Aberrant Expression Of Epstein-Barr Virus Genes In Children With Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE)is aTcell-dependentimmune complex-mediated multisystemautoimmune diseasethat involves multipleorgans including theheart, liver,kidney, and multiple systems includingnerve,blood. SLE can impact onhuman health seriously.SLE can be found in all ages of children,but rarely onset before the age of five.Increased incidence of SLE in adolescence and onset in neonatal can be found.Thepathogenesis, clinical features and laboratory findings inchildren withSLE are similar to the adult with SLE, but there is still a significant difference between the them. The clinical manifestations of SLE in children is less typical and the first symptom is different, so it is prone to be misdiagnosed and is fatal potentially.If it is not treated timely, the prognosis in children with SLE is far more serious than adult. The clinical manifestations of SLE in children are complex and diverse, usually involves multiple organs.The etiology in SLE with childrenis unclear, it may be under the jointactionof genetic, environmental and hormonal, viral infections and other factors,.Then own tissuesor immune cells are altered. losetheirtolerance and result inimmunedysregulation.Recent studies havefound thatEpstein-Barr virus (EBV)infectionmay be onefactor inthe pathogenesis of SLE. EBV was first discovered from the body ofmalignant lymphomainAfrican children, belonging tothe subfamily of Y herpes virus.EBVhas atropismBlymphocytic, temporary clonal proliferation of B lymphocytes can be caused after acuteinfection. The B lymphocyte proliferative responses may soon be suppressed by T lymphocytes including cytotoxic CD8+T cells (CTLs) in individuals with normal immune function,which maintainslifelonglatentEBVinfection status.There are severe immune dysfunction inSLEpatients, so EBV latent infection can not be controlled in SLE patients as effectively as in healthy normal controls. EBV isapersistentthreat and eventually leads to the occurrence of certainpathological conditions.EBVgenomecarries the84open reading frame(ORF),encodes60 kinds ofviral proteins.EBV gene can be divided in 4 groups according to the expression period:latent, immediate early, early and late structural genes. Latent gene includes nuclear antigen gene(EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3CandEBNA-LP), the latency membrane protein family (LMP1, LMP2A and LMP2B), EBV-encoded RNA (EBER1 and EBER2). Immediate early gene is the earliest protein expressed after viral gene activated from latent state, belonging to the family members of transcription factors, starting transcription, translation and expression of other EBV activating genes, including BZLF1 and BRLF1 so on. Early gene is related with viral genome replication enzymes,including encoding helicase, primers and DNA polymerase enzyme, etc. the corresponding gene such as BMLF1, BHRF1, BMRF1, BALF5 and BARF1 and so on.Late structural genes includes the structural gene encoding glycoprotein of structural proteins(gp350, gp85, g110), and capsid protein (VCA) gene such as BCRF1, BLLF1 and so on.EBV has two infection states in vivo including lytic infection and latent infection.Numerous studies indicate that EBV is related with pathogenesis of SLE. 1.EBV serologic abnormalitiesinSLE patients:A large number of researches showed that the EBV seropositive rate and the positive rate of EBV activated early antigen -D antibody, EBV-EA antibody, VCA-IgG antibodies, etc.in SLE patients were significantly higher thanhealthy control groups.2.EBV viral loads inSLE patients were obvious abnormal:EBV viral loads in SLE patients was significantly higher than the normal population, indicating that EBV in SLE patients was out of control.3. EBV-DNA in SLE patients was abnormal. Studies show that EBV-DNA in SLE patients have a higher positive rate.4. Expression of EBV genes are abnormal in SLEpatients. Brian D. Poole et al found that the expression of latent gene LMP-1 andlytic gene BLLF1, BcRFl were significantly increased in SLEpatients,indicating that the expression ofEBV gene is abnormal,EBV may be involved in the pathogenesis of SLE.Recent studiesindicate thatEBV may participate in the pathogenesis of SLE by a variety of ways