1460.
Differential Role of
Liver-targeted Regulatory T Cell to Hepatitis B and C
Virus in Chronically Infected Patients
H. Miyaaki; T. Ichikawa; K. Nakao; H. Shibata; M. Akiyama; S. Miuma; Y.
Motoyoshi; M. Fujimoto; K. Eguchi
Regulatory T cells play a critical role in chronic
virus infection. The role of regulatory T cell in chronic hepatitis B and
chronic hepatitis C is unknown. We examined the distribution and frequency of
Foxp3 positive regulatory T cell in the liver tissues and compared the
clinicopathlogical characters of HBV and HCV.
Methods
Liver needle biopsies were collected from 26 patients
with hepatitis B surface antigen positive (mean age:33.6±10.4,
male: female=23:3) and 27 patients with hepatitis C virus antibody positive
(mean age 57.8±10.6, male: female=
In HBV cases, there was significant correlation
between the ratio of Foxp3 in CD3 and serum ALT level (P=0.025, r=0.402). The
ratio of Foxp3 in CD3 was significantly increase in severe activity group than
in mild activity group (mild:severe=0.08±0.03:0.11±0.04, P=0.04). There were no
significant differences in the ratio of Foxp3 in CD3 in HBV cases among the
other clinical factors (age, gender, platelet, fibrosis, HBV-DNA viral load).
In HCV cases, the variables that were significantly associated with the ratio
of Foxp3 in CD3 were serotype2 (serotype1: serotype2=0.07±0.04:0.15±0.05,
P=0.0002) and male gender (male:female = 0.07±0.03:0.11±0.05, P=0.04). These
factors were easy sensitivity factors for interferon therapy. There were no
significant differences in the ratio of Foxp3 in CD3 in HCV cases among the other
clinical factors (age, ALT level, platelet, activity, fibrosis, HCV viral
load).
Conclusion
In conclusions, these data provided the impaired of
regulatory T cell function cause acute exacerbation in chronic hepatitis B. On
the other hand, regulatory T cell may be related with action of interferon in
chronic hepatitis C.
1467.
Quantitative Detection of
Integrated HBV Surface Gene Sequences in the Liver by Real-Time PCR
A. Laras; A. Kostamena; S. J. Hadziyannis
Introduction
Chronic hepatitis B (CHB) patients
under long-term nucleos(t)ide analogue treatment may achieve profound HBV
suppression with non-detectable serum HBV DNA and low intrahepatic cccDNA
levels. Continuing surface antigen expression observed in these patients could
be attributed to transcribed integrated HBV DNA harbouring intact surface
antigen (S) gene sequences.
Aim
To develop a quantitative
method for the detection of integrated HBV DNA harbouring S sequences in the
liver of CHB patients under suppressed HBV replication and to evaluate its
performance in liver biopsy samples of patients under long-term antiviral
therapy.
Methods
Liver biopsy specimens from
20 CHB patients in profound HBV suppression under nucleos(t)ide analogue
treatment were tested for integrated HBV S gene sequences using specifically
designed real-time PCR assays that simultaneously measure intrahepatic HBV DNA
in the S and the core promoter-precore (CP-preC) regions. HBV integration
occurs predominantly in the CP-preC region of the viral genome leaving S sequences
frequently intact. Cloned and serum HBV DNA were used as controls. Intrahepatic
HBV cccDNA was also measured. HBsAg and HBcAg expression was also assessed in
the same biopsy samples by immunohistochemical methods.
Results
Low HBV cccDNA levels were
found in all patients, median 0.31 (range 0.05-0.84) copies/cell. In fact,
cccDNA was the predominant form of viral DNA representing on the average 79% of
total intrahepatic HBV DNA. These conditions permitted quantitation of low copy
integrated HBV sequences. Total intrahepatic HBV DNA levels measured in the S
region were significantly higher than in the CP-preC region, 0.70 (0.10-2.65)
vs 0.38 (0.05-1.74). In 11/20 patients, as well as in cloned and serum HBV DNA
controls, there was no discrepancy between the two measurements. In 9/20
patients S levels were significantly higher (2-fold to 13-fold) than the
corresponding CP-preC measurments (0.71 vs 0.20). In these patients, integrated
HBV S sequences were detectable in significant levels, 0.51 (0.17-1.4) cp/cell.
Consistent with these findings were the results of histochemical demonstration
of HBsAg proteins in the same liver samples.
Conclusions
1. We have developed a
method for quantitative detection of integrated HBV DNA harbouring S gene sequences
in liver biopsy samples. 2. This method can only be used in situations of
highly suppressed viral replication permitting detection of low copy integrated
S sequences. 3. Integrated HBV S sequences were detectable in approximately 50%
of treated patients with compatible findings in immunohistochemical
observations of HBsAg expression in the liver.
