Influence of Epstein–Barr virus infection in adult T-cell leukemia

Abstract

The involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in about 50% of cases of ATL. Epstein–Barr virus (EBV) infection has often been observed in the clinical course of ATL. In this study, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both lines. As part of the characterization of these cells, an enhanced expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) or ICAM-3 (ICAM-3) (CD50), lymphocyte function-1 (LFA-1) (CD11a/CD18), and Mac-1 (CD11b/CD18) was observed. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor's lymphocytes, an EBV-infected B cell line, Raji, and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was also observed in double transfectants (EBV and HTLV-1). In the clinical course of ATL, LMP-1, EBNA-2, CD50 and CD54 were detected in lymph nodes and skin specimens by immunohistochemical staining. Furthermore, high levels of interleukin-4 (IL-4) were detected in these cell lines and transfectants. The results indicated that coinfection with HTLV-1 and EBV may induce aggressive organ involvement through the enhanced expression of adhesion molecules via IL-4 signaling. A new mechanism of ATL involvement is discussed.     

Systemic lupus erythematosus due to Epstein–Barr virus or Epstein–Barr virus infection provocating acute exacerbation of systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is a rheumatologic disease characterized by an inflammatory destruction of the target organ systems of the body in an unknown way by autoantibodies formed against self-antigens. Infectious agents like Epstein–Barr virus (EBV), cytomegalovirus and parvovirus B19 may have a role in the occurrence or the exacerbation of the SLE. In this report, the clinical follow-up of a 14-year-old girl diagnosed with SLE following an EBV infection with bicytopenia, lymphadenomegaly and hepatomegaly is discussed. This case could support the role of viral infections in the etiology of SLE.     

From the Cover: Efficient replication of Epstein–Barr virus in stratified epithelium in vitro

Epstein–Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi’s-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.Although the association between Epstein–Barr virus and epithelial malignancies has been known for more than three decades, the EBV life cycle within the epithelial milieu is still only poorly understood. In contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and by the ability of some EBV-positive B-cell tumors to give rise to cell lines that maintain restricted programs of latency gene expression similar to those seen in vivo. Although in primary EBV infection the entire complement of EBV latency-associated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP) and membrane proteins (LMPs 1, 2A, and 2B) promote cellular proliferation and survival (Latency III), EBV gene expression must be progressively silenced (Latency II; EBNA1 and LMPs 1 and 2) so that the most restricted program, Latency 0 (in which EBV gene expression is believed to be completely silenced), is achieved in resting memory B cells that serve as the long-term reservoir of latent EBV (1, 2). Because EBNA1 functions to maintain the viral genome during cell division, it alone among the viral protein repertoire is required during periodic proliferation of these memory B cells (Latency I).EBV is strongly associated with subtypes of nasopharyngeal carcinoma and gastric carcinoma, in which it exhibits a Latency I/II program of gene expression (3–5). The presence of latent EBV in epithelial malignancies suggests that EBV might establish a latent infection within primary epithelial cells. In biopsies of oral hairy leukoplakia (OHL), a benign hyperplastic lesion that occurs in immunocompromised individuals, active EBV replication is detected in suprabasal layers of lingual epithelium (6), providing convincing evidence that EBV can infect epithelial cells and undergo productive replication. Similar patterns could be found in biopsies of normal tongue tissue, but in only a small fraction of samples (3 of 217) (7). In contrast, little evidence of latently infected cells is observed, although such cells might be rarer and more difficult to detect. Indeed, possible latently infected epithelial cells are detected in tonsil explants in the presence of acyclovir (an inhibitor of lytic cycle-specific herpesvirus DNA replication), but in less than 0.01% of cells (8).Unfortunately, EBV-infected cell lines cannot be established readily from epithelial tumors, and primary epithelial cells are infected inefficiently in vitro. Nevertheless, EBV infection of primary keratinocytes in monolayer culture results in latency, as evidenced by the expression of Epstein–Barr virus RNA (EBER), with variable expression of EBNA-1 and LMP-1, but the infected cells fail to proliferate (8–10). Although these problems have hindered progress in understanding the life cycle of EBV in epithelial cells, a few EBV-negative epithelial tumor cell lines can be infected with EBV, resulting in Latency II gene expression, and have provided some insight. One notable example is EBV tropism. Although EBV must cross the oral mucosa to gain access to either the B-cell compartment or the oral cavity, whether it infects oral mucosal epithelial cells (with or without a latent infection) to amplify the virus pool or merely crosses the epithelial barrier by transcytosis has been debated (11, 12). However, tropism is controlled during in vitro infection by differences in the glycoprotein profile of virions derived from B versus epithelial cells (13), and virus isolated from the saliva of normal individuals is consistent with that derived from epithelial cells (14). Taken together, these observations suggest that EBV indeed can replicate within the epithelial cell compartment in vivo.To assess better the outcome(s) of EBV infection in an environment more representative of normal epithelium in the host, we examined EBV infection within organotypic (raft) cultures of primary oral keratinocytes. Although these cultures do not contain the complexity of tissues and interactions found in vivo, in all other respects the stratified layers of differentiated tissue generated in vitro resemble the differentiated epithelium, where EBV has been detected in vivo. We demonstrate that EBV is able to infect keratinocytes in raft cultures, and, although we were unable to detect latently infected cells, we readily observed production of virus in the suprabasal layers that was readily disseminated throughout the epithelium, resulting in numerous productively replicating cells. Thus, EBV is able to infect normal epithelial cells and enter the productive cycle to expand the virus pool.     

