Transcript result

title
Gene Expression Signatures of
Peripheral CD4 T Cells
Clearly Discriminate Between Patients with
Acute and Chronic Hepatitis B Infection
( HEPATOLOGY 2009;49:781-790.)
1.Background
2.Methods
3.Result
4.Disscussion
5.Abstract
background
Acute HBV 5% in adults
CHB
Asymptomatic
CHB
cirrhosis /carcinoma
A strong genetic component with modified gene expression seems
to be a major driving force affecting the course of viral hepatitis.
background
T cell hyporesponsiveness/dysfunction
persistent infection
CD4+CD25+ regulatory Tcells (Tregs)
limit excessive
immunopathological damage
facilitate viral persistence
background
the frequency / function
CD4+CD25+ regulatory Tcells
severe CHB infection
≥
mild CHB /early AVH-B infection
background
The molecular mechanisms of host response against HBV
Tregs
is still unknown. ？
idea
CD4 T cells from peripheral blood of patients with AVH-B
and CHB infection in comparison to HCs.
to gain insight on how the status of infection influences the
overall expression pattern of CD4 T cells,
to further analyze their gene expression profiles using
microarray technology.
to choose 350 CD4 T cell–specific genes, customized to
investigate Treg associated genes.
Patients and Methods
Patients and Samples.
● Isolation of CD4 T Cells from PBMCs
and FlowCytometric Analysis.
● Microarray Analysis.
● Statistical Analysis.
●
Patients and Methods
Microarray Analysis
Microarray is a multiplex technology used in molecular
biology and in medicine. It consists of an arrayed series of
thousands of microscopic spots of DNA oligonucleotides,
called features, each containing picomoles of a specific DNA
sequence. This can be a short section of a gene or other
DNA element that are used as probes to hybridize a cDNA or
cRNA sample (called target) under high-stringency conditions.
Probe-target hybridization is usually detected and quantified
by fluorescence-based detection of fluorophore-labeled
targets to determine relative abundance of nucleic acid
sequences in the target .
Patients and Methods
Microarray Analysis
Patients and Methods
AVH-B: ALT>10ULN with no past history and clinical, biochemical, or radiological
evidence, HBsAg +, HBsAb-,HBeAg+, anti-HBeAg–, HBcAg IgM+
CHB: HBsAg + >6m, ALT>1.5ULN, and histological evidence of chronic hepatitis
HCs: with no previous history or current evidence, HBsAb(+)>300 IU/mL
Patients and Methods
Exclusion criteria :
▲ regular alcohol consumption (40 g/day for
the past 5 years), diabetes, severe
systemic illness, pregnancy, hepatocellular
carcinoma
▲ coinfection with human immunodeficiency
virus or other hepatitis viruses
▲ immunosuppressive therapy for other
associated illness.
result
1. Frequency Analysis of Circulating CD4+CD25+ Foxp3 Tregs
·
●
We analyzed peripheral blood from
19 AVH-B patients, 21 CHB patients, and 12 HCs
to determine the percentage of
CD4+ T cells and CD4+CD25+Foxp3 Tregs.
●
Via flow cytometry
result
result
The frequency of CD4+T cells ： similar
(AVH-B, 29%; CHB, 34%; HC, 30%)
The frequency of CD4+CD25+Foxp3 Tregs：different
(AVH-B, 7%; CHB, 3.96%; HC, 2.18%)
result
2. Functional Dissection of Expression Signatures in AVH-B and CHB Patients
CD4T cells were isolated from peripheral blood (92% to 96%
purity) for RNA extraction.
● four (three for AVH-B) individual measures per donor pool
● A unique set of 350 genes to establish an expression
profile of CD4 cells.
● significant expression levels could be detected for 316 genes.
●
result
2. Functional Dissection of Expression Signatures in AVH-B and CHB Patients
Fig: A clear separation of the three CD4pools
by the principal component analysis
The use of this statistical technique allowed us to determine the key
variables in our multidimensional dataset.
result
118/316 differentially in (AVH-B and CHB)/HCs
● 100 genes were assigned to GO terms
using the Source annotation tool
●
result
GO(gene ontology)
是基因本体论联合会所建立的数据库，旨在建立一个适用
于各个物种的，对基因和蛋白功能进行限定和描述的，并随着
研究的不断深入而更新的语言词汇标准。GO是多种生物学
本体论语言中的一种，提供了三层结构的系统定义方式，
用于描述基因产物的功能。
通过GoFigure 网站，可以提交DNA或者蛋白序列，搜查的结果
会清晰具体地注释目标序列在分子功能、生物学途径、细胞组件
三方面的功能。
http://www.geneontology.org/GO.doc.shtml
result
The majority of the genes were related to
cellular processes(24%), biological regulation (14%),
response to stimulus(13%), metabolic processes(12%),
developmental processes(11%), multicellular
organismal processes (8%),and immune system
processes (7%).
result
result
With respect to the microarray-based analysis
of the viral infection process,
we further dissected the GO term
“response to stimulus”(46 genes) (see Fig.)
and “immune system process” (24genes) (see Fig).
result
result
result
Cluster Mapping and Affected Signaling Modules
All genes displayed in the cluster map.