146. Chronic HBV
Evolution Is Associated with Numeric and Phenotypic Changes in Peripheral
HBV-core Epitope-Specific CD4+ T-cells: A Study Using a Novel HBV-core-Specific
HLA-DRB1*0101 Tetramer for the Analysis of Antiviral CD4+ T-cell Responses
During Acute and Chronic Hepatitis B
M. H. Heeg; A. Ulsenheimer; N. H. Gruener; H. M. Diepolder; W. Schraut;
M. Waechtler; R. Zachoval; G. R. Pape; M. C. Jung
A strong HBV-specific CD4+ T cell response is
associated with spontaneous viral clearance in acute hepatitis B but is weak or
short-lived in patients developing chronic disease. So far, this CD4+ T-cell
response could just be investigated by functional assays which lack the
possibility of further T-cell phenotyping and that are unable to identify
dysfunctional cells. HBV-specific HLA-class-II tetramers now can negotiate
those limitations.
Method
To establish a new HBV-core MHC-class-II tetramer we
screened 33 patients with acute HBV infection by overlapping HBV-core peptides
and identified a HLA-DRB1*0101 restricted HBV-core epitope. This epitope was
recognized by all DRB1*0101 patients with acute HBV infection as well as by 77
% (17/22) of all patients with acute HBV. Using this epitope we assembled a new
DRB1*0101 MCH-class-II tetramer. The tetramer specifically stained a
corresponding CD4+ T cell clone. To proof the tetramer sensitivity dilution
experiments of Tet+ clone cells into PBMC were performed and a strong
correlation between added and measured frequencies was found. In six
HBV-negative DR1+ individuals and nine patients with acute hepatitis B lacking
DR1, no CD4+ cells were tetramer positive.
In three HLA-DR1+ patients with acute hepatitis B, HBV-specific
CD4+ T cells were detectable during the acute phase of disease, ranging from
900 to 1680/106 CD4+ T cells during the peak phase. The peak phase was
associated with an ALT peak and viral clearance as well as with a peak level of
IFN-γ secretion. The frequencies of HBV specific CD4+ T-cells decreased
rapidly after viral clearance but were detectable in considerable levels over
several months. In contrast, at different time points of disease 15
investigated patients with chronic hepatitis B showed no or only very low
frequencies of tetramer positive cells. During the course of acute self
limiting hepatitis B, phenotypic characterization demonstrated a rapid increase
of IL7R and CD62L expression on Tet+/CD4+ T-cells.
Conclusion
In conclusion, using a new MHC-class-II tetramer
containing one immunodominant HLA-DRB1*0101 restricted epitope within HBV-core,
we were able to quantify and characterize HBV specific CD4+ T cell responses
patients with acute hepatitis B and chronic hepatitis B. In patients with
chronic disease almost no Tet+/CD4+ T-cells were found indicating a complete
loss of antiviral CD4+ T-cells rather than a loss of function of those cells.
Furthermore those cells showed an impaired capacity to develop a memory
phenotype. So far, tools that allow to distinguish between the absence of
specific cells or their non-function were not available.
87. Dual
Over-expression of Insulin Receptor Substrate-1 and Hepatitis Bx Gene Causes
Pre-malignant Changes in Liver
L. Longato; S. de la Monte; N. Kuzushita; M. Horimoto; B. Slagle; A. Rogers; J. R. Wands
Background
The cellular changes that contribute to the
development of hepatocellular carcinoma (HCC) are poorly understood. Activation
of the IN/IRS-1/Ras/Raf/MAPK and the Wnt Frizzled/β-catenin signaling
cascades is frequent in HCC related to persistent hepatitis B viral (HBV) and
hepatitis C viral (HCV) infection. The aims of this study were to recapitulate
these findings in an animal model system by providing a proliferative stimulus
via IRS-1 in the context of HBx protein over-expression in transgenic mice. The
purpose was to determine if alteration in these genes is sufficient to produce
dysplasia and cellular transformation in the normal liver.
Methods
We generated transgenic mice in which the HBx (ATX;
N=16), IRS-1 (N=15) or both (ATX+/IRS-1+; N=23) genes were over-expressed under
a liver specific promoter. Wild-type litter mates were included as controls
(N=11). We also assessed oxidative damage as well as upregulation of signaling
molecules related to these signal transduction cascades in the liver by “real
time” RT-PCR. Histologic analysis included an assessment of lobular and
periportal inflammation, macro and microsteatosis, polyploidy, dysplasia and
tumor formation.
