The Clinical Diagnosis Methods And Immune Pathogenesis Of Chronic Active Epstein-Barr Virus Infection

BackgroundEpstein-Barr virus(EBV),which was found in the Burkitt's lymphoma cells of African children by Epstein and Barr in 1964,is a kind of wide-spread herpes virus in the world,belonging to herpes virus-4 family.The primary EBV infection of children is very common with the VCA-IgG sero-positive rate up to 90%.Most primary EBV infection is acute infection,manifestating asymptomatic,or infectious mononucleosis (IM),but less frequently presenting as chronic and recurrent IM-like symptom with abnormal EBV antibodies,which is called chronic active Epstein-Barr virus infection(CAEBV).The first report about CAEBV began in the early 1980's.CAEBV mainly occurs in children,less happening to adults,having a highest incidence in Asia, and having a high mortality.The most dangerous thing of CAEBV is that CAEBV is associated with many severe complications,such as EBV-associated T,NK lymphoproliferation syndrome,haemophagocytic lymphohistiocytosis,and malignant lymphoma et al,so more and more attention has been paid to CAEBV by many doctors. Today,correct diagnosis of CAEBV and studying its pathogenesis are two hot researching points.But in the diagnosis process,we found the antibodies of CAEBV presenting diversity,which did not coincidence with the detect of EBV-DNA.Some CAEBV patients lack the elevation of the EBV antibodies if its immunoglobulin was low or had sthenic spleen function,but their EBV-DNA copies increased dramatically.Those having high EBV antibodies may be caused by the cross-reaction of other factors.In addition, the EBV antibody had its up and down during different process time.So,the detect of EBV-DNA is very important for CAEBV,and the further study of the difference between CAEBV,AEBV and normal children helps to the diagnosis of CAEBV.The haematological diagnosis of AEBV include the increasing of WBC,and mainly the elevation of lymphocyte,atypical lymphocyte＞10%,and the increasing of the CD38~+DR~+CD8~+ T lymphocytes when further lymphocyte subsets analysis.But Whether CAEBV have some characteristics on haematology and lymphocyte subsets different from AEBV and normal children which may help the diagnosis of CAEBV hasn't been reported as yet.The same level of EBV-DNA was not equal to the same infection degree in different lymphocyte type.It was reported that EBV T-cell infection related to the high mortality of CAEBV,which needed bone marrow transplantation earlier.The rountine DNA detect method didn't help us to certify the type of EBV-infected cells.So,the study of EBV-infected cell type will help to differentiate CAEBV clinical typing,and may have a significant role on further viral pathogenensis study,individual diagnosis and treatment and prognosis evaluation.On the pathogenesis of CAEBV,it was reported that viral antigen and host immune leading to the EBV immune evasion played an important role.The normal immune response to EBV includes innate immune and acquired(adaptive) immune.It was reported that host adaptive cellular immune response esp.EBV specific CD4~+ T response and CD8~+ T response was very important to clearing EBV and having a good end-result. But CAEBV had cellular immune turbulence,which may be associated with the chronic and active infective process.Nowadays,most study localized in the researching of CD4~+T cell,CD8~+T,and NK cell of CAEBV,but the researching of T lymphocyte functional subsets,stimulatory subsets,na(l|¨)ve and memory subsets and regulatory subsets have not been reported as yet.The pathogenesis on host EBV-specific adaptive cellular immune response in CAEBV is not clear now.It is reported that the decreasing of the number of EBV-specific cytoxic T lymphocyte(CTL) may be an important EBV immune evasion mechanism in CAEBV.However,the number of EBV-CTL does not completely stand for the real function of EBV-specific adaptive cellular immune.Up to now,there is no the study on the function of EBV specific CD8~+ CTL after EBV peptide pools stimulation in CAEBV.Objectives1.To examine the characteristics of CAEBV from the aspects of EBV antigen dignosis,EBV-infected cell type,haemotological and lymphocytic subsets,serum diagnosis and EBV-specific CTL to help the diagnosis of CAEBV.To help the clinician having a deeper understand on the characteristics of CAEBV,and make a correct diagnosis and differential diagnosis,and establish a basis for directing therapy and prognosis evaluation.2.To study the immune evasion pathogenesis of CAEBV from the aspects of EBV-infected cell type,peripheral blood lymphocytic subsets,and the function of the EBV specific adaptive immune response.To definitude the methanism of CAEBV in the cellular and molecular immune levels,and to provide a basic data to the individual diagnosis,therapy and prognosis evaluation.Methods1.collected the serum and PBMC of 10 pediatric patients with CAEBV,13 pediatric patients with AEBV in our hospital between March 2004 and April 2008,12 normal children as a control group.2.Real-time fluorescent quantitative polymerase chain reaction(qPCR) was used to detect the EBV-DNA levels of periphoral blood mononuclear cells(PBMC) in three groups.3.immunomagneticbead cell fractionation and real-time fluorescent quantitative PCR or fluorescent in situ hybridization(FISH) by EBV encoding RNA-1(EBER-1) probe were used in 3 groups to detect the EBV-infected positive cell and EBV-DNA copies in different types of lymphocytes.4.Flow cytometry was used to detect the the peripheral blood NK,B,T lymphocyte subsets and the functional,regulatory,activatory,na(l|¨)ve and