The cluster map organized into six sections
According to gene expression behavior .
The average relative gene expression values were
calculated from four (CHB patients and HCs) and three
(AVH-B patients) individual measures, respectively.
result
Cluster Mapping and Affected Signaling
Modules
result
So:
Different outcome of HBV (CHB/ AVH-B)/
HCs)
different cluster mapping
result
Cluster Mapping and Affected Signaling Modules
All genes displayed in the cluster map were linked to
signaling maps stored in public databases by using MAPP Finder
software.
T cell receptor (TCR) signaling, cell adhesion,
mitogen-activated protein kinase kinase (MAPKK) signaling,
cell cycle/cell proliferation, apoptosis, cytokines,
and chemokines were identified as the most prominently
affected modules (see Fig. ).
result
Fig:Signaling module depicting gene sets activated in CD4 T cells from AVH-B patients
result
TIAM
AVH>CVH
IL-9, IL-4, IL1R2, IFNA1
PECAM1
result
result
Quantitative RT-PCR
Fig: Quantitative RT-PCR analysis of selected genes
discussion
The frequency of CD4+CD25+Foxp3 Tregs：different
(AVH-B, 7%; CHB, 3.96%; HC, 2.18%)
Tregs AVH-B> CHB> HC
discussion
the number of Tregs during the convalescence phase of AVH-B
infection increases before the normal level is restored after resolution
of the infection
discussion
This dynamic modulation of the antiviral immune response via
self-limited action of suppressorTregs might support the healing
process at the site of infection. The activity of Tregs might
prevent the extended tissue damage resulting from an unchecked
proinflammatory response.
On the other hand, a spatiotemporal dysregulation of Treg
activity—that is, uncontrolled establishment of suppressor activity
at the site of infection— could promote viral persistence and
progression to chronicity.
Discussion1:the molecular events are largely unknown
.
discussion
Microarray technology has a tremendous potential for
complementing classic biological and clinical parameters;
therefore, we used a human Treg chip16 to dissect specific
gene networks affected by the action of Tregs on CD4
T cells, which may contribute to the pathogenic stages of
acute and chronic HBV infection.
Disscussion 2:
an extremely large prospective patient cohort will be needed
discussion
T cell receptor (TCR) signaling, cell
adhesion,
mitogen-activated protein kinase kinase
(MAPKK) signaling,cell cycle/cell proliferation,
apoptosis, cytokines,and chemokines
Disscussion3:
the relationship between the related genes with HBV
discussion
The information generated in this study
suggests
that the in-depth investigation of the changes
in major gene programs during the follow-up
of AVH-B and CHB patients would allow us to
elucidate the molecular pathways underlying
the decision-making processes.
Abstract
Abstract
CD4 T and regulatory T cells (Tregs) seem
to play a key role in persistence of
hepatitis B virus (HBV) infection.
However, the molecular events by which
Tregs exert their modulatory activity are
largely unknown.
Abstract
基因表达谱
The transcriptional profiles of CD4 T cells of
healthy controls (HCs) and patients affected by
acute hepatitis B (AVH-B) or chronic hepatitis B
(CHB) infection were established using a custom
expression array consisting of 350 genes relevant for
CD4 T cell and Treg function.
These studies were complemented by real-time
reversetranscription polymerase chain reaction.
Abstract
Peripheral blood mononuclear cells (PBMCs) were
also analyzed for the presence of Tregs,
which Were more abundant in the acute stage of the
disease (7%) than in HCs and CHB infection
(HCs versus AVH-B, P0.003; AVH-B versusCHB, P 0.04).
180 genes (34%) intrinsically differentiate
HBV-infected patients from HCs.
Abstract
Using gene ontology, we identified T cell
receptor signaling and clusterization, mitogenactivated protein kinase kinase signaling, cell
adhesion, cytokines and inflammatory responses
cell cycle/cell proliferation, and apoptosis as the
most prominentaffected modules.
A higher expression of CCR1, CCR3, CCR4, CCR5
and CCR8 was seen in AVH-B than in CHB-infected
patients and HCs.
Annotation of the interconnected functional network
of genes provided a unique representation of global
immune activation during acute infection. Almost all
genes were down-regulated in patients with CHB
infection.
Abstract
Conclusion:
The fingerprints enable clear discrimination
between patients suffering from AVH-B or CHB
infection.
The observed profiles suggest accumulation
of effector Tcells with a potential role in necroinflammation during the acute stage.
Subsequent downregulated effector
functions support the hypothesis of
suppressed CD4 effector T cells favoring viral
persistence in the chronic infection stage.
Tips:
★ 调节性T细胞是热点研究问题
（分子水平）
★ 生物信息学技术 的应用
★ 展望