Results
ATX+/IRS-1+ double transgenic livers had increased
frequency of hepatocellular dysplasia and several developed HCC (N = 3). IRS-1+
and ATX+/IRS-1+ livers had more extensive hepatic steatosis compared to the
other groups. All three transgenic lines had significantly increased IRS-1,
IGF-1, proliferating nuclear cell antigen, Wnt 1 and Wnt 3 mRNA levels, and
increased immunoreactivity for 8-OHdG and HNE indicative of DNA damage and
oxidative stress. The ATX+/IRS+ double transgenic mice were distinguished by
having the highest level of activation of Wnt 3 and Frizzled 7 and selectively
increased expression of IGF-II and aspartyl (-asparaginyl)-β-hydroxylase
(AAH) a downstream insulin responsive gene associated with increased cell
motility and migration.
Conclusion
These results suggest that over-expression of HBx or
IRS-1 can contribute to hepatocyte transformation by promoting cell turnover
and DNA damage but these effects are not sufficient to trigger neoplastic
changes in the liver. However, chronic over-expression of HBx together with
IRS-1 is sufficient to cause hepatocellular dysplasia and HCC in vivo
suggesting that upregulation of both the IN/IRS-1/MAPK and Wnt/β-catenin
signaling cascades are important in the transformation of normal hepatocytes to
the malignant phenotype.
U. Zaid; Z. Zhang; T. Liang
The X protein (HBX) of the hepatitis B virus (HBV) is
important for productive infection in vivo. Although this viral protein is not
essential for viral replication in vitro, HBV negative for HBX replicates less
efficiently in transfected cells. Our previous studies have suggested that the
interaction between HBX and the proteasome complex may underlie the pleiotropic
functions of HBX (Hu et al. JVI 2006; 8-: 1405-1413, Zhang et al. JCI 2001;
108: 1523-1531, Zhang et al. JBC 2000; 257: 15157-15165, Zhang et al. JVI 2004;
78: 4566-4572). We have further demonstrated that inhibition of cellular
proteasome activities enhances hepadnavirus replication in an HBX-dependent
manner.
Aim
In this study, we aim to develop potential anti-HBV
agents by targeting the functions of HBX using a novel screening process.
Methods
A modified yeast two-hybrid disruptor system was
developed to screen a randomly generated library of peptide aptamers displayed
on the bacterial protein Thioredoxin A (TrxA). By screening for a disruption of
the interaction between HBX and the proteasome subunit PSMA7, 367 yeast transformants
were isolated from 1.5 x 10^7 independent yeast colonies of the peptide aptamer
library. On secondary screen against nonspecific interacting pairs, 23 colonies
were confirmed to show specific disruption of the HBX-PSMA7 interaction. The
peptide aptamers from these colonies were isolated, sequenced, and cloned into
a plasmid expression construct for transfection into HepG2 cells for functional
studies. Transcriptional activation assays by HBX demonstrated that most of
these peptide aptamers interfered with HBX transactivation by decreasing the
reporter activities to 20-80% of the control level. When co-transfected with an
HBV replication competent construct, most of the peptide aptamers which
inhibited HBX transactivation also suppressed HBV viral replication by 50-80%.
Very preliminary in vivo murine studies via intravenous injection of one of the
peptides suggest a beneficial effect on inhibition of HBV DNA in HBV transgenic
mice. Further studies are required to confirm this observation.
Conclusion
Our results demonstrate that selection of random
peptide aptamers based on disruption of the HBX-proteasome interaction using a
modified yeast two-hybrid system may identify peptide aptamers as potential
therapeutic drugs for HBV infection. Also, this system may provide a molecular
and structural basis for novel antiviral drug development.
S. Akbar; O. Yoshida; S. Furukawa; Y. Hiasa; N. Horiike; M. Onji
Dendritic cells (DCs) loaded with HBV-related antigens
induce HBV-specific immunity in HBV-transgenic mice. However, the utility of antigen-pulsed
DCs have not been assessed in human in the context of HBV infection. This study
was conducted (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human
blood DCs, (2) to evaluate their safety and (3) to assess their prophylactic
and therapeutic efficacies against HBV.
Methods
Six hepatitis B vaccine nonresponders, who never
developed antibody to HBsAg (anti-HBs) due to immunization with commercial
vaccines for 6-9 times and 5 patients with chronic hepatitis B were enrolled in
this study. Immature blood DCs were prepared by culturing peripheral blood
mononuclear cells with human grade granulocyte-macrophage colony stimulating
factor and interleukin-4 for 7 days. HBsAg-pulsed DCs were prepared by
culturing immature DCs with HBsAg (Hepatvex II, Banyu Pharmaceutical Co, Tokyo,
Japan) for 8 hours. Five million HBsAg-pulsed DCs were administered to each
individual and parameters of generalized inflammation, liver functions, kidney
functions, and autoantibodies were checked at day 0, 1, 3, 7, 14, 28, 90, and
180 and after 2 years. HBsAg-specific T cell responses and anti-HBs were
assessed in all subjects before and after administration of HBsAg-pulsed DCs.
