Fatal familial hemophagocytic lymphohistiocytosis with perforin gene (PRF1) mutation and EBV-associated T-cell lymphoproliferative disorder of the thyroid

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare fatal autosomal recessive disorder of immune dysregulation. The disease presents most commonly in the first year of life; however, symptomatic presentation throughout childhood and adulthood has also been identified. Biallelic mutation in the perforin gene is present in 20%­50% of all cases of FHL. Secondary hemophagocytic lymphohistiocytosis (HLH) in association with hematological malignancies is known; however, whether mutations in HLH-associated genes can be associated with FHL and hematolymphoid neoplasms is not well documented. Also, Epstein­Barr-virus- (EBV) positive systemic T-cell lymphoproliferative disease (SE-LPD) in the setting of FHL is not clearly understood. Here, we present the case of a young boy who presented with typical features of childhood FHL harboring the perforin gene (PRF1) mutation, and had SE-LPD diagnosed on autopsy, along with evidence of recent EBV infection. The patient expired due to progressive disease. Five siblings died in the second or third decade of life with undiagnosed disease. Genetic counseling was provided to the two surviving siblings and parents, but they could not afford genetic testing. One surviving sibling has intermittent fever and is on close follow-up for possible bone marrow transplantation.

Epstein-Barr virus latent genes

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

The following-up study of high-risk and moderate-risk groups defined by EB virus serology test at the nasopharyngeal carcinoma screening programme / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between changes in high-risk populations and screening detected nasopharyngeal carcinoma (NPC) during the three-year follow-up of high-risk and moderate-risk groups at initial EB virus serology screening.</p><p><b>METHODS</b>We tested EB virus VCA-IgA and EBNA1-IgA antibody to identify the probability of suffering from NPC of the crowd. The high-risk and moderate-risk groups at initial screening in one county during 2009 to 2010 were followed-up once a year with EB virus serology testing. All the high-risk people during initial screening and follow-up were conducted with nasopharyngeal fiber endoscopy. Through the follow-up of three years, we analyzed changes in the number of high-risk group, detection rate of NPC in high-risk group, and tumor staging. Firstly detected NPC by screening was defined as screening group, and detected by following-up was defined as following-up group.</p><p><b>RESULTS</b>A total of 404 participants were at high-risk and 1 041 participants were at moderate-risk group, 1 445 persons were in the group. All 404 persons were at high-risk at initial screening, the number of high-risk people during follow-up decreased from 371 to 187, 853 people of the all high-risk group were conducted with nasopharyngeal fiber endoscopy, and 38 cases of NPC were detected. NPC detection rate of high-risk group was 6.2% (25/404), 3.2% (12/371), 0.5% (1/188) and 0 (0/187) during the initial screening and three years follow-up respectively. The cumulative incidence of NPC in the high-risk and moderate-risk group were 7.7% (31/404) ,0.8% (8/1 041) . The early diagnosis rate of NPC in screening group and following-up group was 80% (20/25)and 11/13, respectively. With the primary tumor, the rate of T1 in screening group was higher than following-up group (80% to 38%, 20/25 to 5/13; P = 0.028). However, compared with following-up group, the rate of regional lymph node metastasis in screening group was higher (19/25 to 5/13; P = 0.035 ).</p><p><b>CONCLUSION</b>Along with the high detection rate of early staging NPC in screening group and following-up group, the detection of NPC in high risk people is mainly at initial screening and the first year following-up and NPC detection rate thereafter is dropping significantly.</p>

Epstein-Barr virus in oral mucosa from human immunodeficiency virus positive patients / Vírus Epstein-Barr na mucosa oral de pacientes positivos para o vírus da imunodeficiência humana