Study on the pathogenetic effect of salted pork from a high risk area of stomach cancer in China

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Evasion of affinity-based selection in germinal centers by Epstein–Barr virus LMP2A

Epstein–Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell–specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.Epstein–Barr virus (EBV) is a B-lymphotropic human herpesvirus associated with a variety of hematopoietic cancers, including endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoproliferative disorders in immune-compromised people, nasopharyngeal carcinoma, and some gastric cancers (1, 2). Nevertheless, EBV establishes lifelong latent infection by adolescence in more than 95% of adults worldwide. EBV efficiently infects resting human B cells in vitro, leading to their activation and proliferation. EBV-infected B cells grow in vitro as immortalized lymphoblastoid cell lines (LCL). LCLs express EBV-encoded proteins including the EBV nuclear antigens (EBNAs) EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNALP and the latent membrane proteins LMP1, LMP2A, and LMP2B. Of these EBV gene products, EBNA2, EBNALP, EBNA3A, EBNA3C, and LMP1 are essential for B-cell transformation (3), whereas EBNA1 is necessary for EBV episome persistence (4).LMP2A consists of an N-terminal cytoplasmic domain, 12 membrane-spanning domains, and a short C-terminal cytoplasmic domain. The N-terminal cytoplasmic domain has an immune receptor tyrosine-based activation motif (ITAM), which can recruit the protein tyrosine kinases Syk and Src (5). In LCLs or mouse cells that ectopically express LMP2A, LMP2A suppresses B-cell antigen receptor (BCR) signaling (6–8). In mice lacking BCR expression, transgenic LMP2A can partially rescue B-cell differentiation (9–11), indicating that LMP2A can mimic tonic BCR signals necessary for B-cell survival in vivo (12). Likewise, LMP2A is essential for growth transformation of germinal center (GC)-derived BCR⁻ B cells (13). Depending on its B-cell expression level in murine models, constitutive LMP2A expression alters B-cell development or augments the differentiation and activation of antigen-driven B cells (9, 10, 14–16).In the peripheral blood of persistently infected people, EBV resides exclusively in IgD⁻ memory B cells (17). However, in tonsils, EBV is found not only in memory B cells but also in GC and IgD⁺ resting B cells in which EBNA1, LMP1, and LMP2A are expressed (17, 18). Thereby, EBV may use antigen-driven B-cell activation and differentiation in mucosal secondary lymphoid organs, particularly GC reactions, to establish persistent infection in the memory B-cell pool. Together with LMP1, which constitutively activates CD40 signaling (19, 20), LMP2A may provide a survival advantage for EBV-infected B cells by modifying GC B-cell selection. However, B-lineage–specific expression of LMP1 inhibits GC formation (20) or causes B-cell ablation through immune surveillance (21). LMP2A does not affect affinity maturation of B cells in GCs when expressed in B cells of a transgenic mouse line (22). Constitutive expression in transgenic mice used in these previous studies may be inappropriate for studying the influence of infection-induced virus proteins on B-cell differentiation, particularly in GCs. To date, the impact of LMP2A on differentiation and selection of GC B cells is still controversial.To explore the possibility that LMP2A contributes to persistent EBV infection by modifying GC B-cell selection, we generated an experimental mouse system in which LMP2A is conditionally expressed in GC B cells, similar to LMP2A expression in EBV-infected human B cells. Using this system, we analyzed the effects of LMP2A expression on B-cell entry into and differentiation within GCs.     

