further the functions of these mutations in the view of the cytopathic effects. Methods: A plasmid carrying 1.3-fold the HBV genome obtained from a fulminant hepatitis patient (pBFH2) was constructed, and the mutations at nt 1762/1764, 1862 and 1896 in pBFH2 were converted to the wild type nucleotides using site-directed mutagenesis. Another plasmid was constructed from an acute hepatitis B patient (pBAH2) in the same way. These plasmids were transfected into HepG2 cells, and cell viability was measured by MTS assay four days after transfection. Cytotoxicity was analyzed by LDH assay, and participation of apoptosis was evaluated by measurement of caspase-3/7 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.
Results:
The cell viability of pBFH2-transfected HepG2 was significantly lower than pBAH2, and the release of LDH was increased in pBFH2. In the analysis of mutations in pBFH2, the cell viability was most greatly recovered when nt 1862 mutation was converted to the wild type. Nucleotide conversions in combination revealed that the presence of both nt 1762/1764 and 1862 mutations decreased the cell viability, whereas single mutations showed no effect. Based on these results, these mutations seemed to be associated with the cytopathic effects of this HBV strain. Because caspase-3/7 and TUNEL-positive cells were not increased in pBFH2-transfected cells, necrosis but not apoptosis was thought to lead to the cytopathy. Conclusions: All of the five patients with fulminant hepatitis caused by this HBV strain were more than 60 years old, in which the immune reaction seemed to be relatively low. This fact prompted us to investigate the direct effect of this strain on hepatocytes. In our study, it was suggested that the presence of both core promoter mutations (A1762T/G1764A) and a precore mutation (G1862T) might be associated with the cytopathic effects of this HBV strain, which might lead to the progression of fulminant hepatitis.
Y. Tong; R. Machida; S. Tong; J. R. Wands; J. Li
Background and Aim:
The mechanism of hepadnavirus entry into hepatocytes is not well defined. Duck carboxypeptidase (DCPD) has been identified as a virus docking receptor. In addition, glycine decarboxylase (DGD), a liver specific protein capable of binding to truncated viral pre-S protein with C-terminal dibasic residues (Arg-Arg), is required for a post-binding step. Processing of substrate proteins via dibasic residues such as Arg-Arg is a characteristic of proprotein convertases such as furin and PC7. Therefore, we investigated whether viral envelope proteins can be cleaved by endoproteinases, and whether this cleavage triggers viral entry.
Methods:
Endosome was isolated from duck liver by
sucrose gradient ultracentrifugation. Co-localization of DCPD, DGD and viral
pre-S protein in endosome was documented from DHBV infected liver by Western
blot. Cleavage of viral envelope proteins was examined by Western blot after
incubation of viral particles with the endosomal fraction. The effect of the
envelope cleavage on viral infection was determined in LMH cells, a chicken
hepatoma cell line resistant to viral infection but supportive of viral
replication. Role of furin and PC7 in DHBV infection was investigated using
specific inhibitors. Results: Both DCPD and DGD were found to be co-localized
with the viral pre-S protein in the fraction enriched for EEA1, an early endosome
marker. Interestingly, incubation of virus particles with the endosomal
fractions isolated from Peking duck liver resulted in cleavage of the viral
envelope protein, similar to that observed in vivo in DHBV infected duck liver.
In contrast, no such cleavage could be demonstrated for endosomes isolated from
Conclusion:
Cleavage of viral envelope protein by Furin/PC7 like-convertases may be critical for initiation of productive viral infection.
817. HBx Protein
Is Necessary for HBV Replication in Human Hepatocytes
M. Tsuge; N. Hiraga; M. Imamura; S.
Takahashi; K. Chayama
Background & Aims:
The HBx protein is one of the hepatitis B virus (HBV) proteins that is considered to play an important role in replication of the hepatitis B virus. Previous reports have shown that the X protein is important in the replication of the woodchuck hepatitis virus, but not in the duck hepatitis B virus. Recently, we developed reverse genetics of the HBV using human hepatocyte chimeric mice. In this study, we examined whether or not the X protein of the HBV is dispensable in viral replication in vivo.
Methods:
For expression of the HBx protein, we
constructed plasmids containing the entire HBx protein in pcDNA3 and pcDNA3-HA
plasmid vectors. A stop codon was introduced to the HBx region of the plasmid
containing the entire HBV genome with C1395T nucleotide substitution (
Results:
The HBx protein expression by hydrodynamic
injection of the HBx protein expressing plasmid was confirmed by the Western
Blot analysis, using antibody directed to HA tag. Quantitatively measurable
viremia developed in six of seven HBx injected mice. In contrast, none of the
16 mice without the expression of the HBx developed the measurable viremia (P=
0.000016). The HBV
Conclusions:
Complementation of the HBx protein with hydrodynamic injection rescued the HBx deficient virus infection to the human hepatocyte chimeric mice, and resulted in the emergence of the revertant virus. Our results suggest that the HBx protein is necessary in active replication of the virus in vivo.
G. Reaiche; M. Le Mire; W. Mason; A. R.
Jilbert
Traces of hepatitis B virus persist in the
liver after clearance of acute infections, but the extent to which persistence
depends upon ongoing viral
Six week-old ducks were infected with DHBV and
monitored until recovery. Following recovery, the ducks were treated
continuously with
At the time of initiation of
Hepatocyte proliferation correlated strongly with loss of residual DHBV cccDNA. Assuming loss of cccDNA with cell death, but not with mitosis a mathematical model was used to predict the required turnover of hepatocytes, which would explain the observed half-life of cccDNA. In 5 out of the 6 ducks studied, estimated cell turnover matched that predicted using the model.
Conclusion:
In conclusion, residual DHBV
825. Increased
Innate and Adaptive Immune Pressure Is Responsible for the Reduced
Transcriptional Activity of Intrahepatic Covalently-Closed Circular (ccc)
J. Thompson; J. Chang; N. A. Skinner; S. Lewin; K. Visvanathan; P. V. Desmond; S. Locarnini
Introduction:
The factors regulating the transcriptional
activity (TA) of cccDNA remain unclear. The G1896A (PC) and A1762T/G1764A (
Aim:
To investigate the relationship between
intrahepatic markers of TA, and host innate and adaptive immune reactivity,
comparing patients with HBeAg(+) versus HBeAg(–)
Methods:
Serum and liver tissue were collected from 44
treatment naïve patients with
Results:
HBeAg(–)
Conclusion:
This is the first study to directly examine
the relationship between markers of HBV transcriptional activity and host immunity
within the liver of patients with
(1) Laras,Hepatology,2006,44:694
(2) Volz,Gastroenterology,2007,133:843
831. High
Prevalence of Hepatic Steatosis and its Impact on Fibrosis in Chronic Hepatitis
B
W. Bleibel; G. Hussain; M. Lai; C. Hayne; N. H. Afdhal; D. Lau
Background and Aim:
There is strong evidence that nonalcoholic
fatty liver disease (NAFLD) is associated with increased disease progression in
hepatitis C. The impact of hepatic steatosis and metabolic syndrome on chronic
hepatitis B (
Methods:
All adult patients with treatment naïve
Results:
A total of 220 patients met the inclusion
criteria and they were predominantly male (63%) and Asian American (68%). HBeAg
positive
Conclusion:
There is a high prevalence of hepatic
steatosis in this Asian American predominant
Disease
Progression – Role of Genotypes and Mutations
989. Precore (PC) and Basic Core Promoter (
D. Zoulas; M. J. Schina; A. Kostourou;
H. Theochari; G. Mousoulis; M. Parara
Introduction:
Virological characteristics
of HBV such as efficiency of viral replication and viral genome variability may
explain the possible pathogenic and therapeutic differences among chronic
hepatitis B (
Methods:
321 out of 41,181 blood
donors found to be positive for HBsAg between 2004 and 2007, prevalence 0,78%. 119 from them came for a second test and only 80
subjects with a positive HBV viral load were finally enrolled in the study.