such as molecular modeling, molecular functionsimulation, T lymphocyte immuneresponse, cytokines bypassmechanism, epigenetic.Previous studies based on adults with SLE for the study, while the primary EBV infection mostly occured in childhood, so analysisthe correlationbetween children with SLE and EBV infection may be more helpful to explore the relationship between them.Based on the above research foundation,and further research on correlation between EBV and SLE in children,Our experiments investigated latent and lytic EBV gene expression in peripheral blood mononuclear cells of children with SLE and healthy controls, and attempted to explore the possible pathogenic role of EBV genes in children with SLE, to provide new ideas for SLE pathogenesis and clinical treatment.[Method]1.Peripheral blood mononuclear cells(PBMCs) isolated and partially extracted DNA: PBMCs were isolated from 20 children with SLE and 12 healthy human controls,half of themto be extractedDNA.2.EBVinfected PBMCs forl2days and RNA was extractedusingTrizol(?)reagents.3.Real-time fluorescence quantitativePCR(FQ-PCR) was applied to detect the copies of EBV DNA in PBMCs.4.Reverse transcription PCR (RT-PCR) was applied to detect expression of EBV genes, including LMP1, LMP2, EBNA1, BCRF1, BLLF1 and BILF1 genes.[Result]1. Results of the copies of EBV DNA in PBMCs detected by FQ-PCRCompared with the normal control group (40.1±11.6) copies/μg, a significant increase of EBV DNA copies was observed in SLE patients (658.6±205.7) copies/μg (P<0.01). The EBV DNA copies in the active SLE group (785.2±173.3) copies/μg were significantly higher than those in the nonactive SLE group (586±193.1) copies /μg (P=0.005).2. There is no correlation between EBV DNA copies and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)(The correlation coefficient was 0.03, P=0.826).3. Abnormal latent and lytic EBV gene expression in PBMCs of children with SLE.Significant differences were observed between the patients and controls in the detection rate of LMP1 gene (10/20 vs.1/12, P<0.05).No significant differences were observed between the patients and controls in the detection rate of LMP2 gene (4/20 vs.1/12), EBNA1 gene (13/20 vs.3/12), BCRF1 gene (3/20 vs.1/12) or BLLF1 gene (5/20 vs.2/12) in PBMCs.4. Abnormal latent and lytic EBVgene expression in PBMCs of children with SLEafter co-cultured with EBV.After PBMCs co-cultured with EBV,we found that expression of latent EBV genes and lytic genes were both increased in patients and controls. The latent EBV genes including LMP1, LMP2, EBNA-1 and the lytic genes including BCRF1、 BLLF1 were all increased significantly in patients compared with the controls (1.011±0.141 vs.0.383±0.180,t=10.680,P=0.000; 1.001±0.093 vs.0.208±0.082, t=25.109,P=0.000; 1.012±0.163 vs.0.188±0.129, t=14.591, P=0.000; 1.015±0.167 vs.0.344±0.161, t=11.268, P=0.000; 1.038±0.264 vs.0.117±0.081,1=14.513, P=0.000, respectively)[Conlusion]1. EBV DNA copies is significant increased in children with SLE, EBV DNA copies in the active SLE group were significantly higher than those in the nonactive SLE group, suggesting that EBV is active in children with SLE.2. The expression of latent EBV gene LMP1 in PBMCs of children with SLE is significantly abnormal.3. The expression of latent EBV gene LMP1, LMP2, EBNA-1 and lytic EBVgene BCRF1、BLLFlin PBMCs of children with SLE after co-cultured with EBV are significantly abnormal.4. There is an aberrant expression of EBV genes in PBMCs of children with SLE, and EBV genes may contribute to the development of SLE.The expression of Epstein-Barrvirus latentmembrane proteinl (LMP-1)in kidney ofchildrenwith lupus nephritisnephritisWhenchildren withSLEappearproteinuria,hematuria, red blood cellcasts,white blood cell castsand otherdiversechanges inurinary sediment, as well asedema, hypertension (excluding other factors),abnormalrenalparenchymaldamageperformance, they can be diagnosed with Lupus Nephritis(LN).Childrenwith LNSLE has higherincidence of LN and with the extension of duration ofSLE, The opportunity for children with SLE developing to LN is increasing year by year. Most of the time the child with SLE newly diagnosed is reported about 50% associated with LN, up to 70%-80% within 2 years of onset, more than 90% after four years. If children with SLE are under biopsy, it is almost 100% of the children varying degrees of renal pathological changes. According to international standards, jointly developed by the International Society of kidney disease and kidney pathology (ISN/RPS classification), LN can be divided into: type I (mild mesangial type LN), type Ⅱ (mesangial proliferative LN), type III(focal LN), type IV(diffuse LN), type V (membranous LN) and type VI(terminal sclerosing LN).The etiology andpathogenesis ofLNis not very clear, studies suggest thatpossible mechanismsas following:(1) Circulatingimmune complexesdepositin the kidneytissue.