Results
HBsAg-pulsed human blood DCs expressed DC-related
markers such as HLA DR, CD86 and CD40. In vitro, HBsAg-pulsed DCs induced
proliferation of HBsAg-specific T cells and anti-HBs from HBsAg-specific B
cells. Hepatitis B vaccine nonresponders or patients with chronic hepatitis B
did not develop any adverse effects due to administration of HBsAg-pulsed DCs.
All vaccine nonresponders and patients with chronic hepatitis B did not show
anti-HBs or HBsAg-specific T cells before administration of HBsAg-pulsed DCs.
Anti-HBs and HBsAg-specific T cells were detected in the peripheral blood of
all vaccine nonresponders within 28 days after single administration of
HBsAg-pulsed DCs. HBsAg-pulsed DCs were safe for all patients with chronic
hepatitis B. Three and 2 of these patients exhibited HBsAg-specific T cells and
anti-HBs in the peripheral blood, respectively.
Conclusions
This study has opened a new field of immune
intervention strategy against HBV infection in human in which human consumable
HBsAg-pulsed DCs have been prepared in vitro and their safety and efficacy have
been confirmed in vivo. The principle of this study may be used for development
of cell-based prophylactic and therapeutic vaccines against hepatitis C virus
and other viruses when those cannot be achieved by traditional approaches.
I. E. Vincent; J. Lucifora; I. Chemin; D. Durantel; F. Zoulim; C. Trepo
Background
and Aim
CpG oligodesoxynucleotides (CpG ODN) are synthetic
agonists of Toll-like receptor 9 (TLR9) and potent inducers of innate and
acquired immunity. Antiviral mechanisms of CpG ODN include induction of
antiviral and pro-inflammatory cytokines (INF-α, IL-6), activation of
natural killer cells (INF-γ), thereby promoting strong TH1, CD8 T cell and
humoral responses. Current therapy of chronic hepatitis B infection, either
interferon alpha or nucleoside analogs, fail to control hepatitis B virus (HBV)
replication in most patients. This limitation is mainly due to the slow kinetic
of viral clearance, HBV genetic variability and weak or absent anti-viral
cellular immunity. This provides the rational for the development of
immunotherapeutic approaches combining nucleosid analogs and TLR9 agonists to
accelerate viral clearance and restore anti-HBV specific immune responses.
Our aim was therefore to evaluate the antiviral effect
of CpG-induced cytokines combined with lamivudine on HBV replication in vitro.
Methods
HepaRG cells were infected with purified HBV virions
or transduced with HBV recombinant baculovirus. Peripheral blood mononuclear
cells from naïve individuals were stimulated with CpG ODN and cytokine
secretion (INF-α, INF-γ, IL-6) determined by Elisa. HBV-infected or
-transduced HepaRG cells were treated with CpG-induced cytokines in combination
with lamivudine. Antiviral effects were evaluated 48h post-treatment by
quantification of antigen secretion (HBsAg, HBeAg) and HBV DNA intermediates of
replication by Southern blot.
Results
CpG-induced cytokines inhibited HBsAg and HBeAg
production from infected HepaRG cells. The antiviral effect was demonstrated by
up to 90% inhibition of intracellular HBV DNA intermediates in HBV
transduced-cells. More importantly, the combination of CpG-induced cytokines
with lamivudine reduced by one hundred fold the lamivudine IC50. Overall,
combination was more effective than CpG-induced cytokines or lamivudine
treatment alone.
Conclusion
CpG-induced cytokines with lamivudine represent a
powerful combination to inhibit HBV replication in vitro, most likely by
inhibiting different steps of viral replication. The use of TLR9 agonists
combined with nucleoside analogs should be evaluated in vivo in order to assess
restoration and duration of anti-HBV specific immune responses. The development
of such novel immunotherapeutic combinations provides promising approaches to
decrease the risk of liver disease progression in chronic hepatitis B patients.
A. C. Jenke; V. Orth; H. Lipps; S. Wirth
Introduction
and Aim
In the past, it has been shown that RNA interference
(RNAi) has the potential to suppress hepatitis B virus (HBV) replication. For
further gene therapy an optimal vector system free of common side effects as
for example induction of mutagenesis has still to be found. The aim of this
study was to test the non viral episomal vector system pEPI-1 as a tool for
inhibition of HBV replication.
Methods
Recombinant vectors pEPI/HBV1 and pEPI/NonSense were
constructed and transfected into HepG2.2.15 cells. After selection of
transfected clones, cells were grown for 3 months in the absence of selection
pressure and harvested at 2 and 12 weeks. The episomal status of the plasmid
was confirmed by Southern analysis. The level of HBsAg mRNA was measured by
RT-PCR and the concentration of HBsAg in the supernatant was determined using
ELISA.