Objective: the detection rate of Epstein-Barr virus (EBV) is higher in people living with human immunodeficiency virus (HIV). In an attempt to contribute to our epidemiological understanding of this coinfection and to investigate the activity of EBV in normal oral mucosa, we performed a cross-sectional study with HIV-positive patients. Methods: oral smears from 145 HIV-positive patients were collected between March 2010 and March 2011. Nested polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) were used to genotype EBV and to detect EBNA-2 expression, respectively. Results: EBV DNA was detected in 48.3% of the study participants, of whom 32.85% were EBV-1 and 45.71% were EBV-2 carriers. Additionally, 14.28% were coinfected with both types. EBNA-2 mRNA was expressed in 45.7% of the EBV -positive samples, including 20.0% with EBV-1 only, 20.0% with EBV-2 only and 1.4% with both genotypes. Immune status affected the overall EBV infection, and EBV-2 positivity was significantly correlated with sexual lifestyle of the participants. EBV co-infection with both viral types was dependent upon HIV viral load and the activity of the EBNA-2 gene. Conclusion: we report a high prevalence of active EBV in the oral mucosa of asymptomatic HIV-seropositive individuals. This study addresses the need for monitoring and treatment of HIV-infected patients with EBV reactivation. .

Objetivo: a taxa de detecção do vírus Epstein-Barr (EBV) é alta em pacientes vivendo com o vírus da imunodeficiência humana. Com o objetivo de contribuir para o entendimento epidemiológico e investigar a atividade do EBV na mucosa oral, foi realizado um estudo de coorte com pacientes HIV positivos. Métodos: esfregaços orais de 145 pacientes HIV positivos foram coletados entre março de 2010 e março de 2011. A reação de cadeia de polimerase (PCR) internalizada e a PCR reversa (RT-PCR) foram usadas para genotipar o EBV e detectar a expressão do EBNA-2, respectivamente. Resultados: o DNA do EBV foi detectado em 48,3% dos participantes, dos quais 32,85% eram portadores do EBV-1 e 45,71% de EBV-2. Adicionalmente, 14,28% eram co-infectados por ambos os tipos. O mRNA do gene EBNA-2 foi expresso em 45,7% das amostras positivas para EBV, incluindo 20% por EBV-1 somente, 20% por EBV-2 somente e 1,4% por ambos os genótipos. O estado imune afetou a infecção por EBV, e a positividade para EBV-2 foi significativamente correlacionada com o comportamento sexual dos participantes. A co-infecção por ambos os genótipos de EBV foi dependente da carga viral de HIV e da atividade do gene EBNA-2. Conclusão: registrou-se uma alta prevalência de EBV em atividade na mucosa oral de indivíduos assintomáticos soropositivos para HIV. O estudo focaliza a necessidade de monitoramento e tratamento de pacientes infectados por HIV com reativação pelo EBV. .

Construction and immunological responses of recombinant adenovirus containing Epstein-Barr nuclear antigen 1 in mice / 病毒学报

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.

Characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2010 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2.</p><p><b>RESULTS</b>Type A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively.</p><p><b>CONCLUSION</b>EBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.</p>