Age, sex and nationality were recorded. HBsAg, HBeAg and anti-HBe were assayed
using standard EIAs (AXSYM, Abbott) while HBV viral load was determined by
real-time
Results:
All but six were Greek. 79%
male, mean age 33,96 ±10,83 years. Median HBV
Conclusions:
In general, Greek population
genotype D is the predominant and eAg-
868. Increasing Frequency of Acute Hepatitis
B with Imported Strain Genotype A in
Y.
Tamada; K. Yano; A. Komori; S. Abiru; K. Migita; M. Nakamura; H. Fujioka; H.
Yatsuhashi; H. Ishibashi
Background and Aim:
An increasing trend of acute hepatitis B
(AH-B) in
Methods:
A total of 457 cases of AH-B were enrolled in
a nationwide prospective survey on acute hepatitis, in which 28 national
hospitals participated, between 1991 and 2007. The diagnosis of AH-B was made
by positivity for IgM-HBc and HBsAg with an elevation of
Results:
Of 457 cases, gt A, B, and C accounted for 93
(20.4%), 40 (8.8%), and 320 (70.0%), respectively. Other gts,
such as gt D, E, G and H, were detected in 1 case (0.2%) each. Gt A accounted
for 6.4%, 9.2%, 23.1%, 38.5% in 1991-1994, 1995-1999, 2000-2004, and 2005-2007,
respectively. The prevalence in the recent 3 years was significantly higher
than those in the first and second period each (p<0.0001, p<0.01,
respectively). Particularly in 2007, gt A accounted for as much as 52.0% of
AH-B. In Kanto region, where the capital locates and the population flux is
highest in
Discussion and Conclusion:
Gt A infection, which was considered rare in
869. Non-A
Hepatitis B Genotypes are Associated with More Liver Fibrosis in HIV/HBV
Patients
D. Y. Dao; H. Yuan; R. Joshi; N. Attar;
W. M. Lee; M. K. Jain
Background:
Approximately 10% of HIV-infected patients are co-infected with hepatitis B virus (HBV). Few studies have evaluated the prevalence of HBV genotypes and its impact on fibrosis. Evidence exists that genotypes are important in the progression of chronic liver disease in HBV mono-infected patients. The purpose of this study was to determine the prevalence of HBV genotypes and their association with advanced liver fibrosis in co-infected patients. Method: We examined a cohort of 141 HIV and HBV surface antigen (HBsAg) positive patients, for whom sera were available from a large inner city HIV clinic. HBV genotyping was done by direct sequencing. Baseline demographics and current liver function tests were obtained by chart review. Non-invasive serum biomarkers (APRI and Fib-4) were used to assess fibrosis stage at the last clinical follow-up. APRI and Fib-4 scores were dichotomized to examine scores indicating stage 1 fibrosis or ≥ stage 2 fibrosis.
Indeterminate values were not assessed. Categorical variables were analyzed by chi-square and continuous by Wilcoxon Rank Sum test. Results: HBV genotype A was the most frequent genotype (n=100; 75%) while non-A genotypes were present in 25% (n=41). The frequency of the non-A genotypes were the following: 13% (n=17) genotype G, 3% (n=4) genotype A/G mix, 6% genotype D (n=8) and 1.5% (n=2) 2 each were identified for genotypes F and H. Seven patients could not be genotyped. African Americans (AA) composed 47% (n=66) and Caucasians 37% (n=51) of the study population, and the median time of follow-up was 37 months. African Americans were more likely to be genotype A than non-A compared to Caucasians (AA 83% genotype A vs. 67% in CA, p=0.06).
In the non-A genotypes 7/17 (41%) had advanced fibrosis compared to 6/64 (9%) in genotype A, p=0.0015. Similarly, Fib-4 showed 8/24 (33%) of non-A genotypes with advanced fibrosis compared to 9/68 (13%), p=0.03. Median ALTs were higher in non-A genotypes [65 (7-193)] compared to genotype A [30 (8-159)], p<0.0001. In addition, HBeAg was positive in 18/20 (90%) of non-A genotypes compared to 44/62 (71%) of genotype A, p=0.08).
Conclusion:
This cross-sectional analysis reveals that HBV genotype appears to play a role in the natural history of the HIV/HBV co-infected patients. Genotype A is the most prevalent but there is a high prevalence of genotype G in HIV/HBV co-infected population. Non-A genotypes are associated with more liver fibrosis on follow-up evaluation. HBV genotypes may be an important tool to predict those who may be at greatest risk for liver disease progression in the HIV/HBV co-infected population.
870. Higher Levels of HBV-
H. Krarup; P. Madsen; A. Bentzen-Petersen; J. M. Møller; N. Weis
Hepatitis B viral factors such as viral load and HBV genotype have been suggested to influence progression of chronic hepatitis B.
Aim:
To explore relations between 1) genotype and viral load, 2) gender and viral load, 3) gender and genotype and 4) genotype and country of origin.
Material:
Samples from 802 consecutive HBV-
Results:
Male/female ratio 427 to 375, age given as median (25;75 percentiles) was for males 37 (27;46) and females 30 (24;38) years. In total 195 samples could not be genotyped; 106 due to lack of sufficient amount of material and 89 due to various reasons, mostly too low viral load. Of 607 samples 11.2% were genotype A, 14.3% B, 19.4% C, 50.3% D, 2.3% E, 0.5% F and 1.5% genotype G, 0.8% had more than one genotype.
1) A significant difference was observed in viral load in genotype A compared to B and C (p values <0.008) and genotype D compared to B and C (p values <0.007), but no difference was observed in viral load between A and D or B and C. The viral loads in genotypes A to D were 1.2 x 10(5) (4.4 x 10(3); 3.8 x 10(7)) IU/mL, 1.4 x 10(7) (2.9 x 10(4); 1.4 x 10(8)) IU/mL, 1.4 x 10(7) (8.5 x 10(4); 1.4 x 10(8)) IU/mL and 1.0 x 10(5) (4.0 x 10(3); 5.9 x 10(7)) IU/mL respectively.
2) No difference in viral load among gender was observed either overall or in different genotypes.
3) A significant difference in distribution of gender over the 4 dominant genotypes was observed: more women had genotypes B or C while more men had genotypes A or D (p values <0.001). Women were significantly younger than men, 30 years versus 41 years (p value <0.001) and 30 years versus 35 years (p value <0.004) in genotypes B and D respectively, whereas no significant age difference was observed in genotypes A or C, men 32 years versus 35 years and 29 years versus 31 years respectively.
4) More than 65% of patients with genotype A
originated from Africa.73% and 95% of patients with genotypes B and C
originated from South East
Conclusion:
We found the median of serum HBV-
C. Kim; S. Yoon; C. You; J. Jang; S. Bae; J. Choi; J. Yang; J. Han; S.
Choi; N. Han; C. Lee; Y. Lee
Background:
'a' determinant, which is in major hydrophilic
region (
Methods:
Serums from total 122 patients with chronic
HBV infection were used for HBV
Results:
A total of 122 patients, infected all with HBV
genotype C (100%), were composed of 78 chronic hepatitis B, 24 cirrhosis and 20
The frequency of T126 as another wild type
instead of I126 showed significant relationship with disease progression from
chronic hepatitis via cirrhosis to
Conclusions:
126 aa of HBsAg is
important position in variations of HBsAg. The frequency of T126 showed
significant relationship with disease progression associated with HBV (genotype
C) from chronic hepatitis via cirrhosis to
T. Sakamoto; Y. Tanaka; M. Sugiyama; F.
Kurbanov; K. Matsuura; A. Kusakabe; N. Shinkai; F. Sugauchi; M. Mizokami
Background:
Hepatitis B virus genotype G (HBV/G) has stop codons in the precore region and is usually being detected in coinfection with the other genotypes, typically HBV/A2/Ae (Europe/USA). HBV/G reportedly accelerates liver fibrosis in patients with HIV infection (Lacombe K, et al. AIDS 2006). As recently reported, HBV/G monoinfection in chmeric mice resulted in viral replication at very low levels, but the replication increased markedly when animals coinfected with HBV/A2 and induces fibrosis, that concurs with findings among immunosuppressive patients. However, it is still unclear how the interaction with other genotypes enhances the replication of HBV/G.