Results
Transfection of HepG2.2.15 cells with pEPI/HBV1
resulted in more than 90% suppression of HBsAg mRNA 2 weeks after transfection
compared with the control plasmid pEPI/NonSense. This effect was even more
pronounced after 12 weeks, when almost no HBsAg mRNA could be detected.
Likewise, the secretion of HBsAg was inhibited by more than 95%. Also the
number of DNA copies within the culture medium was significantly decreased.
Conclusion
The non viral episomal vector system pEPI-1 allows
efficient long term suppression of HBV replication in the absence of selection
pressure and without the risk for insertional mutagenesis or expression of
additional viral proteins.
T. Liu; H. You;
M. Cong
Rep78, the large rep gene product of adeno-associated
virus (AAV), is a highly multifunctional protein, which is required for AAV DNA
replication and the trans-regulation of AAV gene expression. Apart from these
essential functions prerequisite for the life cycle of AAV, Rep78 is able to
inhibit the replication and gene expression of some DNA virus by binding to the
promoter, such as HPV and HIV. The aim of this study was to determine whether
Rep78 can bind to the promoter of HBV DNA and then inhibit the replication of
HBV DNA.
Methods and
Results
Here, we demonstrated that Rep78, as a chimeric with
the maltose binding protein, binds to both the full length of HBV X promoter
and the full length of HBV C promoter, as analyzed by electrophoretic mobility
shift assay. To map the key region of binding, sequentially smaller substrates
from the full length of promoter were synthesized and tested for recognition by
Rep78. It is demonstrated that the binding between Rep78 and the full length of
HBV X promoter was specific, but not with Rep78 and the smaller substrates from
the full length. It was therefore possible that multiple binding sites were
needed or the secondary structure of the full length of HBV X promoter was
needed for specifically binding with Rep78. The smaller substrates from the
full length, B1, the active region of HBV C promoter can bind to Rep78
specifically. Furthermore, investigating the biological implications of these
interactions, Rep78 inhibited both the HBV X promoter and HBV C promoter
activity in vitro transcription assays in Hela nuclear extracts, and the
inhibition of X promoter was stronger than that of C promoter. In addition, to
investigate whether the binding between Rep78 and HBV DNA promoters could
inhibit the replication of HBV DNA, we constructed the plasmid containing the
AAV2 rep78 gene and transfected the plasmid to the HepG2.2.15 cell line, the
concentration of HBV cccDNA was decreased indicating that the replication of
HBV was inhibited.
Conclusion
The ability of Rep78 to interact with the HBV X
promoter and HBV C promoter and down-regulate corresponding transcription and
replication suggests the mechanism by which AAV may modulate the HBV life
cycle.
D. Xi; S. Gao;
L. Li; C. Zhu; J. Guo; X. Luo; Q. Ning
Background
and Aims
Our previous studies have shown an anti-sense plasmid
to mouse fgl2 prothrombinase (mfgl2) gene significantly reduced mfgl2 gene
expression in vivo, markedly ameliorated inflammatory infiltration, fibrin
deposition and hepatocyte necrosis, prolonged the survival time period and
elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3
(MHV-3) induced fulminant hepatitis. This study was designed to explore the
opportunity of RNA interference technique in the inhibitory application of
human fgl2 prothrombinase (hfgl2) expression, which has been reported to be
involved in a variety of disease development including fulminant viral
hepatitis, acute rejection of allo/xeno transplantation and fetal loss
syndrome.
Methods
A plasmid named p-hfgl2shRNA complementary to the
sequence of hfgl2 was constructed, meanwhile irrelevant shRNA plasmid with a
random combination of the hfgl2shRNA sequence was used as control. A plasmid
named pEGFP-hfgl2 expressing hfgl2-EGFP fusion protein was also constructed for
the screening of the effect of p-hfgl2shRNA on the hfgl2 expression. By
cotransfection of p-hfgl2shRNA and pEGFP-hfgl2 or pcDNA3.1-hfgl2 expression
construct into CHO cells, the inhibition of hfgl2 expression by hfgl2shRNA was
analyzed by direct observation through fluorescent microscopy, FACS, real time
PCR and immunohistochemistry staining. Results: The experiments showed the
significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48h
post-transfection by observation of green fluorescent cells, FACS, real time
PCR and immunohistochemistry staining as well in CHO cell lines with the
inhibitory efficiency reached as high as 85.5%.
