Epstein–Barr virus infection in non-carcinomatous gastric epithelium

Gastric tissue specimens from 20 patients with chronic atrophic gastritis, one of whom also had an early gastric carcinoma, were studied for evidence of Epstein–Barr virus (EBV) infection by Southern blot analysis, DNA and RNA in situ hybridization, and immunohistochemistry for the presence of the EBV-determined nuclear antigen 1 (EBNA-1) and the latent membrane protein 1 (LMP-1). EBV DNA was detected in two cases with chronic atrophic gastritis and in the case with early gastric carcinoma by Southern blot hybridization. DNA in situ hybridization showed EBV genomes in the epithelial cells of two other cases with chronic atrophic gastritis and in non-carcinomatous and carcinomatous epithelial cells of the early gastric carcinoma case. EBNA-1 was detected in all cases. LMP-1 was detected in areas of intestinal metaplasia in eight patients with chronic atrophic gastritis. EBV-encoded small RNA 1 (EBER-1) expression was limited to carcinoma cells. These results show that gastric epithelium is frequently infected with EBV and suggest that prolonged EBV persistence may contribute to the development of gastric carcinoma. © 1997 John Wiley & Sons, Ltd.     

Epstein–Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti‐DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities – anti‐dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two‐thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two‐thirds of SLE patients reacted to a large spectrum of self‐molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein–Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.     

Epstein–Barr virus infection induces genome‐wide de novo DNA methylation in non‐neoplastic gastric epithelial cells

Epstein–Barr virus (EBV)‐positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome‐wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low‐methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non‐neoplastic epithelial cells remains unclear. Here we performed post‐infection time‐series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome‐wide analysis using Illumina's Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post‐infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC‐specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV‐positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well‐ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower‐CpG peripheral regions and extending to higher‐CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non‐neoplastic cells within a month in a spatiotemporally well‐ordered manner. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cytomegalovirus and Epstein–Barr virus coinfection in three toddlers with prolonged illnesses

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) usually cause primary and latent infections during childhood; thus, coinfection with these viruses occurs occasionally in children. However, its clinical impact has not been established, and may be underestimated. Three cases of coinfection involving these two viruses in toddlers are described: a 14‐month‐old male with infectious mononucleosis, an 18‐month‐old female with hemophagocytic lymphohistiocytosis, and a 13‐month‐old female with acute hepatitis. All three patients had prolonged illnesses. Serial serological testing and quantitation of viral DNA for CMV and EBV using peripheral blood from the patients suggested primary infections with both viruses. In all three cases, the viral load of EBV and CMV in the early stage of disease exceeded 6.4 × 10³ and 8.8 × 10² copies/ml of whole blood, respectively, suggesting that the viruses were associated with the clinical condition. Recognizing that coinfection with these viruses may modulate the clinical course of disease is important. J. Med. Virol. 81:1399–1402, 2009. © 2009 Wiley‐Liss, Inc.     

Epstein–Barr virus in Reed–Sternberg G-like cells in non Hodgkin's lymphomas

In the course of our study on Hodgkin's disease (HD), ten cases of non-Hodgkin's lymphomas (NHL) containing Hodgkin and Reed-Sternberg-like (MRS) cells were encountered. Many of these cases had initially been diagnosed as HD, but on careful review of the histology, with the aid of immunophenotyping studies, they were reclassified as NHL. The presence of Epstein–Barr virus (EBV) in these HRS-like cells was investigated using a combination of EBER in situ hybridization (ISH) and immunostaining for the detection of EBV-encoded latent membrane protein (LMP). HRS-like cells in four cases (two lymphoplasmacytoid lymphomas, one Richter's transformation of lymphoplasmacytoid lymphoma, and one immunoblastic lymphoma of T-cell type) were found to be EBV-positive. In two of these cases, a second biopsy taken up to 10 years later also contained EBV in the HRS-like cells. In three of the four cases, HRS-like cells expressed the activation antigen CD30, but the expression of B- or T-cell antigens was variable. All cases of T-cell-rich B-cell lymphomas were negative for EBV. In conclusion, EBV may play a role in the development of HRS-like cells i some cases of NHL. The relationship of HRS-like cells to HRS cells of HD is discussed.     