(2) Immune complex is formedin situ. (3) Autoantibodies damage to the kidneysdirectly. (4) Localcomplementactivation, resulting in self-chemokines, causing local inflammationdamage.It can also form themembrane attack complexdirectly attackthe basement membrane,resulting inan increasein glomerularcapillary permeabilityandcausingkidney damage.(5) Tcell-mediatedimmune responseinvolved in regulatingthe secretion ofautoantibodies of B cells, caused kidneydamage. (6)Other factors such asbradykinin\bradykininsystem, monocytes,macrophagesalso play arole inthe LN.Foreign research showed that LMP-1 could aggravate the autoreactivity of SLE by a variety of mechanisms, including activate of B cells directly, enhanced activation costimulatory signal of B cell, stimulate the pruductin of B cell activating factor (BAFF), break the self-help tolerance and so on. LMP-1 could combinate with CD40L,recruittumor necrosis factor receptor-associated factor (TRAF) family members on the cell membrane and activate a signal transduction pathway, so EBV was abnormal cloned andinduced B cell activation and amplification.Bishop showed that LMP-1 expression could result in B lymphocyte activation, a large number of autoantibody production and kidney damage of individuals with a genetic predisposition through animal experiments. Tumor necrosis factor receptor binding factor-6 (TRAF-6) playde an important role in the process ofLMP-1-induced B cell activation. LMP-1 can directly activate the expression of apoptotic protein Bcl-2,can also upregulated the expression of bcl-2in infected B lymphocytes througha number of signaling pathways,inhibit apoptosisof B cell and promote the survival of B cells,produce antibodies continuely, lead to the development of SLE.Studies on EBVantigen,EBVgenomeand serological of EBV, showed thatEBVinvolved in the pathogenesisof SLE.The firstpart of the studyfound thatEBV-LMPlgeneplays an important roleinthe pathogenesis of SLE, But it was unclearwhetherLMP1 involved in the pathogenesisof LN.80%of children with SLE would develop to LN,while only 60 percent of adultswith SLE would develop to LN.So we picked children with LN as research subjectsin our experiment,we reserchedcorrelation betweenLN clinical symptoms, renal pathology grading and antibody expression and the expression of LMP-1,trying to explore correlation between the LMP-1 and the incidence of LN in children.[Method]Experimental group were from Hainan MCH dermatology and Children’s Medical Center, Second Xiangya Hospital, Central South University Pediatric kidney specialist,51 cases of LN in children since January 1,2009 to November 2013 were treated as the research objects complete clinical datas with patients werecollected.Wedetected the expression ofLMP-1 in kidney tissue in LN and normal group and performed the correlation analysis with clinical course, renal pathology grade, clinical symptoms and antibody expression.[Result]1.The expression of LMP1 in the renal tissues of young patientsOf the 12 samples detected, only 2 (16.7%) was positive for LMP1 in the normal controls. However, there were 40 (78.4%) samples were positive for LMP1 in total 51 patient with LN. The positive rate of LMP1 in young patient with LN was significantly higher than in normal controls (P=0.000).2.The expression of LMP1 is not associated with the disease course of LNOf the 15 samples from initial onset patient,12 (80.0%) were positive for LMP1. There were 28 (77.8%) samples were positive for LMP1 in the 36 samples from relapse patients. There was no statistical difference between the initial onset and relapse young patient with LN (P=1.000).We further detected the expression of LMP1 