Effect of Epstein-Barr virus nuclear antigen 1 on cell proliferation and cell cycle in nasopharyngeal carcinoma cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effect of Epstein-Barr virus nuclear antigen 1 (EBNA1) on cell proliferation and cell cycle in nasopharyngeal carcinoma (NPC) cells.</p><p><b>METHODS</b>Recombinant lentivirus that encoded EBNA1 short hairpin RNA (shRNA) was prepared. The C666-EBNA1 (CE) cells were transduced with lentivirus and selected by fluorescence activated cell sorting (FACS) to repress EBNA1 expression. The protein expression levels of EBNA1 were examined by Western blot. The effect of EBNA1 silence on cell proliferation was analyzed by MTT assay and cell growth assay, respectively. Cell cycle was assessed by flow cytometry. The mRNA and protein levels of cell cycle regulators were examined by real-time PCR and Western blot.</p><p><b>RESULTS</b>Recombinant lentivirus encoded EBNA1 shRNA was successfully constructed. The EBNA1 expression in CE cells was significantly reduced by lentivirus-mediated RNA interference. The results of cell counting and MTT assay showed that EBNA1 down-regulation significantly inhibited cell growth in CE-shRNA EBNA1 cells (P < 0.05). Compared with the control group, the percentage of cells in G0-G1 phase was increased from (62.43 ± 6.62)% to (89.66 ± 0.64)% (t = -7.091, P = 0.002), and that in S phase was decreased from (34.93 ± 7.36)% to (7.82 ± 2.44)% (t = 6.095, P = 0.004). The mRNA expressions of c-myc, CDK4, CDK6 and pRb were decreased by 65.60%, 34.06%, 41.05% and 55.29% respectively with the similar results in protein expression levels.</p><p><b>CONCLUSIONS</b>Suppression of EBNA1 may inhibit the growth of nasopharyngeal carcinoma cells in vitro and induce a G1-phase cell cycle arrest, which might be mediated by down-regulation of c-myc, CDK4, CDK6 and pRb.</p>

Prediction and research on homology of B-cell epitopes of Epstein-Barr virus nuclear antigen-1 / 生物医学工程学杂志

We predict in this paper B-cell epitopes of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and analyze the results matched with the related autoantigens sequence of human. We selected EBV-1 standard strain NA-1 amino acid sequence as the basis. We predicted B-cell dominant epitopes of EBNA-1 with the methods of SOPMA, GOR and HNN, combined with the multi-parameter analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenicity index, polarity and average flexibility. The blastp method was adopted to analyze the matched results between the predicted B-cell epitopes of EBNA-1 and the related autoantigens sequence of human. The results have shown that the possible B-cell dominant epitopes of EBNA-1 were located in the N terminal regions of 16-23, 35-78, 332-337, 340-357, 398-404, 419-432 and 620-637, in which different regions gained higher scores when matched with small nuclear ribonucleoprotein SmB, SmD, ribonucleoprotein SSA, heterogeneous nuclear ribonucleoprotein hnRNP A1, hnRNP G, respectively. It was available to predict B-cell dominant epitopes of EBNA-1 with multiparameter methods and to analyze the same or similar autoantigens sequences of human, which laid a theory foundation for the study of pathogenesis, diagnosis and treatment of autoimmune diseases.

Role of cytokines and gene expression characteristics in cultured lymphocytes ex vivo for adoptive immunotherapy / 中国实验血液学杂志

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.

Early NPC detection through serologic risk stratification and clinical follow-up of high risk subjects / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#Nasopharyngeal Carcinoma (NPC) can be successfully treated by radiotherapy, if the tumor is confined to nasopharynx, but clinical onset is usually delayed to more advanced stages, when prognosis is poor. The objective is to determine efficacy of a new program for early NPC detection, which entails stratification of the NPC risk of target population according to serum levels of 3 Epstein Barr Virus (EBV) antibodies.@*METHOD@#The sera of 1373 healthy adult residents from Zhongshan were collected and analyzed in this study from Mar 16, 2007 to Dec 31, 2007. The levels of EBNA1/IgA, zta/IgG and EBNA1/IgG were tested by ELISA. To stratify the subjects of 1373 adults into high, moderate and normal NPC risk groups by regression analysis of the levels of the EBV antibody. The high-risk groups of nasopharyngeal carcinoma risk could be followed-up every 3-6 month.@*RESULT@#NPC risk of 1379 adults was stratified according to serum levels of the 3 EBV antibodies. Eleven (0.8%) were identified to be of high risk for NPC, having high levels of all three antibodies and/or IgA EBNA level > 3 rod. Clinical examination of high risk subjects detected 5 NPC cases, 3 cases detected in the first instance and 2 in follow-up examination 3 to 6 months hence. Three cases were diagnosed with UICC Stage I tumor (60%), one in the first instance and 2 in follow-up, and the 5 cases account for all NPC cases detected from the entire cohort over 28 months(100%).@*CONCLUSION@#The new program affords an efficient and efficacious means for early NPC detection.

Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis

INTRODUCTION: Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations. OBJECTIVE: To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis. METHODS: In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis. RESULTS: Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82). In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9). CONCLUSION: Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking and shared epitope alleles were correlated with anti-cyclic citrullinated peptide-antibody-positive rheumatoid arthritis. Of the risk factors, only anticyclic citrullinated peptides antibodies were independently associated with rheumatoid arthritis susceptibility.

Relationship between clinical stages of nasopharyngeal carcinoma and Epstein-Barr virus antibodies Rta/IgG, EBNA1/IgA, VCA/IgA and EA/IgA / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the relationship between the clinical stages of nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) antibodies Rta/IgG, EBNA1/IgA, VCA/IgA and EA/IgA.</p><p><b>METHODS</b>Serum samples obtained from 211 untreated patients with NPC categorized by the project of 92' stage were examined for the presence of the EBV antibodies Rta/IgG and EBNA1/IgA by enzyme-linked immnunosorbent assay (ELISA) and for VCA/IgA and EA/IgA by immunoenzymatic assay. The positive rates and antibody levels in the NPC patients in different TNM stages and clinical stages were analyzed statistically.</p><p><b>RESULTS</b>No significant difference in Rta/IgG rA value was found in the NPC patients in different TNM or clinical stages (P>0.05). The EBNA1/IgA rA value was significantly lower in stage T1, N0, and clinical stage I than in the other corresponding T stages, N stages and other clinical stage (P<0.05). The antibody titers of VCA/IgA and EA/IgA differed significantly between the N stages and the clinical stages (P<0.05).</p><p><b>CONCLUSION</b>The expression of EBV Rta/IgG is not associated with NPC stage. The expression of EBNA1/IgA is relatively low in early NPC. The antibody level of VCA/IgA and EA/IgA are significantly correlated to the degree of neck lymph node metastasis, and might be helpful to classify the clinical stages of NPC.</p>

Clinicopathologic features and protein expression study of Epstein-Barr virus-associated gastric carcinoma in Guangzhou / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of Epstein-Barr virus (EBV)-associated gastric carcinomas in Guangzhou, their clinicopathologic features and related protein expressions including DNMT1, p16, and cyclin D1.</p><p><b>METHOD</b>A total of 676 cases of EBV-associated gastric carcinoma were included in the study. The presence of EBV-encoded small RNA1 (EBER1), a marker for EBV infection, was analyzed by in-situ hybridization using formalin-fixed and paraffin-embedded tumor samples. Expression of EBV-encoded proteins, DNMT1, p16 and cyclin D1 were detected by immunohistochemistry.</p><p><b>RESULTS</b>Forty-five of 676 gastric carcinomas showed EBER intranuclear positivity in all tumor cells. EBV involvement was significantly more frequent among the male than the female patients, especially in tumors of less differentiated types (diffuse type) and involving the upper stomach (P < 0.05). EBNA1 and LMP2A expression were detected in 42 (93.3%) and 24 (53.3%) cases, respectively. None expressed EBNA2, LMP1, and ZEBRA. Among 45 cases of EBV associated gastric carcinomas, DNMT1, p16 and cyclin D1 expression were seen in 35 (77.8%), 10 (22.2%), and 29 (64.4%) cases, respectively. In contrast, among 40 EBV negative gastric carcinomas, expression of the three proteins were 20 (50.0%), 25 (62.5%) and 12 (30.0%), respectively. The difference of expression of the three proteins between the two groups was significant (P < 0.05). Expression of p16 correlated with the depth of the tumor invasion. Correlated protein expression was seen between LMP2A and DNMT1, between DNMT1 and p16, and between p16 and cyclin D1 (P < 0.05).</p><p><b>CONCLUSIONS</b>EBV associated gastric carcinoma accounts for 6.7% of gastric carcinomas in Guangzhou with the Latency I pattern in some cases and between Latency I and II in others. The correlated expression of LMP2A, DNMT1, p16 and cyclin D1 may contribute to the pathogenesis of EBV associated gastric carcinomas.</p>