Conclusion
The study demonstrated that the construct of
p-hfgl2shRNA successfully interferred hfgl2 expression in vitro and this
provides the foundation for further investigation of this construct’s application
in vivo and further more as a therapeutic strategy for a targeting intervention
in the disease control to which the gene fgl2 contributed. This work was
supported by the NSFC30571643,30672380 and National Key Basic Research Program
of China(2005CB522901).
M. H. Heeg; A.
Ulsenheimer; N. H. Gruener; H. M. Diepolder; W. Schraut; M. Waechtler; R.
Zachoval; G. R. Pape; M. C. Jung
A strong HBV-specific CD4+ T cell response is
associated with spontaneous viral clearance in acute hepatitis B but is weak or
short-lived in patients developing chronic disease. So far, this CD4+ T-cell
response could just be investigated by functional assays which lack the
possibility of further T-cell phenotyping and that are unable to identify
dysfunctional cells. HBV-specific HLA-class-II tetramers now can negotiate
those limitations.
To establish a new HBV-core MHC-class-II tetramer we
screened 33 patients with acute HBV infection by overlapping HBV-core peptides
and identified a HLA-DRB1*0101 restricted HBV-core epitope. This epitope was
recognized by all DRB1*0101 patients with acute HBV infection as well as by 77
% (17/22) of all patients with acute HBV. Using this epitope we assembled a new
DRB1*0101 MCH-class-II tetramer. The tetramer specifically stained a
corresponding CD4+ T cell clone. To proof the tetramer sensitivity dilution
experiments of Tet+ clone cells into PBMC were performed and a strong
correlation between added and measured frequencies was found. In six
HBV-negative DR1+ individuals and nine patients with acute hepatitis B lacking
DR1, no CD4+ cells were tetramer positive. In three HLA-DR1+ patients with
acute hepatitis B, HBV-specific CD4+ T cells were detectable during the acute
phase of disease, ranging from 900 to 1680/106 CD4+ T cells during the peak
phase. The peak phase was associated with an ALT peak and viral clearance as
well as with a peak level of IFN-γ secretion. The frequencies of HBV
specific CD4+ T-cells decreased rapidly after viral clearance but were detectable
in considerable levels over several months. In contrast, at different time
points of disease 15 investigated patients with chronic hepatitis B showed no
or only very low frequencies of tetramer positive cells. During the course of
acute self limiting hepatitis B, phenotypic characterization demonstrated a
rapid increase of IL7R and CD62L expression on Tet+/CD4+ T-cells.
Conclusion
In conclusion, using a new MHC-class-II tetramer
containing one immunodominant HLA-DRB1*0101 restricted epitope within HBV-core,
we were able to quantify and characterize HBV specific CD4+ T cell responses
patients with acute hepatitis B and chronic hepatitis B. In patients with
chronic disease almost no Tet+/CD4+ T-cells were found indicating a complete
loss of antiviral CD4+ T-cells rather than a loss of function of those cells.
Furthermore those cells showed an impaired capacity to develop a memory
phenotype. So far tools that distinguish between the absence of specific cells
or their non-function were not available.
H. L. Janssen;
J. N. Stoop; A. M. Woltman; R. S. Binda; J. G. Kusters; P. E. Zondervan; E. J.
Kuipers; R. G. van der Molen
CD4+CD25+FoxP3+ regulatory T cells (Treg) play a key
role in the impaired immune response observed in patients with a chronic
hepatitis B virus (HBV) infection. Most studies concerning Treg and HBV are
performed with cells isolated from peripheral blood, while the liver is the
main site of infection. To gain more insight in the role of Treg in chronic HBV
we have monitored intra-hepatic Treg.
Methods
Peripheral blood and liver biopsy samples were
obtained from 40 patients with a chronic HBV infection. The liver tissue was
collected in culture medium and was digested with collagenase for 15 minutes to
create a single cell suspension. The percentage of Treg was determined by
flowcytometry using antibodies to CD4, CD25 and FoxP3.
Results
The results show that patients with a high viral load
of >1 x 105 geq/mL have a significantly higher percentage of intra-hepatic
Treg as compared to patients with low viral load <1 x 105 geq/mL (8.20 % ±
0.50% vs. 6.18% ± 0.54% respectively; p< 0.05), while the percentage of peripheral
blood Treg was similar in both groups (9.38% ± 0.39% vs. 8.94% ± 0.43%
respectively). When patients were divided into two groups based on liver
inflammation, indicated by their serum ALT levels (ALT < 2 x ULN and ALT
> 2 x ULN) no differences in percentages were observed between the two
patient groups for intra-hepatic Treg (7.61% ± 0.56% vs. 7.03% ± 0.52%
respectively) as well as for peripheral blood Treg (9.34% ± 0.31% vs. 8.89 ±
0.60% respectively). There was also no relation observed between the proportion
of Treg and the Metavir Fibrosis score. Next to CD4+CD25+FoxP3+ Treg the liver
also contained a large population of CD4+CD25-FoxP3+ cells (2.40% ± 0.21%
(intra-hepatic) vs. 0.89 % ± 0.06% (peripheral blood); p < 0.05).