in young patients with or without concurrent infection. There were 16 (80.0%) samples positive for LMP1 in 20 samples without concurrent infection and 24 (77.4%) samples were positive for LMP1 in 31 samples with concurrent infection. There was no statistical difference between the positive rates of LMP1 in the young patients with or without concurrent infection (P=1.000).3.The positive correlation of LMP1 with the classification of LNTo determine whether the classification of LN is associated with the infection of EBV, we detected the positive rates of LMP1 in the patients with different classification of LN. The positive rate of LMP1 was 57.1% in class II and 66.7% in class Ⅲ. However, the positive rate of LMP1 increased to 80% in class IV and 83.3% in class Ⅲ+Ⅴ. It was 91.7% in class IV+V, which is higher than most of other classes. Spearman correlation analysis LMP-1 expression in LN patients and renal pathology grading indicates that the Spearman correlation coefficient=1, P <0.01.These results indicate that thepositive rate of LMP1 was positive correlated with the classification of LN. And EBV infection is likely involved in the pathogenesis of LN.4.Association of LMP1 expression with clinical symptoms in the renal tissues from patients with LNWe also examined the positive rate of LMP1 in the renal tissues from young patients with LN and different clinical symptoms. The positive rates of patients with Anaemia, Hypercoagulation, Hypoproteinemia or Hypocomplementemia are not obviously different in the LMP1 positive or negative samples (P>0.05). However, the positive rate of patients with Renal failure is significantly higher in LMP1 positive samples compared with LMP1 negative controls(P<0.01). And the positive rates of patients with Haematuria, Proteinuria or both with Haematuria and Proteinuria are also significantly higher in the LMP1 positive samples than the LMP1 negative controls(P<0.05).5.Association of LMP1 expression with autoantibody production in patients with LNWe detected the expression of autoantibodies and LMP1 in the young patients with LN. The results indicate that only the positive rate of anti-Sm was statistically higher in LMP1 positive samples compared with LMP1 negative samples(P=0.009). While there were no detectable difference for the positive rates of serum ANA, anti-RNP, anti-Jo-1, anti-dsDNA, anti-SSA and anti-SSB between the LMP1 positive and negative groups (P>0.05).[conlusion]1.LMP-1correlated withthe incidence ofLN,butregardless of thediseasestatusandinfection.2.LMP-lmay have some relevance to the level of kidney damage and type in children with LN.3. The positive rates of patients with Renal failure, hypocomplementemia,Haematuria, Proteinuria or both with Haematuria and Proteinuria are also significantly higher in the LMP1 positive samples than the LMP1 negative controls. These results suggest that EBV infection may aggravate the clinical manifestations of LN.4. The positive rate of anti-Sm was statistically higher in LMP1 positive samples compared with LMP1 negative samples. These results indicate that EBV infection may aggravate LN through increasing Anti-Sm antibodies.Comparison among three methodsto detectexpression of EBvirus latent membrane proteinl inchildrenwith lupus nephritispatientsObjectiveTo discussthe role of latent membrane protein 1 (LMP-1)in the pathgenesis of Juvenile Lupus nephritis (JLN) through investigating expressi on of LMP-1 byRT-PCR,immunohistochemistry and ELISA.Methods RT-PCR,Im munohistochemistry and ELISA were applied to detect expression of LMP-1 in PBMCs, kidney tissue and serum from 20 patients with LN and 12 healthy c ontrol subjects respectively. Results ① There was signifcant difference between JLN patients and healthy controls by RT-PCR, Immunohistochemistry and ELI SA (positive rate was 65.9% vs 8.3%,95.1% vs 16.7%,22.0% vs 8.3%, res pectively) (P<0.05). ② Positive rates of LMP-1 in JLN was significant diff erence among three methods and the positive rate detected by immunohistoche mistry was higher than the other two methods (P<0.05). Conclusion The resu Its demonstrate that there are aberrant expression of LMP-1 gene in JLN, thep ositive rate of LMP-1 in kidney tissue is highest and suggest that LMP-1 ge ne may contribute to the pathgenesis of JLN.