memory subset of T lymphocytes in 3 groups.WBC and lymphocytes was deteced by blood routine detection.5.ELISA and heterophil agglutination tests were used to detect all kinds of EBV-antibodies in 3 groups,and EA-IgG and EBNA-1-IgG also were detected by the antibody dilution test.6.ELISPOT was used to detect the level of IFN-γsynthesized and secreted by EBV peptide pools-specific cytoxic T lymphocyte(CTL) in the in vitro cultivated PBMC of 3 groups.7.Statistic methods:Statistic analysis was performed by SPSS version 13.0 software. The statistic significance was evaluated by one-way ANOVA for multi-group and non-parameter test or one-sample-T-test for two groups.The statistic significance was defined as p＜0.05.Results1.The average EBV-DNA level of CAEBV patients' PBMC was 6.81×10~7±1.1×10~8 copies/ml,while that of AEBV patients' PBMC was 1.32×10~6±1.6×10~6 copies/ml. The average EBV-DNA level of CAEBV patients' PBMC was dramatically higher than that of AEBV patients' PBMC(P＜0.01).2.The cell fractionation and FISH,qPCR in 7 CAEBV patients showed:EBV in CAEBV patients infected not only B cells,but T and NK cells,and these patients presented recurrent and persistent IM-like symptom.Six CAEBV patients infected mainly T cells,in which one,infected mainly in CD8~+ T cells,died from the fulminant and deadly T lymphocytes proliferative syndrome,except presenting firstly high fever,enlargment of the liver,spleen,lymphnode and the severe decreasing of one or three kinds of blood cells.One CAEBV patients,infected mainly in NK cells, presented hypersensitivity to mosquito biting and high IgE level(2500 U/ml).But EBV in seven AEBV patients infected only B cells who presented only IM for one time and no EBV-infected PBMC were found in six normal children.3.Compared with the other 2 groups,the number of white blood cells,lymphocytes,NK,total T,CD4~+ T cells and CD8~+ T cells in CAEBV patients decreased significantly(p＜0.01),while the number of B cells in CAEBV patients had no difference with the normal group,but was less than AEBV group(p＜0.01).4.Functional subsets of T cells:The proportion of CD28~+ CD8~+ in CAEBV wasn't dramatically different from normal group except the proportion of CD28~+ CD4~+ in CAEBV group was lower than normal group(p＜0.01) but still higher than AEBV group(p＜0.01).5.Treg subsets(regulatory t lymphocyte subset):Although the proportion of CD25~+CD4~+ in CAEBV group was higher than normal group(p＜0.01),but the proportion of CD25~+CD4~+ in CAEBV was dramatically lower than AEBV group(p＜0.01).6.Na(l|¨)ve/memory subsets:In CAEBV,the proportion of CD4~+/CD8~+ CD62L~+ CD45RA~+ (naive T cells) was lower than that in normal group(p＜0.01),but the proportion of CD4~+/CD8~+ CD62L~- CD45RO~+(effective memory T cells) in CAEBV group was dramatically lower than that in AEBV group(p＜0.01).The proportion of CD4~+ CD62L~+ CD45RO~+(central memory T cells) in CAEBV group was higher than that in the other 2 groups(p＜0.01),the proportion of CD8~+ CD62L~-CD45RA~+(fake na(l|¨)ve T cells) in CAEBV was higher than that in the other 2 groups(p＜0.01) and the proportion of CD8~+ CD62L~+ CD45RO~+(central memory T cells) in CAEBV group was higher than that in the other 2 groups(p＜0.01 compared with normal group, p＜0.05 compared with AEBV group).7.Activatory subsets:The proportion of CD38~+CD8~+ or DR~+CD8~+ in CAEBV group was higher than that in normal group(p＜0.01) but still lower than that in AEBV group(p＜0.01). 8.Compared with normal group,the levels of VCA-IgA,EA-IgA and high-affinity EA-IgG in CAEBV elevated dramatically(P＜0.01)9.Both AEBV and CAEBV have lower EBNA-1 IgG than the normal group(P＜0.01), but CAEBV's EBNA-1 IgG had higher affinity than AEBV's.10.Compared with normal group,CAEBV has a lower function of EBV-peptide pools-specific CTL(P＜0.01).Conclusions1.Real-time fluorescent quantitative PCR is a very sensitive and specific molecular biological method for the diagnosis of CAEBV and the survey of the different end-results of EBV infection.2.CAEBV was characterized by infecting simultaneously not only B cells,but also CD4~+ T,CD8~+ T and NK cells in different degree,having a trend of multiclonal and oligoclonal proliferation,and these patients presented recurrent and persistent IM-like symptom and had a worse prognosis.AEBV patients infected only B cells who presented only IM for one time and had a better outcome.Detecting the type of EBV-infccted lymphocytcs play an important role for CAEBV individual dignosis trcatment and development evaluation.3.There is an imbalance in lymphocyte subsets and turbulence in cellular immunity in CAEBV patients,which may be associated with EBV chronic active infection process. Detecting the peripheral haematology and lymphocyte subsets helps to the diagnosis and the differential diagnosis of CAEBV.4.The increasing of VCA-IgA,EA-IgA,high affinity EA-IgG and the decreasing of EBNA-1 IgG are the characteristics of the EBV-spccific adaptive antibody immune response in CAEBV.Detecting the levels of VCA-IgA,EA-IgA,EA-IgG and EBNA-1 IgG and the antibody affinity could help the diagnosis and differential diagnosis of CAEBV.5.The decreasing of EBV-specific CTL function(compared with VCA-IgG positive normal children) is the characteristics of the EBV-specific adaptive cell immune response in CAEBV.6.CAEBV has many characteristics of EBV specific adaptive immune response different from AEBV and VCA-IgG positive normal children,which may help to the diagnosis and differential diagnosis of CAEBV in one hand,and in the other hand, may be associated with the immune evasion mechanism of CAEBV and EBV chronic active infection process.