Aim:
To investigate which viral proteins of HBV/A2 contribute in the replication of HBV/G in course of their coinfection. Methods: Four HBV/A2 recombinant plasmids were constructed (HBV/A2/S, HBV/A2/C, HBV/A2/pol and HBV/A2/X) in which only one of the four viral proteins (large surface, precore/core, polymerase or X, respectively) was selectively translated whereas translation of the other three was prevented by introduction of the corresponding stop codons. Huh7 cells were co-transfected with HBV plasmids encoding replication-competent 1.24-fold HBV genome of HBV/G and the recombinant HBV/A2 plasmids in following five protocols: (1) HBV/G alone; (2) HBV/G and HBV/A2/S; (3) HBV/G and HBV/A2/C; (4) HBV/G and HBV/A2/pol; and (5) HBV/G and HBV/A2/X. HBsAg and HBeAg in culture supernatant and core-related antigens (HBcrAg) in cell lysates were determined by CLEIA. Viral replication was estimated by Southern blot hybridization.
Results:
Comparing with HBV/A2, HBV/G produced barely
detectable intracellular HBV
Conclusion:
Low replication of HBV/G would be contributed by low synthesis or dysfunction of HBV/G core protein due to weak core promoter activity. HBV/G might take advantage of core protein of the other genotypes to replicate efficiently on coinfection with them, and the enhanced replication might result in disease progression under immunocompromised states.
N. Yamada; H. Yotsuyanagi; H. Miyoshi; T. Tsutsumi; H. Fujie; Y. Shintani; K. Moriya; C. Okuse; M. Suzuki; F. Itoh; K. Yasuda; S. Iino; K. Koike
Background:
Most HBe antigen (HBeAg)-positive asymptomatic carriers (AsC) suffer active liver injury in their adolescence, the mechanism of which is not clear yet. To elucidate contributing viral factors, we analyzed the structure and full nucleic/amino acid sequences of cloned HBV recovered from the sera of HBeAg-positive AsC and patients with active hepatitis.
Methods:
Nine HBeAg-positive
HBV carriers persistently infected with HBV of genotype C (4 AsC, 5 chronic
hepatitis (CH) patients with an acute exacerbation) were studied. The
full-length genome of HBV was amplified using a single-step
Results:
1) Surprisingly, HBV clones with large deletions at from the end of core to the middle of preS-S region or and with those in the core region were detected.
2) 2) In AsC, the HBV deletion mutants circulated as minor clones (median 7.0%), whereas those circulated as major clones (median 67.6%), in CH patients.
3) 3) In CH patients, amino acid substitutions in the core region at AA13, 87 and 97 were detected more frequently than in AsC (AA13: 14.7% vs. 0.0%, AA87: 55.6% vs. 0.0%, AA97: 55.6% vs. 0.0%).
4)
4) Double mutations in the
basal core promoter (
5) 5) Time-course analysis of the sera showed that the HBV deletion mutants emerged on the exacerbation and dominated along with seroconversion to anti-HBe.
Conclusions:
The clinical course of HBeAg-positive carriers
with genotype C HBV is accompanied by the appearance of large deletions in the
core and preS-S regions and amino acid substitutions in the core region in
addition to nucleic acid substitutions in the
863. Drifting of HBV-specific
S. Ma; M. Liang; X. Hu; Y. Yu; J. Chen; L. Yang; C. Li; X. Xu; Z. Wang; J. Hou
Background:
HLA-A2 restricted HBcAg18-27V (C18-27V)
specific cytotoxicity T lymphocyte (
Aims:
The aims of this study are to profile the
difference between C18-27V/I mutants in the prevalence and immunogenicity in
Results:
We found that C18-27I motif was the
predominant virological background in 80 genotype B strains, 41 genotype C1, 78
genotype C2 and 26 CD recombinants (97.5%, 82.9%, 78.1% and 100%), whereas
total 5 genotype D strains showed C18-27V. The proportion of C18-27I specific
Conclusions:
It suggests that C18-27I is an immunodominant peptide than C18-27V under prototype C18-27I virological background. It will be further exploited in the development of the diagnostic and vaccine in the control of HBV infection.
F. Sugauchi; Y. Tanaka; T. Sakamoto; A.
Kusakabe; K. Mtsuura; M. Sugiyama; S. Nojiri; J. Takashi; M. Mizokami
Background and Aim:
Virologic characteristics of hepatitis B virus (HBV) on the reactivation of occult hepatitis B in patients who received hematopoietic stem cell transplantation (HSCT) or cytotoxic chemotherapy for the hematological malignancy are not well defined. Influences of HBV genotype and viral mutations on reactivation of occult hepatitis B in patients with hematological malignancy were investigated.
Methods:
Twenty eight HBsAg-negative patients who
received HSCT for the hematological malignancy and 138 HBsAg-negative patients
treated for malignant lymphoma (ML) with cytotoxic chemotherapy containing
rituximab at the
Results:
Three of the 28 patients (10.7%) received HSCT and 1 of the 138 (0.72%) patients treated for ML with cytotoxic chemotherapy developed de novo HBV hepatitis. The four patients with de novo hepatitis B had the mean age of 49 + 10 years including 3 men, and anti-HBc was detected in 4 and anti-HBs in 2 patients before commencement of HSCT or chemotherapy. HBV genotype Bj was detected in 2 (50%) and C in 2 (50%).They all possessed wild type sequences (A1762/G1764) in core promoter region, and the precore stop mutation (G1896A) was detected only in a patient with genotype Bj who developed fulminant hepatic failure.
HBV
Conclusions:
Fulminant outcome on reactivation of occult hepatitis B in patients received HSCT or cytotoxic chemotherapy for the hematological malignancy is associated with genotype Bj accompanied by high replication due to the G1896A mutation, which is supported by in vitro replication model.
M. Sugiyama; Y. Tanaka; I. Maruyama; T.
Shimada; S. Takahashi; T. Shirai; K. Hino; I. Sakaida; M. Mizokami
Background:
Hepatitis B virus (HBV) genotypes/subgenotypes indicate different clinical features, i.e. genotype C is associated with hepatocellular carcinoma, and Bj with precore mutation (PCm) is associated with fulminant hepatitis. We previously showed the difference of each genotype in vitro and partially in vivo.
Aim:
Our purpose of this study is to investigate the difference of viral replication, protein production and liver damage among HBV genotypes in vivo.
Methods:
Sera of six HBV carriers [three each for
Bj_wild-type (from acute HB patients) and Bj_PCm (fulminant HB patients)] were
prepared, and the other sera were obtained from pre-infected mice which were
inoculated with culture medium produced by transfection with plasmids (3
strains each for Ae, Ce, Bj_wild-type and Bj_PCm) carrying HBV genome in Huh7
cells. These 18 sera were inoculated into uPA/SCID mice harboring human hepatocytes
(chimeric mice). HBV
Results:
Chimeric mice inoculated with HBV/C or Bj_PCm
for 6 months showed that human hepatocytes had somewhat ground-glass appearance
and fibrosis, while chimeric mice inoculated with HBV/Ae or Bj_wild-type were not
induced. Immunostaing analysis showed strong staining of α-SMA around
fibrosis.
Conclusion:
Using humanized in vivo model, we demonstrate
that different HBV genotypes and even particular mutation resulted in different
virological and histopathological outcomes of infection, indicating that distinct
HBV variants may be directly cytopathic in immunosuppressive conditions. This
would represent a novel mouse model for human liver fibrosis associated with
Disease
Progression – Development of Cirrhosis and Liver Cancer
867. Accumulation of Hepatitis B Virus
A. Tamori; T. Hayashi; M. Shinzaki; S. Kobayshi; S. Iwai;
H. Morikawa; M. Enomoto; H. Sakaguchi; S. Shiomi; S. Kubo; N. Kawada
Background and Aim:
Hepatocellular carcinoma (
Patients and Methods:
Operable
Results:
Anti-HBc was positive in 10 (66.7%) of 15
patients with
Conclusion:
Occult HBV infection might be one of the most
important factors in
847. Future
Hepatitis Activation and Hepatocarcinogenesis in HBeAg-Negative HBV Carriers
with Persistently
T. Nakazawa; Y. Shibata; A. Takeuchi; Y.
Okuwaki; K. Ono; Y. Miura; H. Hidaka; Y. Tanaka; J. Takada; M. Watanabe; A.