Additionally, approximately 40% of liver Treg expressed the immunoregulatory
marker programmed death-1 (PD-1) while peripheral blood Treg hardly express
PD-1. PD-1 expression does not seem to be specific for Treg, since the liver
contains more PD-1+CD4+ T cells compared to peripheral blood.
Conclusion
Liver CD4+ T cells have a higher expression of the
immunoregulatory receptor PD-1. There was no relation between previous liver
damage or liver inflammation and the presence of Treg. On the other hand, an
increased proportion of intra-hepatic Treg was observed in patients with a high
viral load, suggesting the importance of Treg in HBV tolerance and persistence
of viral replication.
O. Yoshida; S. Akba; T. Miyake; M. Hamada; Y. Hiasa; K. Michitaka; M.
Onji
Purpose
The magnitudes of both innate and adaptive immunities
are decreased in chronic infections and cancers. Accordingly, immune therapy
against these diseases have been formulated to activate innate immunity
targeting natural killer (NK) cells and adaptive immunity targeting dendritic
cells (DC). This study was conducted to develop insights about crosstalk
between NK cells and DCs and to assess if antigen-pulsed DCs can activate both
innate and adaptive immunity.
Methods
Both normal C57BL/6 mice and hepatitis B virus
transgenic mice (that express hepatitis B surface antigen (HBsAg), HBV DNA, and
hepatitis B e antigen (HBeAg) are used. NK cells were depleted by
administrating anti-asialo GM1 antibody, 100 microliter/mouse, intraperitoneal,
twice at an interval of 48 hours. Normal C57BL/6 mice and NK-depleted mice were
immunized with hepatitis B vaccine containing HBsAg, intraperitoneally and the
levels of antibody to HBsAg (anti-HBs) and HBsAg-specific T cells were
estimated 28 days after immunization. Spleen DCs from normal C57BL/6 mice and
hepatitis B virus transgenic mice were cultured with HBsAg to prepare
HBsAg-pulsed DCs and administered to NK-depleted mice, twice, at an interval of
2 weeks. We assessed if HBsAg-pulsed DCs activated innate immunity in NK-depleted
mice by inducing proinflammatory cytokines (interleukin-12p70,
interferon-gamma, tumor necrosis factor-alpha, and interleukin-6), 3 days after
administration of HBsAg-pulsed DCs. Also, we evaluated if HBsAg-pulsed DCs
induced anti-HBs and HBsAg-specific lymphocytes in NK-depleted mice.
Results
Flow cytometric analyses revealed that injection with
anti-asialo GM1 in mice resulted in depletion of >90% NK cells from liver,
spleen and bone marrow. Compared to normal C57BL/6 mice, DCs from NK-depleted
normal C57BL/6 mice produced significantly lower levels of all proinflammatory
cytokines (p<005) and could not activate HBsAg-specific lymphocytes in vitro
indicating that NK cells are essential for proper functioning of DCs. High
levels of anti-HBs were detected in the sera of all normal mice (10 of 10,
100%), but very low levels of anti-HBs were found in only 2 of 10 NK-depleted
mice due to immunization with HBsAg (p<0.05). Also, the levels of
HBsAg-specific T cells were significantly lower in NK-depleted mice compared to
those in normal C57BL/6 mice (p<0.001). Administration of HBsAg-pulsed DCs
induced higher levels of all proinflammatory cytokines, anti-HBs and
HBsAg-specific lymphocytes in NK-depleted mice.
Conclusion
This study provides the rationale for using
antigen-pulsed DCs for treatment of chronic infections when both innate and
adaptive immunities are impaired.
N. T. Pati; S. Bose; S. Baveja; H. Syed; S. K. Sarin
Background
The Notch signaling plays a key role in cell-fate
determination and differentiation, functioning in a cell- and tissue-specific
manner. Both up-regulated and down-regulated Notch 1 signaling is seen in
carcinogenesis of different tissues. Whereas, in stem cells, notch activity
inhibits differentiation and promotes carcinogenesis but in primary epithelial
cells high notch activity leads to exit from the cell cycle and commitment to
differentiation. Notch 1 exerts specific protective effects against HPV-virus
and down-modulation of Notch 1 was observed in the late stages of HPV-induced
carcinogenesis.