Transient localized hemophagocytosis in pleural effusion

Epstein-Barr virus [EBV] is one of the most important causes of hemophagocytic syndrome. We report a 76-year-old man who presented with pneumonia like symptoms and pleural effusion following upper respiratory tract infection. He underwent thoracentesis and pleural fluid cytology revealed large number of histiocytic macrophages that had phagocytosed RBCs and other inflammatory cells like lymphocytes and neutrophils. Pleural fluid analysis showed Epstein-Barr virus Antigen [EBNA] as the causing agent and corticosteroid therapy was initiated. A few days later, pleural effusion subsided and further cytologic examinations revealed no trace of hemophagocytosis

Epstein-Barr virus nuclear antigen-2 detection and typing in immunocompromised children correlated with lymphoproliferative disorder biopsy findings

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, plays a significant role as a cofactor in the process of tumorigenesis, and has consistently been associated with a variety of malignancies especially in immunocompromised patients. Forty-four children and adolescents (21 liver transplant patients, 7 heart transplant, 5 AIDS, 3 autoimmune hepatitis, 2 nephritic syndromes, 2 medullar aplasia, 2 primary immunodeficiency disorder patients, 1 thrombocytopenic purpura and 1 systemic lupus erythematosus) presenting with chronic active EBV infection (VCA-IgM persistently positive; VCA-IgG > 20 AU/mL and positive IgG _ EBNA) had peripheral blood samples obtained during clinically characterized EBV reactivation episodes. DNA samples were amplified in order to detect and type EBV on the basis of the EBNA-2 sequence (EBNA2 protein is essential for EBV-driven immortalization of B lymphocytes). Although we have found a predominance of type 1 EBNA-2 virus (33/44; 75 percent), 10 patients (22.73 percent) carried type 2 EBNA-2, and one liver transplant patient (2.27 percent) a mixture of the two types, the higher proportion of type 2 EBV, as well as the finding of one patient bearing the two types is in agreement with other reports held on lymphoproliferative disorder (LPD) patients, which analyzed tumor biopsies. We conclude that EBNA-2 detection and typing can be performed in peripheral blood samples, and the high prevalence of type 2 in our casuistic indicates that this population is actually at risk of developing LPD, and should be monitored.

Presence of Epstein- Barr virus in oral Lichen planus and normal oral mucosa

Oral lichen planus [OLP] is a chronic immunologic disease. The etiology of OLP is unknown, viral antigens [for example Epstein-Bar virus] have been proposed as etiologic agents. OLP may get transformation to malignancy so research on the presence of EBV in OLP lesions seems to be necessary. The aim of this study was to ascertain if EBV acted as etiologic factor in pathogenesis of OLP. Tissue specimens of 22 patients with clinical diagnosis and histopathological confirmation of OLP were used as case group. And that of 22 persons without OLP served as control group. Polymerase Chain Reaction [PCR] method was used. Each sample was tested twice. All biopsy specimens from patients and controls were negative for EBV presence. In spite of the fact that the presence of EBV in OLP in these two small groups of Iranian population was not confirmed with PCR method, but due to different ideas and reports in this field, proving or disproving of presence or etiological role of EBV in OLP is continuously a question and needs to be examined in further studies

Infecção pelo vírus Epstein-Barr em pacientes com lúpus eritematoso sistêmico / Epstein-Barr virus infection in patients with systemic lupus erythematosus