Shibuya
Background/Aims:
To elucidate the incidence of future hepatitis activation and hepatocarcinogenesis in HBeAg-negative HBV carriers with persistently normal alanine aminotransferase (PNALT), long-term observation was performed in a prospective cohort.
Methods:
Ninety-nine consecutive untreated
HBeAg-negative carriers with PNALT (40IU/L) were prospectively included in the study.
The physical examinations, laboratory tests, and abdominal ultrasound
sonography were performed every 6 months after the enrollment. The incidence of
hepatitis activation (
Results:
The mean follow-up period was 65.3±36.8
months. Thirteen carriers (13%) revealed hepatitis activation, and
hepatocarcinogenesis was observed in 4 (3.7%). The cumulative rate of hepatitis
activation was 10.5% and 14.9% at 2 and 5 years, while the cumulative rate of
hepatocarcinogenesis was 2.9% and 12.3% at 5 and 8 years. In statistical
analyses for future hepatitis activation, the univariate analyses revealed
high-normal
Conclusions:
HBeAg-negative HBV carriers with high-normal
845. Clinical
Significance of Low Viral Load in Antiviral Treatment-naive HBsAg Positive
Patients
H. Woo; J. Choi; A. Min; J. Kwon; J. Kim;
J. Jang; S. Bae; S. Yoon; S. Cho; J. Yang; Y. Lee
Background/Aim:
The low limit of HBV
Methods:
All HBsAg-positive subjects with HBV
The following patients were excluded; those with prior
antiviral treatment, with prior or concomitant chemotherapy or
immunosuppressive treatment, with other acute or chronic viral infection and
with diagnosis of intrahepatic or extrahepatic malignancy. Liver cirrhosis was
defined when pathology was confirmed or two of 3 following criteria are met;
Platelet <100,000/mm3, definitive cirrhotic figure by ultrasonography, and
variceal change by endoscopy.
Results:
Of the 1025 samples, 241 could not be genotyped: 137 due to lack of material and 104 for other
reasons, mostly very low viral load.
We found no gender difference in viral load, neither
overall nor in genotype A, B, C and E. A slight difference was observed in genotype
D (p=0.048)
Distribution of gender over the 4 dominant genotypes
differed markedly (p<0.001) while more men had genotypes A (p=0.015) or D
(p<0.001).
Women were younger than men, 28 years vs. 39 years (p=0.001)
and 30 years vs. 33 years (p=0.026) in genotypes B and D respectively. No age difference was observed in genotypes A
or C, men 32 years vas. 35 years and 29 years vs. 31
respectively.
·
62% o patients with genotype A originated from
·
82% with genotype B and 95% with genotype C from
·
62% with genotype D from
the
·
75% with genotype E from
Conclusion
The median of serum HBV-NDA levels was 100 times
higher in genotypes B, C and E compared to genotypes A and D.
·
Men had a slightly higher viral load in genotype D compared
to women. A similar difference is not
seen in the other genotypes.
·
More women than men had genotypes B or C, as opposed to
genotypes A or D.
·
The distribution of genotypes was in keeping with the origin
of the patients.
R. Muroyama; N. Kato; M. Otsuka; J. Chang; M. Omata
Background/Aim:
Hepatitis B virus (HBV) is a major risk factor
for hepatocellular carcinoma (
Methods:
1)
Using
cassette-ligation-mediated
2) 2) We silenced the expression of mRNA from HBV integrant with siRNA in Hep3B cells, and established stably knocked-down clones (KD cells). Using KD cells, we investigated the expression of fusion HBx by immunofluorescence staining.
3) 3) Using KD cells, we evaluated the effect of fusion HBx on cell growth and invasiveness by MTT/BrdU assay and matrigel invasion assay.
4) 4) We compared the transactivation activity and the ability to transform cells between wild HBx and fusion HBx by luciferase assay and soft agar assay.
Results:
1) We identified full-length mRNA from HBV integrant with the length of 3725bp containing 1877bp human sequences at 3’ end, and fusion HBx (3’-truncated HBx + human peptides) was supposed to be translated.
2)
2) Nearly 90% reduction of
fusion mRNA expression by siRNA was confirmed using Realtime
3) 3) KD cells demonstrated significant reduction in cell proliferation and invasion ability compared to parental cells.
4) 4) Although fusion HBx had significantly decreased transactivation ability compared to wild HBx, fusion HBx had anchorage-independent growth ability in soft agar whereas wild HBx did not have.
Conclusion:
Fusion HBx from HBV integrant may play an important role in the development and progression of hepatocellular carcinoma.
841. Pattern of
serial HBV
J. Kwon; J. Choi; H. Woo; J. Kim; C. You; S. Bae; S. Yoon; N. Han; C. Lee; Y. Lee
Aims:
Hepatitis B virus (HBV)
Methods:
Among 352 patients who diagnosed a newly HBV
related
Results:
There was no difference of age, sex, HBe Ag
status, antiviral therapy status and Child–Pugh class in
Conclusions:
The cirrhosis patients with persistently high
pattern (≥104 C/mL) of HBV
Disease
Progression : Reactivation of Infection with
Chemotherapy
848. Rituximab Leads to
Reactivation of Hepatitis B in Individuals with Resolved Infection
F. Metzler;
Background
Rituximab is a chimeric anti-CD20 antibody
causing a massive reduction of B-cells without affecting their progenitors. It
is indicated for the treatment of B-cell Non-Hodgkin Lymphoma (
Aim:
The aim of this study was to analyse the course of hepatitis B in patients with rituximab treatment.
Patients & Results
A total of 709 courses of rituximab (range
1-11) were given to 258 hospitalized patients at the
One patient remained an inactive HBsAg carrier
with detectable HBsAg, low HBV viremia at 1.1x103 IU/mL and normal
Conclusions
Our results show that reactivation of
hepatitis B can occur. Decline and subsequent loss of neutralizing anti-HBs
antibodies and reappearance of HBV
R. Lee; K. Vu; H. Shah; C. Bell; L. K.
Hicks
Reactivation of hepatitis B
(HBV) is a recognized complication of chemotherapy associated with substantial
morbidity and mortality. Prophylaxis with lamivudine in chronic HBV carriers
prior to chemotherapy can reduce the incidence and severity of reactivation
hepatitis. Published guidelines recommend screening all patients receiving
chemotherapy for HBV prior to initiation. There is currently no such policy at
St. Michael’s Hospital in
Aim:
The objectives of this study
are to 1) determine the frequency with which HBsAg is tested prior to
intravenous (IV) chemotherapy at St. Michael’s Hospital, and 2) to compare the
incidence of HBsAg testing pre-chemotherapy, with the incidence of cardiac
function testing prior to potentially cardiotoxic chemotherapy.
Patients who were started on
IV chemotherapy at St. Michael’s Hospital between March 2006
to March 2007 were retrospectively identified using existing pharmacy
records. The frequency of HBsAg and
Results:
Despite the recognized
importance of HBV testing prior to chemotherapy, the proportion of patients
being tested for HBV status prior to chemotherapy at our academic institution
is low. In contrast, cardiac function is routinely determined prior to
cardiotoxic chemotherapy. The high rate of testing for cardiac function
indicates that programs aimed at predicting and preventing side effects to
chemotherapy can have a high compliance. Barriers to universal HBV testing
likely include oncologists’ perception of the importance of testing as low and
lack of a systematic requirement to test prior to chemotherapy. A quality
assurance intervention will be performed in response to this data to determine
whether the frequency of HBsAg testing can be increased to 90% or more at our
academic institution.
Experimental
Therapies
949. Serine Palmitoyltransferase Inhibitor
Suppresses Hepatitis B Virus Replication
K. Tatematsu; Y. Tanaka; M. Sugiyama; M. Mizokami
Background:
Serine palmitoyltransferase
(SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin
is an inhibitor of SPT and suppresses replication of the hepatitis C virus
(HCV) replicon. However, it is still unknown whether this SPT inhibitor can
suppress HBV replication.
Aim:
To investigate the anti-HBV
effect of myriocin against intact HBV using Huh7 cells transfected with HBV (in
vitro) and uPA/SCID mice with the liver replaced by human hepatocytes (chimeric
mice) with HBV (in vivo).