The effect of Epstein–Barr virus status on clinical outcome in Hodgkin’s lymphoma

The association of Epstein–Barr virus (EBV) with Hodgkin’s lymphoma (HL) has been investigated over the last few years. The impact of EBV on clinical outcome is still controversial, however. In this study, we investigated the effect of EBV status on clinical outcome of HL patients. Between January 1986 and September 2004, fifty-six patients, diagnosed as having HL, were included in the analysis. Clinical data were reviewed retrospectively from the patients’ records. Tissues from 56 patients were analyzed for the presence of EBV using the in situ hybridization (ISH) for EBV-encoded RNA (EBER) and immunohistochemistry for latent membrane protein (LMP)1. EBV infection was identified in 41.1% of cases by EBER ISH, 26.8% by LMP1 expression, and 26.8% by LMP1 and EBER ISH. EBER-positive HL were significantly more frequent in mixed cellularity (MC) subtype (P=0.014) and advanced stage (P=0.034). There was a trend toward shorter overall survival in EBER-positive patients without statistical significance (P=0.238). LMP1 expression also correlated with MC subtype (P=0.006) and advanced stage (P=0.007), although it did not significantly influence the survival outcome. In subgroup analysis, both EBER and LMP1 positivities were associated with longer progression-free survival in patients with age <25 years old (P=0.045). Reverse trends were shown in patients ≥25 years old. In this study, we demonstrated that the impact of tumor EBV status on prognosis may be age dependent and young patients with latent EBV infection have favorable prognosis.J.M. Kwon, Y.H. Park, J.H. Kang contributed equally to this work.     

Epstein–Barr virus status is a promising biomarker for endoscopic resection in early gastric cancer: proposal of a novel therapeutic strategy

Journal of Gastroenterology - Epstein–Barr virus-positive gastric cancer (EBVGC) is associated with a low prevalence of lymph node metastasis (LNM); however, EBV status is not considered in...     

Epstein–Barr virus and methotrexate-related CNS lymphoma in a patient with rheumatoid arthritis

Abstract

Patients with rheumatoid arthritis (RA), especially those treated with methotrexate (MTX), might have an increased risk of lymphoproliferative disorders that are associated with Epstein–Barr virus (EBV). We describe a case of EBV-associated central nervous system (CNS) lymphoma (diffuse large B-cell lymphoma) in a patient with RA on a short course of MTX treatment. The neoplastic cells express the B-cell surface markers (CD20, Pax-5 and CD30), and EBV-encoded RNA was demonstrated by in situ hybridization. The patient’s lymphoma did not recur for the 8-year follow-up period after the tumor resection and cessation of MTX. MTX may promote EBV-positive CNS lymphoma in RA patient due to its immunosuppressive properties as well as reactivating latent EBV infection.     