OBJETIVO: Verificar a associação entre a atividade do lúpus eritematoso sistêmico (LES) e a avidez das imunoglobulinas IgG anti-EBV. MÉTODOS: Analisou-se o sangue periférico de 66 pacientes distribuídos em dois grupos: 22 pacientes com LES em atividade e 44 pacientes com doença inativa. A presença e o índice de avidez de anticorpos IgG anti-EBV foram determinados pela técnica ELISA. (Enzygnost anti-EBV - Dade Behring). RESULTADOS: Identificou-se positividade no teste de detecção de IgG para EBV em 21 (95,5 por cento) pacientes do grupo LES ativo e em 40 (90,9 por cento) do grupo LES inativo. O índice de avidez alcançou valores 40 em 54 (88,5 por cento) pacientes, sendo 34 (85 por cento) do grupo LES inativo e 20 (95,2 por cento) do grupo LES ativo; em cinco (12,5 por cento) pacientes do grupo LES inativo, este índice ficou entre 20 e 40 e foi inferior a 20 em dois (3,3 por cento) pacientes. Adotando-se 20, 30 ou 40 como ponto de corte do índice de avidez, para diagnóstico de reativação da infecção por EBV, foram classificados como infecção reativada, nos grupos LES ativo e inativo, respectivamente: 1 (4,8 por cento) x 5 (12,5 por cento) pacientes, 1 (4,8 por cento) x 4 (10 por cento) pacientes e 1 (4,8 por cento) x 5 (12,5 por cento) pacientes. CONCLUSÃO: No presente estudo, não foi demonstrada associação entre a atividade do LES e a reativação do EBV. Esse fato parece indicar que a não eliminação dos linfócitos B infectados se deve à falha no mecanismo de apoptose ou à ação de linfócitos T citotóxicos, permitindo assim a progressão do LES.

OBJECTIVE: To verify the association of SLE activity to the avidity of IgG anti-EBV immune globulins. METHODS: Peripheral blood of 66 patients was analyzed, 22 had active SLE and 44 had inactive SLE. Presence and avidity index of IgG anti-EBV antibodies were determined by the ELISA method (Enzygnost® anti-EBV/IgG - Dade Behring). RESULTS: IgG anti-EBV test was positive for 21 (95.5 percent) patients in the active SLE group and 40 (90.9 percent) in the inactive group. The avidity index was 40 for 54 (88.5 percent) patients of which 34 (85 percent) belonged to the inactive SLE group and 20 (95.2 percent) to the active group. For 5 (12.5 percent) inactive SLE patients, the avidity index reached values ranging from 20 to 40; while for only 2 (3.3 percent) patients this index was lower than 20. Adopting 20, 30 or 40 as a cutoff point of the avidity index for diagnosis of reactivation of the EBV infection, the author classified as having reactivated infection, for active and inactive SLE groups, respectively: 1 (4.8 percent) x 1 (2.5 percent) patient; 1 (4.8 percent) x 4 (10 percent) patients and 1 (4.8 percent) x 5 (12.5 percent) patients. CONCLUSION: Association between EBV activity and SLE was not demonstrated. This appears to indicate that persistence of infected B lymphocytes may be due to failure in the apopotosis mechanism or to the action of T cytotoxic lymphocytes, permitting evolution of SLE.

Epstein-barr nuclear antigen-1 (EBNA-1) in diffuse large B-cell lymphoma and its relationship to the bcl-2 protein.

AIM: To determine EBNA-1 expression in the tissue of patients with diffuse large B-cell lymphoma and its relationship to bcl-2 expression. METHODS: Paraffin-embedded tissue from 24 cases of diffuse large B-cell lymphoma were stained immunohisto chemically with monoclonal antibody anti-EBNA-1 and anti-bcl-2 using Streptavidin Biotin Complex method. The bcl-2 expression was measured as negative (no staining), positive 1 (weak staining), and positive 2 (moderate to strong staining). The relationship between the immunohisto chemistry results was examined. RESULTS: From the 24 samples, 12 (50%) were EBNA-1 positive and 14 (58.3%) showed expression of bcl-2. Eleven of the 14 samples (90%) showed expression of bcl-2 were EBNA-1 positive, while only 3 of these samples (10%) were EBNA-1 negative. The relationship between bcl-2 expression and EBNA-1 was statistically significant (Chi square-uncorrected 11.2571, p= 0.0036). CONCLUSION: Our results showed that 50% of diffuse large B-cell lymphoma were EBNA-1 positive. Furthermore, EBNA-1 seemed to be correlated with bcl-2 expression.