Method:
Huh7 cells were transfected with
plasmids carrying 1.24-fold the HBV genome of genotype C which is the most
common in
Result:
We succeed in reducing the HBV
Conclusion:
Myriocin could inhibit HBV replication in Huh7 cells. Although
myriocin monotherapy was not effective, combined treatment with
M. Kumar; A. Follenzi; S. Gupta
Despite the success of interferon alfa (IFNα) and nucleoside/nucleotide-based antiviral therapies for HBV, treatment failures and emergence of drug-resistant HBV strains pose problems. Also, HBV infection of the graft after orthotopic liver transplantation may produce aggressive liver disease and transplant failure. Therefore, alternative approaches to control HBV replication are required.
Method:
To identify optimal strategies for suppressing
HBV replication, in initial studies, we used two published shRNAs sequences
inserted in a miR-30 backbone to target HBV. These sequences were cloned either
individually or in combination in lentiviral vectors (LVs) under control of CMV
promoter upstream of green fluorescent protein (GFP) to track gene expression.
Anti-HBV LVs were designated LV-miK-GFP (HBVno.6 sequence from Kay et al. Nat
Biotech. 2003;21:639), LV-miSH-GFP (pSUPERcore
sequence from Shaul et al. Hepatology 2003;37:764) and LV-miK+SH for the
combined sequence. Another
Conclusions:
Suitable antiviral shRNAs could be combined to
effectively limit HBV replication in cells. The ability of fetal human
hepatoblasts to spontaneously suppress HBV replication coupled with their known
avidity for
877. Drug-resistant and Wild-type Strains of HBV Are Inhibited Equally by Simvastatin
T. Bader; B. Korba; M. Bronze
The purpose of this report is to compare the
in vitro inhibition of a panel of clinically relevant drug resistant viruses of
HBV by simvastatin (
Methods:
Cultures of HBV-producing HepG2 2.15 cells
were maintained as previously described (Antivir.Res.19:55-70, 1992). Confluent
cultures of 2.2.15 cells were maintained on 96-well flat-bottomed tissue
culture plates. Cultures were treated with nine consecutive daily doses of the
test compounds. Activity against 3TC-resistant and
Discussion:
The safety of
Conclusion:
Comparable efficacy against a panel of
clinically relevant drug resistant viruses is yet another indicator of the
potential usefulness of
|
|
Simvastatin
(μM) |
Lamivudine
(μM) |
Adefovir
(μM) |
|||
|
HBV STRAIN |
EC50 |
EC90 |
EC50 |
EC90 |
EC50 |
EC90 |
|
Wild-type |
8.2 |
35 |
0.2 |
0.7 |
1.5 |
6.5 |
|
L180M |
9.5 |
32 |
5.2 |
20 |
1.9 |
8.5 |
|
M204V |
9 |
39 |
>100 |
>100 |
1.8 |
7 |
|
M204I |
8.4 |
40 |
>100 |
>100 |
1.3 |
7.2 |
|
LM/MV |
8.8 |
44 |
>100 |
>100 |
1.3 |
7.8 |
|
N236T |
9.3 |
45 |
0.2 |
0.6 |
8.1 |
32 |
EC50, EC90 are concentrations for 50% and
90% viral inhibition, respectively.
I. Otano; J. Crettaz; L. Ochoa; A.
Benito; A. Paneda; I. Aurrekoetxea; A. Astudillo; F. Kreppel; S. Kochanek; J.
Ruiz; S. Menne; J. Prieto; G. Gonzalez-Aseguinolaza
Background and Aims:
Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-2 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus (HBV) infection. We tested the therapeutic potential of IL-12 gene therapy in woodchucks with chronic woodchuck hepatitis virus (WHV) infection, a condition that closely resembles chronic hepatitis B.
Methods:
Eight woodchucks treated by intrahepatic injection of a helper-dependent adenoviral vector encoding murine IL-12 under the control of a liver-specific RU486-responsive promoter. IL-12 expression was induced for 42 days. Five animals with viremias over 10e11 viral genomes/ml (vg/ml) received combination therapy with nucleos(t)ide analogues for four weeks and IL-12 gene therapy for 42 days.
Results:
All woodchucks with viral load below 10e10 vg/ml showed a marked and sustained reduction of viremia accompanied by loss of WHVe and WHV surface antigens and improved liver histology. In contrast, none of the woodchucks with viremia higher than 10e10 vg/ml responded to therapy. In the responder animals, the antiviral effect was associated with induction of T cell immunity against viral antigens and reduction of hepatic expression of Foxp3, the nuclear factor that characterizes regulatory T cells. The animals that received combination therapy experienced a transient decrease of viremia with relapse after cessation of therapy.
Conclusions:
IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection. This therapy is able to break immunotolerance and to control viral replication in all cases with viral load below 10e10 vg/ml, which is the situation of most patients with chronic hepatitis B.
S. Woo; C. Kim; S. Nam; J. Choi; S. Cho; J. Yang; J. Han; Y. Lee
Background:
In chronic hepatitis B virus (HBV) infection, antiviral drugs are the effective way of restraining disease progression, but small number of cases showed surface antigen (HBsAg) seroconversion by antiviral drugs. Though some reports showed the effect of immune cell therapy in which sensitized lymphocyte induced HBsAg seroconversion, the effect of immune cell therapy is still uncertain. The aim of this study was to investigate the effect of allogenic hematopoietic stem cell transplantation (allo-HSCT) on HBsAg seroconversion in patients with chronic HBV infection.
Methods:
HBsAg and antibody to HBsAg (anti-HBs) were surveyed retrospectively in total 537 patients who underwent allo-HSCT from January 2000 to June 2007 in single bone marrow transplantation center. Forty-five (8.4%) of 537 allo-HSCT patients showed HBsAg positive/Anti-HBs negative. HBV serologic markers and clinical variables were inspected in these 45 HBsAg positive allo-HSCT recipients and their donors before and after allo-HSCT. Median observational period was 36.6 months (range 3~80 months) after transplantation.
Results:
Among 45 HBsAg positive patients, 21 were
transplanted with hematopoietic stem cells (
Conclusion:
The rate (19.0%) of HBsAg seroconversion in chronic HBV infection patients who underwent allo-HSCT is so high compare to natural HBsAg seroconversion. This high rate of HBsAg seroconversion shows a possibility of immune cell therapy in chronic HBV infection.
823. Efficacy of
Hepatitis B Vaccine Against Antiviral
Drug-resistant Hepatitis B Virus Mutants in the Chimpanzee Model
S. Kamili; T. Sozzi; G. Thompson; K. Campbell; C. M. Walker; S. Locarnini; K. Krawczynski
Hepatitis B virus (HBV) mutants resistant to treatment with nucleoside or nucleotide analogues and those with the ability to escape from HBV neutralizing antibody have the potential to infect HBV-vaccinated individuals. To address this potential serious public health challenge, we tested the efficacy of immunity induced by a commercial hepatitis B vaccine against a tissue culture-derived, clonal HBV polymerase mutant in HBV seronegative chimpanzees. The polymerase gene mutant, generated in vitro by genetic engineering technology, contained a combination of three mutations (rtV173L+rtL1806M+rtM204V) two of which resulted in changes to the overlapping viral envelope of the hepatitis B surface antigen (sE164D+sI195M).
The in vitro generated polymerase gene mutant inoculum was proven infectious in three HBV seronegative chimpanzees. Cloning and sequencing experiments determined that the three mutations in the polymerase gene mutant remained stable. Two naïve chimpanzees were vaccinated with a pediatric dose of hepatitis B vaccine, which resulted in both humoral (steady levels of >75 mIU/L anti-HBs) and cellular immune responses against HBsAg in the vaccinated chimpanzees. The two vaccinated chimpanzees were challenged with infectious polymerase gene mutant inoculum; following challenge anti-HBs titers increased more than 100 fold, anti-HBc was detected on day 7 in one chimpanzee and on days 7, 10 and 17 in the second chimpanzee. Analysis of cellular immunity in one chimpanzee also provided evidence of productive infection with the polymerase gene mutant. Specifically, a transient T cell response targeting the polymerase protein was detected on day 28 post-infection by the interferon-γ ELISpot assay. T cell lines specific for the HBV polymerase protein derived from the peripheral blood of the chimpanzee on day 28 were CD4+, suggesting that the response was mediated in part by T helper cells.
