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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