Virus de Epstein-Barr y su relación con el desarrollo de enfermedades linfoproliferativas / Epstein-Barr virus and itïs association with lymphoprolipherative diseases

El Virus de Epstein Darr (VED) es un y herpes virus que infecta al huésped y persiste durante toda la vida del mismo. Se asocia con un espectro variado de desórdenes clínicos desde infecciones agudas y crónicas hasta neoplasias linfoides y epiteliales y se cree que contribuiría en la patogénesis de los mismos. La asociación del VEB con varias neoplasias linfoides es bastante contundente, indicando un rol etiopatogénico en su desarrollo. Sin embargo, dado que la infección por el VED es prácticamente ubicua en adultos sanos, es difícil establecer su rol causal en la génesis de linfomas. La infección primaria generalmente es asintomática aunque en adolescentes y adultos jóvenes frecuentemente se manifiesta con el síndrome de mononucleosis infecciosa. Luego del primer contacto, el huésped controla al virus a través de una respuesta T específica. Sin embargo, el VEB ha desarrollado estrategias que le permiten minimizar o eliminar su potencial patogénico para mantener la infección y la sobrevida del huésped en el cual persiste. Al desregular el crecimiento celular, favorece la aparición de alteraciones gen éticas con la consiguiente transformación celular y desarrollo de linfomas.

Progress of EBNA1/oriP-based plasmid applied in gene therapy / 生物工程学报

The nonviral gene delivery systems are usually not very effective in transferring gene into target cells, and the intensity and duration of the gene expression is very poor. The EBNA1/oriP maintain EBNA1/oriP-based plasmids as episome, contribute to nuclear transport of the plasmid and transcriptional up-regulation of target gene. The EBNA1/oriP based plasmid enhances the transfection rate as well as magnitude and longevity of gene expression. This article reviews recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, the EBNA1/ oriP based plasmid encoding the HSV1-TK suicide gene was combined with a cationic polymer to transfer into HCC cell line. The expression level of TK gene was 100- to 1000-fold higher than the conventional plasmid. The sensitivity of HCC to ganciclovir (GCV) elevated several hundred-fold. The EBNA1/oriP based plasmid equipped with tumor specific promoter, such as CEA promoter, enabled targeted killing of CEA-positive tumor cell. Transfection of EBNA1/oriP based plasmid carrying IL-12 and IL-18 gene either locally, or systemically, induced therapeufic antitumor immune responses including augmentation of the cytotoxic T lymphocyte and natural killer activities and growth retardation of tumors. For gene therapy of congenital diseases and chronic diseases, the EBNA1/oriP based plasmid encoding the adenosine deaminase gene was transfered into human hematopoietic progenitor cells. The ADA activity was elevated 1.5-to 2-fold. Intracardiomuscrlar transfer of the EBNA1/oriP based plasmid encoding the beta-AR gene may be useful for the treatment of severe heart failure. Human tumor necrosis factoralpha (hTNFalpha) is one of the most important inflammatory cytokines. It has been implicated in many autoimmune and inflammatory diseases. sTNFR can efficiently neutralize the bioactivities of hTNFalpha. In primary study we cloned the chimeric protein sTNFR II-IgG Fc and expect to use it in the gene therapy of the inflammatory disease relative to TNF. In summary, The EBNA1/oriP based plasmid shows advantage in gene therapy of cancer, congenital and inflammatory diseases. Moreover, the EBNA1/oriP element may greatly contribute to the engineering of a human artificial chromosome, the ultimate device for controllable gene therapy.