Epstein–Barr virus (EBV) provides survival factors to EBV+ diffuse large B‐cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+ DLBCL

Diffuse large B‐cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non‐Hodgkin lymphomas. Epstein–Barr virus (EBV) ‐positive DLBCL of the elderly is a newly recognized subtype that accounts for 8–10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL‐derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin‐4 and interleukin‐21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4‐ or CCR7‐mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant‐negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV‐positive lines. These results show that EBV plays an important role in EBV‐positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV‐carrying DLBCL cells and might have therapeutic implications .     

Disappearance of the Epstein–Barr virus in a relapse of Hodgkin's disease

Hodgkin's disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case of nodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negative in the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This finding implies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not required to maintain neoplastic growth of HD tumour cells. © 1997 John Wiley & Sons, Ltd.     

Frequent detection of Epstein–Barr virus-infected B cells in peripheral T-cell lymphomas

Although Epstein–Barr virus (EBV) positivity has been described in peripheral T-cell lymphomas (PTCLs) in Chinese patients, the cellular lineage of EBV-harbouring cells is unknown. Forty-four cases of PTCL were therefore studied by in situhybridization (ISH) for EBV-encoded small non-polyadenylated RNA 1 and 2 (EBER), and the lineage of the EBER+ cells was determined by double labelling. The findings were further correlated with the clonality of EBV and the genotype of these EBER+ tumours. The results for the detection of EBV by ISH show that 23 of the 44 cases were EBER+. In 5/23 of the EBER+ cases, EBER was found in around 50 per cent of atypical cells and in 18/23 cases, EBER was found in a subpopulation of atypical cells. Amongst the EBER+ cases, all 15 tested showed clonal T-cell receptor gene rearrangement by Southern blot hybridization. Double labelling was successfully done in 11 EBER+ cases, and by comparison, EBER+/CD20+ B cells outnumbered the EBER+/CD3+ T cells in all these cases. EBV clonality analysis revealed that EBV was monoclonal in six EBER+ cases and biclonal in three cases. With the predominance of EBV+ B cells over EBV+ neoplastic T cells being observed in most of these cases, it is possible that the EBV-infected clonal population may be of B-cell lineage. This was supported in some cases where a faint clonal band was seen over a background smear in the gene rearrangement study of immunoglobulin heavy chain gene by polymerase chain reaction (PCR), indicating a minor B-cell clone. It is concluded that in EBV+ PTCL, EBV is preferentially localized in B cells rather than neoplastic T cells. The neoplastic T cells may support the clonal proliferation of a subpopulation of EBV+ B cells in PTCLs. © 1998 John Wiley & Sons, Ltd.     

Antigen‐specific over‐expression of human cartilage glycoprotein 39 on CD4+CD25+ forkhead box protein 3+ regulatory T cells in the generation of glucose‐6‐phosphate isomerase‐induced arthritis

Human cartilage gp‐39 (HC gp‐39) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the exact localization, fluctuation and function of HC gp‐39 in RA are unknown. Therefore, using a glucose‐6‐phosphate isomerase (GPI)‐induced model of arthritis, we investigated these aspects of HC gp‐39 in arthritis. The rise in serum HC gp‐39 levels was detected on the early phase of GPI‐induced arthritis (day 7) and the HC gp‐39 mRNA was increased significantly on splenic CD4⁺T cells on day7, but not on CD11b⁺cells. Moreover, to identify the characterization of HC gp‐39⁺CD4⁺T cells, we assessed the analysis of T helper (Th) subsets. As a result, HC gp‐39 was expressed dominantly in CD4⁺CD25⁺ forkhead box protein 3 (FoxP3)⁺ refulatory T cells (T_reg), but not in Th1, Th2 or Th17 cells. Furthermore, to investigate the effect of HC gp‐39 to CD4⁺T cells, T cell proliferation assay and cytokine production from CD4⁺T cells using recombinant HC gp‐39 was assessed. We found that GPI‐specific T cell proliferation and interferon (IFN)‐γ or interleukin (IL)‐17 production were clearly suppressed by addition of recombinant HC gp‐39. Antigen‐specific over‐expression of HC gp‐39 in splenic CD4⁺CD25⁺ FoxP3⁺ T_reg cells occurs in the induction phase of GPI‐induced arthritis, and addition of recombinant HC gp‐39 suppresses antigen‐specific T‐cell proliferation and cytokine production, suggesting that HC gp‐39 in CD4⁺ T cells might play a regulatory role in arthritis.     