Conclusion:
The data from this study showed vaccination of naïve chimpanzees with commercial hepatitis B vaccine did not confer sterilizing immunity against a clonal rtV173L+rtL1806M+rtM204V HBV polymerase gene mutant.
824. New Insights
into HBV Pathogenesis from Kinetic Analysis of HBeAg Titre Decay During Potent Antiviral
Therapy.
A. J. Thompson; E. Shudo; R. Ribeiro; K. Visvanathan; P. V. Desmond; G. K. Lau; A. S. Perelson; S. Locarnini
Background:
HBeAg seroconversion is one of the major therapeutic
endpoints used when treating HBeAg-positive chronic hepatitis B (
Methods:
Mathematical modelling was used to analyze and
compare the kinetics of the decline in serum HBV
Results:
The pattern of HBV
Conclusion:
This is the first mathematical analysis of the
kinetics of HBeAg titre during potent antiviral therapy. A lag phase was
identified prior to HBeAg titre decline, consistent with a drug-induced
reduction in cccDNA level through inhibition of the intracellular recycling
pathway. Furthermore, the occurrence in a number of patients of a prolonged lag
phase despite 2nd phase HBV
(1)
Nowak M, PNAS, 1996, 93(9): 4398
(2)
Lau G,
M. Mizrahi; G. Lalazar; A. Ben Ya'akov; T. Adar; L. Zolotarov; Y. Ilan
Latency-associated peptide (LAP) is an amino-terminal domain of the TGF-beta precursor peptide and remains non-covalently associated with TGF-beta. after cleavage, forming the latent complex. The presence of membrane-bound LAP on the surface of regulatory T cells (Tregs) is associated with their regulatory function. Tregs responses may be either beneficial or detrimental to those infected with HBV, by either limiting liver immunopathology or suppressing protective T-cell responses.
Aim:
To determine the ability to enhance the anti-HBV immunity by promoting LAP+ Tregs.
Methods:
Thirteen groups of mice were vaccinated with
HBV vaccine along with oral (
Results:
A five fold increase in spleen-derived CD4+LAP+
lymphocytes was induced by oral administration of GC with the HBV vaccine (0.62
vs.0.12%, for GC vs. controls, respectively, p<0.001), and was associated
with a three fold elevation of the anti-HBV antibody titer (945 vs. 312 mIU/ML,
for GC vs. controls, respectively, p<0.02). Mode of adjuvant administration
determined the increase in CD4+LAP+ and anti HBV antibody titer. Spleen levels
of CD4+LAP+ increase with oral, IP, and IN administration of GC, but to a much
lower extent following IR administration (0.62, 0.78, 0.64, and 0.14%, for GC,
PO, IP, IN, and IR, respectively, p<0.001). These data correlated with the
anti-HBV antibody titers (604, 945, 620, and 283 mIU/ML, for GC,
Diagnostic Tools
S. C. Raftopoulos; E. Rossi; B. De Boer; G.
P. Jeffrey; D. J. Speers; G. C. MacQuillan; L. A. Adams
Staging liver fibrosis in chronic hepatitis B
(
Methods:
Patients with
Results: 70 patients (73% male) with a mean ±SD age of 42.5 ±12.6 years underwent biopsy. The majority were Asian (59%) and eAg negative (60%). The median time between serum collection and biopsy was 0.7 months (range 0-6). Thirty two (46%) had significant fibrosis, 21 (30%) had advanced fibrosis and 12 (17%) were cirrhotic. Hepascore had an area under the ROC curve for significant fibrosis, advanced fibrosis and cirrhosis of 0.91 (95% CI 0.84-0.98), 0.94 (95% CI 0.89-0.99) and 0.92 (95% CI 0.85-0.99) respectively. A cutoff of 0.62 provided sensitivity, specificity, positive and negative predictive values (NPV) of 85%, 84%, 82% and 87% respectively for significant fibrosis. The same cutoff provided a 100% NPV for advanced fibrosis and cirrhosis. Among 18 patients who were monitored over a median of 1.7 (range 0.4-6.8) years without anti-viral treatment, mean Hepascore tended to increase over time (0.59 ±0.30 to 0.72 ±0.29, p=0.09). Among 21 patients who had anti-viral therapy over a median of 2.9 (0.8-7.0) years, mean Hepascore did not change significantly (0.65 ±0.29 to 0.61 ±0.35, p=0.56). The annual rate of change of Hepascore was significantly higher in those that did not receive anti-viral therapy compared to those who did (0.07 ±0.15 vs -0.04 ±0.13, p=0.04).
Conclusion:
Hepascore is an accurate predictor of liver
fibrosis in patients with
F. Bonino; B. Ofenloch; W. Melchior; B. Upmeier; D. Rössler; W. van der Helm
Background/Aims:
Recent data suggest that on-treatment monitoring of HBsAg during peginterferon alfa-2a therapy in patients with chronic hepatitis B may be useful for predicting response to therapy. We hypothesized that it may be possible to use an available qualitative HBsAg screening assay to determine HBsAg quantitatively. By selection of appropriate dilutions, limitations of the measuring range can be overcome, allowing quantification of HBsAg in a suitable linear range. We evaluated the feasibility of a simple algorithm for quantification of serum HBsAg based on the Elecsys® HBsAg II screening assay (Roche Diagnostics).
Methods:
A total of 29 patient serum samples were tested according to the following algorithm. Samples were tested at a dilution of 1:400 in serum negative for HBsAg and anti-HBs. If the cut-off index (c.o.i.) was ≥1, results were expressed as c.o.i x 400. If the c.o.i was <1, the sample was retested undiluted. Samples giving a value >300 c.o.i. were re-tested with 1:8000 dilution and results were calculated as c.o.i x 8000. Quantitative results (IU/mL) were determined by reading off resulting c.o.i. values from a master calibrator curve (target values assigned with WHO standard material).
Results:
There was a good correlation between the c.o.i and IU/mL and this was linear over the entire range tested (5–6 logs) [Figure]. The concentration of the samples was in the range <0.05–46382 IU/mL. 21/29 samples gave a value <1 c.o.i. after 1:400 dilution and were re-tested undiluted. 2/29 samples gave a value >300 c.o.i. and required retesting at 1:8000 dilution.
Conclusions:
This simple testing algorithm used in connection with the qualitative Elecsys®HBsAg II screening assay appears to be suitable for the effective quantification of serum HBsAg in clinical samples. This assay may provide useful information during on-treatment monitoring of HBsAg levels to guide patient management.
939. Changes in Liver Stiffness During
Entecavir Therapy in Patients with Chronic Hepatitis B
S. Lim; J. Cheong; S. Cho
Background/Aims:
Liver stiffness (LS)
measurement is a non-invasive method for assessment of fibrosis in patients
with liver disease. The usefulness of LS measurement for the follow-up of
fibrosis in hepatitis B patients receiving antiviral therapy is unknown. The
aim of this study was to evaluate changes in LS and factors associated with LS
changes in chronic hepatitis B (
Methods:
Study population included 24
Conclusions:
LS is
significantly reduced during entecavir therapy. Regression of hepatic
inflammation and fibrosis during therapy may result in reducing LS.
964. Rapid, Reliable and Cost-effective
Detection of Hepatitis B Virus in Human Plasma
C. M. Habibullah; Y. Naresh; C. Madhavi;
H. Aejaz
A number of common mutations
have been described in HBV strains and recombinant genomes have been reported.