Hepatitis B virus genotypes,phylogeny and occult infection in a region with a high incidence of hepatocellular carcinoma in China

AIM:To determine the genotypes and phylogeny of hepatitis B viruses(HBVs)in asymptomatic HBV carriers,and the prevalence of occult HBV infection in Long An County,Guangxi Zhuang Autonomous Region,an area with a high incidence of hepatocellular carcinoma. METHODS:A nested polymerase chain reaction(nPCR) was used for detection of HBV DNA in serum samples from 36 blood donors with asympmatic HBV infection,and in serum samples from 52 HBsAg negative family members of the children who did not receive hepatitis B vaccination in Long An County.PCR products were sequenced,and the genotype of each HBV sequence was determined by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases using the BLAST programme.Phylogenetic trees were constructed by the quartet maximum likelihood analysis using the TreePuzzle software. RESULTS:Twenty(55.56%)of 36 HBV asymptomatic carriers were positive for HBV DNA.They were all genotype C by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases.The full-length HBV DNA sequence isolated from the sample No.624 contained 3 215 bases.No interesting mutations were found in this isolate.The homology analysis showed that this strain was closer to the Vietnamese HBV genotype C strain,with a homology of 97%,compared its relation to the same genotype of HBV isolated in Shanghai.Six(11.5%) of the 52 HBsAg negative family members were positive for HBV DNA.A point mutation was found in the sample No.37,resulting in the substitution of amino acid glycine to arginine in the"a"determinant.Other samples with positive HBV DNA did not have any unusual amino acid substitutions in or around the"a"determinant,and were attributed to the wild-type HBV. CONCLUSION:The HBVs isolated from asymptomatic carriers of Long An County were all identified as genotype C,and the prevalence of occult HBV infection in the population of the county is as high as 11.5%.It is suggested that genotype C and persistent occult HBV infection may play an important role in the development of HCC in the county.     

Sulfasalazine-induced hypersensitivity syndrome and hemophagocytic syndrome associated with reactivation of Epstein–Barr virus

A 58-year-old woman with rheumatoid arthritis (RA) developed fever, skin eruptions, leukocytopenia, and thrombocytopenia, 3 weeks after treatment with sulfasalazine. A skin biopsy showed hydropic degeneration of keratinocytes and lymphocytic infiltrate. A bone marrow aspiration demonstrated an increased number of macrophages with hemophagocytosis. Although serologic tests for Epstein–Barr virus (EBV) indicated a previous infection, EBV deoxyribonucleic acid was detected in her serum by polymerase chain reaction. Cessation of sulfasalazine and administration of steroids led to dramatic improvement. This case illustrates that the hemophagocytic syndrome associated with reactivation of EBV can occur as part of drug hypersensitivity reactions in RA patients taking sulfasalazine.     

A case of acute cholecystitis without cholestasis caused by Epstein–Barr virus in a healthy young woman

Epstein–Barr virus (EBV) is known to be one of the causes of viral hepatitis, but its association with cholecystitis is known to be rare. Cholestasis by EBV-induced hepatitis might be a cause of acute cholecystitis in all of the recently reported cases. In contrast, we experienced the case of a 20-year-old woman who was infected with EBV and presented with acute cholecystitis without cholestasis.     

Clinical features of adult-onset chronic active Epstein–Barr virus infection: a retrospective analysis

We performed a retrospective analysis of patients with adult-onset chronic active Epstein–Barr virus infection (CAEBV). First, we analyzed five patients (aged 28–72) diagnosed at our hospitals with EBV-infected clonally proliferating T cells. Four patients were administered cyclophosphamide/doxorubicin/vincristine/prednisone (CHOP) chemotherapy, but no remarkable decrease of viral load was observed in three of the patients. The other patient died 19 days after initiation of CHOP treatment due to disease progression. Addition of high-dose cytarabine to the regimens of two of the patients was discontinued shortly after administration, due to the development of grade 4 pericardial effusion. Together, these regimens may be insufficient for treating adult-onset CAEBV. We next reviewed 23 adult-onset CAEBV patients, adding 18 previously reported patients to the five patients described in the present study. T cells were frequently infected (87%), whereas NK- and T-cell types are known to be almost equally prevalent in childhood-onset cases. The time duration from the onset of disease to initiation of treatment averaged 20 months. Reports showed that 12 patients died; seven patients died at an average of 8 months after initiation of treatment. Patients’ disease courses seemed to be rapidly progressive and more aggressive than those of childhood-onset cases. More cases must be studied to clarify clinical features and establish an optimal treatment strategy.     