Aim
To study notch signaling in different stages of HBV
induced liver disease and its role in liver disease progression. Patients and
Methods: PBMCs of chronic hepatitis B (CHB) (n=10) (age 29.4±13.2 yr.,
M:F::8:2) patients and healthy controls (n=10) (age 27.9 ± 4.6 yr., M: F ::7:3)
and liver biopsies from normal liver – 3 (age 49±3.8 yr.M:F::2:1), CHB patients
(age 39±12.8, M:F::3:1) HBV cirrhosis -4 (age-36.2±20.0, M:F::3:1) and HBV
related HCC –3(age-55.5±7.2 yr., M:F::3:0) were studied. Total RNA was
extracted from PBMCs and biopsies and Notch1, Hes1, Jagged1, and NFKB genes
were analyzed by quantitative RT-PCR.
Results
Compared to PBMCs of CHB patients, the liver biopsies
showed significantly lower expression of Notch1 (p=0.034), Jagged 1 (p=0.021)
but not Hes 1 (Table 1). The reduction in expression of these genes was
significantly greater in HCC patients compared to CHB and controls. In
comparison to control biopsies and PBMCs in CHB, cirrhotic tissue showed
significantly lower expression of NFKB (p=0.02).
Conclusion
In different stages of liver disease due to HBV, the PBMCs
show higher expression of notch signaling molecules, but in the target liver
tissue, the expression of these molecules is significantly inhibited, maximum
being in CHB and HCC. This expression pattern of Notch1 signaling suggests that
this protein may play a permissive or tumor-promoting function in chronic HBV
infection.
|
|
CHB PBMCs |
CHB Bx |
P |
Cirrhotic Bx |
P |
HCC Bx |
P |
|
Notch1 |
0.39 |
-0.006 |
0.034 |
-0.03 |
NS |
-0.06 |
0.05 |
|
Hes1 |
0.82 |
-0.26 |
0.4 |
-0.29 |
NS |
-0.5 |
NS |
|
Jagged1 |
0.63 |
0.057 |
0.021 |
0.02 |
NS |
-0.34 |
0.034 |
|
NFKB |
2.2 |
-0.3 |
0.027 |
-0.42 |
0.021 |
-0.25 |
0.034 |
M. Sakaki; K.
Hiroishi; T. Baba; M. Kushima; M. Imawari
Background/Aims
Regulatory T cells (Tregs) maintain immunological tolerance
and suppress autoreactive immune responses. We evaluated the intrahepatic
status of Tregs in patients with autoimmune hepatitis (AIH), primary biliary
cirrhosis (PBC), chronic hepatitis C (CH-C), or chronic hepatitis B (CH-B).
Methods:
We analyzed 85 patients (20 AIH, 22 PBC, 27 CH-C, and
16 CH-B) and 14 controls. Using liver tissue samples obtained by needle biopsy
or from marginal parts of resected metastatic liver tumors in controls,
immunohistochemical analyses of forkhead box P3+, which is a specific marker
for Tregs, CD4+, and CD8+ cells were performed.
Results
Intrahepatic Tregs were significantly infiltrated in
patients with liver diseases than in controls. There were significantly fewer
intrahepatic Tregs in AIH patients than in PBC patients (p=0.037). Patients
with a low frequency of intrahepatic Tregs were detected significantly less in
the AIH and CH-B groups than in the PBC and CH-C groups. We found significantly
less infiltration of CD4+ T cells in AIH than in other diseases (p<0.05).
Liver-infiltrating CD8+ T cells were detected more frequently in the CH-B group
than in other groups (p<0.003).
Conclusion
Intrahepatic Tregs were increased in both patients
with autoimmune liver diseases and those with viral hepatitis. In autoimmune liver
diseases, intrahepatic Tregs in AIH patients were fewer than in PBC patients.
Frequency of intrahepatic FOXP3+, CD4+, and CD8+ cells
in patients with each liver disease.
|
|
AIH |
PBC |
CH-C |
CH-B |
Control |
|
%FOXP3+ |
5.2±2.4*,** |
7.2±3.5 *,** |
6.2±3.7 ** |
6.3±1.6 ** |
1.1±1.1 ** |
|
%CD4+ |
37.8±11.0† |
47.4±11.0 † |
44.0±9.3 †, ¶ |
50.6±8.7 †, ¶, Ω |
41.0±10.9Ω |
|
%CD8+ |
32.2±12.2‡ |
33.3±10.4 ‡ |
30.5±7.0 ‡ |
44.2±9.3 ‡ |
32.0±1.9‡ |
The values
are means ± SD. *p = 0.037 (AIH vs PBC); **p < 0.001 (Patients vs Control)
†p = 0.007, 0.045, 0.001 (AIH vs PBC, CH-C, CH-B); ¶p = 0.026 (CH-C vs CH-B);
Ω p = 0.013 (CH-B vs Control); ‡p = 0.003, 0.002, 0.001, 0.001 (CH-B vs
AIH, PBC, CH-C, Control).