These include variability within the basic core promoter, precore/core and the
pre-S1 gene, which shows great heterogeneity. Designing primer and probe sets
to detect and quantify all HBV variants by real-time
Materials
and Methods:
A total of 250 samples
consisting of HBV positive (n=100); HBV, HCV and HIV negative (n=100); HCV
positive (n=25) and HIV positive (n=25) were collected from out patient unit,
Centre for Liver research and diagnostics. Complete Human HBV
We constructed a plasmid
with x region using p-GEMT easy vector for being used as a control for deriving
standard curve. Out of 100 negative samples screened by ELISA and standard RT-
Conclusion:
This assay is reproducible
showing limited inter and intra assay variability and good amplification
efficacy in different HBV genotypes and serotypes. We demonstrate that the
results of our assay correlated well with the standard kit for HBV viral load
monitor. In this study we eliminated the drug inducible mutation and variable
regions for selecting the conserved portion of viral genome for effective oligo
designing and evaluated the same.
947. Development of Kinetic Amplification-Based Assay
for Detection and Quantification of HBV
C. L. Wong; L. Ku; A. Ying; A. Jacobson; S. K. Aung; Z. Li; D. G. Sherman; L. Shen
Background:
Hepatitis B virus (HBV) can cause
lifelong infection, cirrhosis of the liver, liver cancer, liver failure and
death. Worldwide, an estimated 400 million people are persistently infected
with HBV. HBV quantification is useful for therapeutic monitoring of disease
progression and detecting resistance to antiviral therapy. The authors are
developing a highly sensitive, automated, kinetic amplification assay for the
detection and quantification of HBV
Method:
The kinetic amplification-based
HBV assay was performed using the VERSANT kPCR Molecular System*, which is
under development by Siemens Healthcare Diagnostics. The HBV assay includes HBV
primers and probe sequences, a noncompetitive internal control (IC) to monitor
the sample preparation process, and dUTP/UNG to eliminate chemical
contamination during a heat-labile
Results:
The assay dynamic range was 28 copies/mL to 1.0E+09 copies/mL in plasma or serum of
HBV-infected individuals, with a sensitivity of below 28 copies/mL of HBV
Conclusion:
The automated HBV kinetic
amplification-based assay currently in development demonstrated a wide dynamic
range of 28 copies/mL to 1.0E+09 copies/mL, with
sensitivity of 28 copies/mL. In addition, the assay performed with excellent
specificity and good precision. The incorporation of UNG eliminates the
potential for contamination up to 1.0E+9 amplicons per reaction. A
* System and assay not
commercially available
T. Tanwandee; P. Charatcharoenwitthaya;
V. Viboolsirikul; W. Chotiyaputta; S. Chainuvati; M. Maneerattanaporn; V.
Prachayakul; S. Pongprasobchai; S. Manatsathit; S. Leelakusolvong; N.
Pausawasdi; W. Srikureja; U. Kachintorn
Background:
Transient Elastography (TE) is a reliable
non-invasive predictor of hepatic fibrosis but data on TE in patients with
chronic hepatitis B (
Aim:
To prospectively evaluate
the accuracy of TE for diagnosis of hepatic fibrosis in patients with
Methods:
One hundred and four consecutive patients with
Results:
Patients had a mean age of 44 years and 63% were male. Mean body mass index at baseline (standard deviation) was 23.6 (4.2) kg/m2. The HBeAg was positive in 27 (26%) patients, and 77 (74%) patients were HBeAg-negative. Mean level of serum aspartate aminotransferase, albumin, and platelet count was 49 U/L, 4.4 g/dl, and 219,000/ml, respectively. The median liver stiffness was 6.9 kPa (range 3.3-46.4 kPa). The median liver stiffness measurement value correlated well with histological fibrosis grade (r=0.719, p<0.001) and good with necroinflammation activity (r=0.656, p<0.001). AUROC for diagnosis of significant fibrosis was 0.757 (95% CI: 0.66, 0.84) and advanced fibrosis was 0.793 (95% CI: 0.70, 0.87). Optimal liver stiffness value was 6.9 kPa for diagnosis of significant fibrosis, which yielded a sensitivity of 70%, specificity of 79%, positive predictive value (PPV) of 82%, and negative predictive value (NPV) of 66%. Optimal liver stiffness value was 7.3 kPa for diagnosis of cirrhosis, which provided a sensitivity of 93%, specificity of 61%, PPV of 31%, and NPV of 98%.
Conclusion:
Liver stiffness is a reliable predictor of
hepatic fibrosis in patients with
M. Wong; T. Poon; V. W. Wong; J. Yu; Y.
Chan; K. Yu; J. Sung; H. Chan
Most noninvasive predictive
models of liver fibrosis are complicated and have suboptimal sensitivity. Serum
proteomic fingerprinting by SELDI ProteinChip technology is a potential
approach for detecting liver fibrosis in patients with chronic hepatitis B
virus (HBV) infection. Unfortunately, the diagnostic SELDI peak cannot be
directly recovered for protein sequence analysis. To overcome this problem, a
similar mass spectrometry-based technology which is based on chromatographic
magnetic beads has been developed by our team. This technology allows
simultaneous analytic profiling and obtaining preparative fractions for
subsequent protein identification experiments. Serum samples were collected
from 100 treatment-naïve chronic HBV infected patients [76 males and 24
females; mean (SD) age, 35.0 (10.3) years] who underwent liver biopsy as per
protocol procedure for clinical trials. 57 patients had minimal fibrosis (Ishak
score = 0 or 1); 22 had mild fibrosis (Ishak score = 2); 9 had moderate
fibrosis (Ishak score = 3 or 4), 6 had severe fibrosis (Ishak score = 5); and 6
had probable/definite cirrhosis (Ishak score = 6). The mean alanine
aminotransferase, HBV
Linear regression method
(forward stepwise) was used to develop a diagnostic model for predicting the
degrees of liver fibrosis from the serum proteomic profiles, clinical data,
serological data, and biochemical data. Two proteomic features and platelet
counts were included in the final diagnostic model. Other factors including
liver biochemistries or HBV
Conclusion:
At a specificity of 89%, the
sensitivity was 67%, suggesting that combined use of serum proteomic
fingerprinting and platelet count could avoid about 65% of liver biopsy for
detection of liver fibrosis in the
975. Do Serum
J. Kim; J. Choi; J. Kwon; C. You; S.
Bae; S. Yoon; S. Cho; J. Yang; J. Han; N. Han; S. Choi; Y. Lee; C. Lee; E. Jung
Previous studies of
antiviral therapy in patients with decompensated cirrhosis showed that the nucleos(t)ide analog was well-tolerated and could improve clinical
outcomes. The aim of this study was to assess the relationship between
pretransplantational serum
Serum
Conclusion:
In conclusion, although the
patients with decompensated cirrhosis show normal
987. Clinical Usefulness of Liver Stiffness
Measurement in HBeAg-positive Chronic Hepatitis B Patients with
J. Choi; D. Kim; J. Park; S. Ahn; K. Yoon;
J. Lee; J. Kim; Y. Paik; K. Lee; B. Moon; C. Chon; K. Han
Background/Aims:
In patients with HBeAg
positive chronic hepatitis B (
Subjects
& Methods:
Liver biopsy and LSM were
carried out in patients with
Results:
Forty-eight patients
underwent liver biopsy and LSM. Their mean age was 41.7±13.3 years and 28
(58.3%) were men. The mean body mass index (
Discussion:
LSM using FibroScan® was
effective for predicting inflammatory activity and fibrosis in patients with
A. Kusakabe; Y. Tanaka; T. Yamaguchi; M.
Segawa; F. Kurbanov; M. Sugiyama; F. Sugauchi; M. Hijikata; K. Shimotohno; M.
Mizokami
Background:
We reported development of clinical hepatitis in a healthy individual with antibody to hepatitis B virus (HBV) surface antigen (HBsAg) and isolation of a strain with sG145R amino acid replacement (J Gastrol 2008), suggesting that mutations within the ‘a’ determinant may affect antigenicity and enable HBV to evade the neutralizing antibodies. Investigations on vaccine escape mutants (VSM) have been hampered by the absence of accessible in vitro infection modeling tools. Recently, we applied three-dimensional (3D) principle to culture immortalized human hepatocytes (HuS-E/2) and reported on ability of this system to maintain HBV infection and replication (AASLD 2007).