Nonmuscle myosin heavy chain IIA mediates Epstein–Barr virus infection of nasopharyngeal epithelial cells

EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection.EBV is a nearly ubiquitous human γ-herpesvirus that causes B-cell lymphomas and nasopharyngeal carcinoma (NPC), indicative of tropism for both cell types (1–3). Until recently, the molecular mechanisms of EBV infection of B lymphocytes were better understood than the mechanisms of epithelial cell infection (4). EBV attachment to the B-cell membrane is mediated by interactions between EBV glycoprotein 350 (gp350) and complement receptor type 2 (CR2 or CD21) (5) or CD35 (6). EBV gp42 binding to HLA class II triggers EBV fusion with B cells in the presence of EBV glycoprotein B (gB) and gH/gL (7, 8). For epithelial cells, gH/gL and gB are important for EBV infection (4, 9, 10). Epithelial cells lack HLA class II expression; thus, gp42 cannot trigger EBV and cell fusion. Instead, gp42 inversely suppresses the infection (11), and an antibody against gp350 can enhance infections of CD21/CD35-negative epithelial cells (12). The gH/gL heterodimer is required for virus entry (4) and may be involved in binding (13), as well as fusion of EBV (14–17). However, the crystal structure of EBV gH/gL does not show any known fusion domain (18). It is now thought that gH/gL regulates the fusion function of gB (19). Binding of gH/gL to a subset of αv integrins (e.g., αvβ₅, αvβ₆, or αvβ₈) provides the initial trigger for gB-mediated fusion (16, 20, 21). However, E1D1(gH/gL) antibody or CL59(gH) antibody, with a different epitope, can impair epithelial cell infection (20, 22). Thus, multiple gH/gL domains are critical to EBV infection, and gH/gL may interact with proteins in addition to integrins. Direct interaction of EBV gB amino acids 23–431 with neuropilin 1 (NRP1) and its associated tyrosine kinases is critical for EBV infection of nasopharyngeal epithelial cells (NPECs). NRP1 knock-down or EBV pretreatment with soluble NRP1 suppresses EBV NPEC infection, whereas NRP1 overexpression enhances EBV infection (10). Confocal microscopy and experiments with inhibitors of macropinocytosis indicate that EBV enters NPECs through macropinocytosis and not through clathrin-mediated endocytosis (10).The principal obstacle to identifying factors that may enable more efficient EBV infection of NPECs and better understanding of the role of EBV in NPC is that EBV is remarkably inefficient in infection of primary or B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs. As a polycomb complex protein, BMI1 is a proto-oncogene. BMI1 has an important role in regulating proliferation, senescence, and self-renewal of stem cells (23, 24). BMI1 overexpression immortalizes human epithelial cells and mouse embryonic fibroblasts (25, 26). By optimizing the growth of BMI1-immortalized NPEC cultures, we found that BMI1-immortalized NPECs seeded at a 10-fold higher density than used in previous protocols and grew initially as a monolayer. “Spherical cells” then grew above the monolayer. Surprisingly, the spherical cells consistently supported an EBV infection efficiency of ∼20–30%, using an estimated EBV multiplicity of infection (MOI) of 300. Using this more efficient in vitro EBV infection protocol, we identified an interaction between nonmuscle myosin heavy chain IIA (NMHC-IIA) and gH/gL on the cell surface, which was critical for more efficient EBV NPEC infection.