Role of DNA methylation in the development of Epstein–Barr virus‐associated gastric carcinoma

The frequencies of DNA methylation of certain tumor‐related genes are higher in Epstein–Barr virus (EBV)‐associated gastric carcinomas than in EBV‐negative gastric carcinomas. EBV‐associated gastric carcinomas have distinct clinicopathological features; however, there are no case‐control studies comparing methylation frequency between EBV‐associated gastric carcinomas and controls that have been adjusted according to the clinicopathological features of EBV‐associated gastric carcinomas. This study evaluated 25 EBV‐associated gastric carcinomas that were positive for EBV‐encoded small RNA 1 (EBER‐1) by in situ hybridization and 50 EBV‐negative gastric carcinomas that were matched with the EBV‐associated gastric carcinomas by age, sex, histology, depth of tumor invasion, and stage. Methylation status of 16 loci associated with tumor‐related genes was analyzed by methylation‐specific polymerase chain reaction (PCR) to identify genes in which DNA methylation specifically occurred in EBV‐associated gastric carcinomas. Methylation frequencies of 12 of the 16 genes were higher in EBV‐associated gastric carcinomas than in EBV‐negative controls, and the frequency of methylation of 6 specific loci (MINT2, MINT31, p14, p16, p73, and RUNX3) was significantly higher in EBV‐associated gastric carcinomas than in EBV‐negative controls. There were no significant differences in the methylation frequencies of the other genes. The mean methylation index in EBV‐associated gastric carcinomas was significantly higher than that in EBV‐negative controls. DNA methylation of tumor suppressor genes that regulate the cell cycle and apoptosis specifically occurred in EBV‐associated gastric carcinomas. Aberrant DNA methylation might lead to the development and progression of EBV‐associated gastric carcinoma. J. Med. Virol. 85:121–127, 2012. © 2012 Wiley Periodicals, Inc.     

Modulation of interleukin-6 expression in Hodgkin and Reed–Sternberg cells by Epstein–Barr virus

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein–Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed–Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce interleukin (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (31 of 37, 84 per cent) as compared with LMP1-negative HD cases (35 of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD. © 1997 John Wiley & Sons, Ltd.     

Detection of Epstein–Barr virus‐specific memory CD4+ T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Approaches to evaluate T‐cell responses to Epstein–Barr virus (EBV) include enzyme‐linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon‐γ secretion upon antigen stimulation. However, evaluation of expandable EBV‐specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV‐specific T‐cell precursors with high proliferative capacity by using a peptide‐based cultured interferon‐γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15‐mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV‐specific T‐cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T‐cell responses quantified by cultured ELISPOT were mainly mediated by CD4⁺ T cells and a marked pattern of immunodominance to latent‐phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV‐specific T‐cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung‐transplanted patient with EBV‐associated diseases. Analysis of T‐cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV‐related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.     

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients. Copyright © 1999 John Wiley & Sons, Ltd.     

CD21−/low B cells associate with joint damage in rheumatoid arthritis patients

Depletion of B cells is beneficial in rheumatoid arthritis (RA) patients with autoantibodies to citrullinated proteins (ACPA) and/or the Fc portion of immunoglobulins (rheumatoid factor [RF]), suggesting a role for B cells in disease pathogenesis. To date, however, the identity of specifically pathogenic B cell subsets has not been discovered. One candidate population is identified by the low expression or absence of complement receptor 2 (CD21^?/low B cells). In this study, we sought to determine whether there was any correlation between CD21^?/low B cells and clinical outcome in patients with established RA, either ACPA⁺/RF⁺ (n = 27) or ACPA^?/RF^? (n = 10). Healthy donors (n = 17) were included as controls. The proportion of the CD21^?/low CD27^?IgD^? memory B cell subset in peripheral blood (PB) was significantly increased in ACPA⁺/RF⁺ RA patients compared with healthy donors, and the frequency of this subset correlated with joint destruction (r = 0.57, P < 0.04). The levels of the chemokines CXCL‐9 and CXCL‐10 were higher in synovial fluid than in plasma, and PB CD21^?/low cells expressed the receptor, CXCR3. In synovial fluid, most of the B cells were CD21^?/low, approximately 40% of that population was CD27^?IgD^?, and a third of those expressed the pro‐osteoclastogenic factor receptor activator of the nuclear factor κB ligand (RANKL). This subset also secreted RANKL, in addition to other factors such as IL‐6, even in the absence of stimulation. We interpret these data as reason to propose the hypothesis that the CD27^?IgD^? subset of CD21^?/low B cells may mediate joint destruction in patients with ACPA⁺/RF⁺ RA.