Aims:
To examine the infectivity of the sG145R
mutant in comparison to the wild type of the clone in the 3D culture system,
and to evaluate ability of PreS2 monoclonal antibody (anti-PreS2) (PNAS 1986)
to prevent the infection. Methods: The 3D culture system was prepared by
injecting HuS-E/2 cell suspension into hollow fiber bundles. The fibers were
then cultured in serum-free medium. A point mutation leading to sG145R
amino-acid substitution was engineered by site-directed mutagenesis on the
basis of 1.24-fold HBV/Ce genomic (C-AT wild) replicative construct, and
infectious virions were propagated in Huh7 cells transfected with the mutant or
wild construct. The culture supernatant aliquots containing different amount of
the virions (3, 2, 1 and 0 log copies) were inoculated into the 3D culture. In
infection-block experiment, infectious aliquots (2 log copies) were incubated
at 37 degrees C in presence of anti-PreS2 at different concentrations (1, 10,
20 and 40μg/mL) for 2 hours before inoculation into the 3D culture.
Anti-PreS2 was added every 3 days when culture medium was changed. HBV
Conclusion:
In the 3D culture system, we demonstrated that the infectivity of sG145R clone was comparable with that of C-AT wild and that anti-PreS2 might be protective against infections with VSM. The new modification of the 3D culture system using HuS-E/2 appears to be useful in examining of viral replication capacity and phylaxis ability of antibody.
850. Limited Value of Single
E. Heathcote; P. Marcellin; I. M. Jacobson;
M. L. Shiffman; H. N. Trinh; K. J. Peschell; D. Goodwin; J. Sorbel; E. A.
Fagan; F. Rousseau
Background/Aims:
Current practice guidelines for chronic
hepatitis B (
Methods:
Patients with ≥ 2
Results:
Group 1 and 2 patients were similar in age (~
40 yrs old) and Asian ethnicity (~ 40% Asian). Compared to Group 2, Group 1 patients
were less likely to be male (63% vs. 76%), and had lower Baseline HBV
Conclusion:
These results indicate that
846. Usefulness
of Serum Markers for Predicting Liver Cirrhosis in Patients with Chronic
Hepatitis B
K. Lee; Y. Seo; H. An; E. Jung; B. Keum; Y. Kim; H. . Yim; Y. Jeen; H. Chun; C. Kim; H. Ryu; S. Um
Background and Aims:
Liver biopsy is still considered as the gold standard method for the evaluation of liver cirrhosis. However, there are several limitations. It is an invasive method that is associated with patient discomfort and in some case with serious complication of bleeding and infection. In addition, its accuracy is limited by sampling errors and inter- or intra-observer variability. Recently, various serum markers were proposed for non-invasive methods for predicting liver fibrosis stage. However, most of the studies were performed about chronic hepatitis C patients and the studies about chronic hepatits B patients are insufficient. This study was performed to evaluate the significance of various noninvasive markers for predicting cirrhosis in chronic hepatitis B patients.
Methods:
Liver biopsy was performed in 125 chronic
hepatitis B patients. In this patients, WBC, platelet count, prothrombin time
(PT), AST,
Results:
Among 125 patients with chronic hepatitis B,
34 patients (27.2%) were diagnosed as liver cirrhosis on liver biopsy. Age,
platelet count, WBC count, AST,
Conclusion:
The PAHA index including platelet count, AST, haptoglobin and apo-A1 was useful for predicting liver cirrhosis in patients with chronic hepatitis B. The PAHA index might be useful for planning treatment and reducing the need for liver biopsy.
973. Usefulness of Liver Biopsy in Chronic
HBV with
T. Goebel; U. Heinzel-Pleines; C. Poremba; S. E. Baldus; M. Herwig; M. Molderings; D. Haussinger; A. Erhardt
Introduction:
In chronic HBV-infection
indication for antiviral treatment depends on
Patients
and Methods:
We retrospectively analysed
consecutive 252 patients with chronic, replicative hepatitis B who underwent
liver biopsy. Patients with HIV/HDV or HCV-coinfection were excluded. Normal
transaminases were defined as
Results:
Of the 252 patients, 214 had elevated and 38 normal
Discussion:
The present study shows that in patients of a non-Asian origin with normal or
slightly elevated transaminases, advanced liver fibrosis or even liver
cirrhosis is frequently found. Therefore, liver biopsy should be considered
even in patients with normal or near normal transaminases.
|
Parameter |
Normal |
(n=84) |
(n=130) |
|
Significant liver fibrosis [≥F3] |
16/38 (42%) |
45/84 (54%) |
82/130 (63%) |
|
Liver cirrhosis |
9/38 (24%) |
21/84 (25%) |
28/130 (22%) |
|
Significant inflammation [≥G2] |
7/27 (26%) |
34/66 (52%) |
71/104 (68%) |
|
HBeAg-positivity |
17/38 (45%) |
24/84 (29%) |
61/130 (47%) |
|
Male/Female [%] |
53/47 |
73/27 |
79/21 |
|
HBV- |
1782101 |
788698 |
1869311 |
Y. Seo; E. Jung; H. An; B. Keum; Y. Kim; H. Yim; Y. Jeen; H. Chun; C. Kim; H. Ryu; S. Um
Background and Aims:
Various non-invasive methods were reported to
be very useful for predicting fibrosis stage in patients with chronic hepatitis.
However, most published studies focused on the patients with chronic hepatitis
C. Therefore, it is still uncertain which non-invasive method is most accurate
for fibrosis assessment in patients with chronic hepatitis B (
Methods:
We enrolled patients with
Results:
Eighty-five patients with
Conclusions:
LSM was useful method for predicting
significant fibrosis in patients with
840. Is Liver Biopsy
Required for All Patients (pts) with HBeAg-negative (HBeAg-) Chronic HBV
Infection and HBV
G. V. Papatheodoridis; E. K. Manesis; S. Manolakopoulos; I. Elefsiniotis; I. Giannousis; G. Kafiri; A. J. Archimandritis
Background/Aim:
The optimal HBV
Methods:
From 9/2001-12/2007, liver biopsy was
performed in 105 HBeAg- pts with increased
Results:
There was no significant difference in any
characteristic between Group A1 and A2, except for a trend for worse fibrosis
(stage:2.8±1.7 vs 2.2±1.7, P=0.087) or more frequent
severe fibrosis/cirrhosis (stage 4-6:37% vs 19%, P=0.079) in group A1 than A2
pts. Group A compared to Group B pts did not differ in age, sex,
Conclusions:
Our results demonstrate that a) there are no
substantial differences between HBeAg- chronic HBV pts with elevated
958. Quantitative HBsAg Titer Correlates Well
with HBV
T. Su; C. Hsu; C. Liu; Y. Huang; C. Liu;
P. Chen; J. Kao; D. Chen
Background
and Aims:
Serum HBV
Methods:
A total of 209 chronic
hepatitis B patients without previous anti-HBV treatment were enrolled as
treatment-naïve group. Serum HBsAg was quantified by a commercial kit
(ARCHITECT, Abbott laboratories,
Results:
In treatment-naïve group, a
Pearson correlation coefficient of 0.59 (p<0.01) was found between log
(HBsAg) and log (HBV
Conclusions:
Quantitative HBsAg assay is a useful and economic tool
to monitor serum HBV
Children
923.
High Prevalence of HBV-
S.
Gehring; T. Gumbrich; F. Zepp; U. Kullmer
Introduction
& Aim:
The goal of therapy for chronic hepatitis B virus
(HBV) infection in children is to eliminate HBV replication and prevent the
progression of liver disease to cirrhosis. Interferon-alpha (IFN-α) and
Lamivudine (LMV) have been approved for treatment in children, Adefovir is
currently investigated in a multi-centre trial. In western countries, treatment
with IFN-α has been found to lead in up to 58% of children to the loss of
HBV-
Methods:
Forty pediatric patients ages 4 – 22 years (average 13,4) were evaluated. 24/40 children underwent a treatment
with IFN-α. 8/40 children received treatment with LMV. Patients were
monitored with liver function tests (LFT), complete blood cell count,
auto-antibody screening (LKM,
Results:
Eighty percent of the children in our study group were
infected by neonatal transmission. Ethnic background included 23
South/Eastern-European (
Conclusions:
Key elements of a successful treatment in HBV infected
individuals (sustained response) are seroconversion from HBeAg to anti-HBe and
/ or loss of HBV-