Epstein-Barr Virus Lytic Reactivation Induces IgG4 Production by Host B Lymphocytes in Graves' Disease Patients and Controls: A Subset of Graves' Disease Is an IgG4-Related Disease-Like Condition.

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a newly recognized systemic fibroinflammatory disease with characteristic histological findings and high serum IgG4 levels. Epstein-Barr virus (EBV) is a persistent herpesvirus in B lymphocytes, and we previously reported EBV reactivation-induced Ig production. We showed that EBV reactivation induced the production of thyrotropin receptor antibodies, the causative antibodies of Graves' disease. In the present study, we investigated whether EBV reactivation induced IgG4 production and if EBV-positive B cells or IgG4-positive plasma cells are present in the thyroid tissues of Graves' disease patients with lymphoplasmacytic infiltration. EBV-encoded small RNA1 (EBER1) in situ hybridization and immunohistochemistry for IgG and IgG4 were performed on seven resected thyroid tissues with lymphoplasmacytic infiltration collected from the thyroids of 11 Graves' disease patients. We then cultured the lymphocytes of 13 Graves' disease patients and 14 controls and induced EBV reactivation to measure IgG4 levels in culture fluids. We detected EBER1-positive cells and IgG4-positive plasma cells in the same area of thyroid tissues. EBV-reactivated cells with IgG4 on their surface were observed in culture cells, and IgG4 production was detected in culture fluids. The IgG4/IgG percentage was higher than that in normal serum level. A subset of Graves' disease is an IgG4-RD-like condition, not an IgG4-RD. EBV reactivation stimulates IgG4 production, which may result in high serum IgG4 levels and promote IgG4-positive plasma cell infiltration. EBER1 needs to be examined when an increase in IgG4-positive plasma cell numbers is noted.

Aberrant expression of AID and AID activators of NF-κB and PAX5 is irrelevant to EBV-associated gastric cancers, but is associated with carcinogenesis in certain EBV-non-associated gastric cancers.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is a distinct subtype of gastric cancer characterized by clinicopathological features including lymphoepithelioma-like histology. Aberrant expression of activation-induced cytidine deaminase (AID) as a genomic modulator was demonstrated through pathogen-related nuclear factor κB (NF-κB) signaling in Helicobacter pylori-associated gastric cancer. To elucidate whether or not AID expression is relevant to carcinogenesis in EBVaGC, immunohistochemical expression of AID and AID-regulatory factors between EBVaGC and EBV-non-associated gastric carcinoma (GC) were evaluated, each using 15 cases of GC with lymphoid stroma (GCLS) and other types of GC. Aberrant expression of AID, NF-κB and paired box 5 (PAX5) were significantly decreased in EBVaGC (0/11, 1/11 and 1/11) compared with in EBV-non-associated GC (7/19, 12/19 and 11/19) (P=0.025, 0.005 and 0.01, respectively); however, no significant difference in c-Myb proto-oncogene expression was identified. AID expression was also decreased in EBV-associated GCLS (0/10) compared with in EBV-non-associated GCLS (3/5). Unexpectedly, decreased expression of NF-κB and PAX5 was observed in GCLS (1/15 and 2/15) compared with in GC without LS (12/15 and 10/15) (P<0.001 and P=0.003, respectively). Decreased AID expression observed in EBVaGC is consistent with the reported molecular characterization of hypermethylation and rare somatic gene mutation in EBVaGC. Only PAX5 was identified to be significantly associated with venous invasion (P=0.022). The results of the present study suggest that pathogen-induced AID expression may be irrelevant to carcinogenesis of EBVaGC, whereas it contributes to carcinogenesis in certain types of EBV-non-associated GC.

Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

Presence of Epstein-Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves' disease patients and in healthy individuals.

Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.

EBNA-2 -deleted Epstein-Barr virus from P3HR-1 can infect rabbits with lower efficiency than prototype Epstein-Barr virus from B95-8.

OBJECTIVES: To clarify characteristics on rabbit in vivo infection with type 2 EBV nuclear antigen (EBNA-2)-deleted Epstein-Barr virus (P3HR-1-EBV) and compare infectious efficacy of P3HR-1-EBV with previously reported prototype type 1 EBV from B95-8. METHODS: Twelve Japanese White rabbits were inoculated with P3HR-1-EBV via intranasal or intravenous routes and autopsied on day 70-84. RESULTS: In only 2 of 12 P3HR-1-EBV-inoculated rabbits, EBV-DNA was detected in peripheral blood mononuclear cells (PBMCs). BamHI M rightward reading frame (BMRF)-1, EBNA-1 and BamHI Z leftward reading frame (BZLF)-1-mRNA were intermittently expressed in PBMCs. In 1 infected rabbit with continuous detection of EBV-DNA in PBMCs, many EBER-1-positive lymphocytes were observed in germinal centers and/or marginal zones in some follicles of the appendix, and for the first time a lymphocyte with EBER-1 expression infiltrating in the squamous cell layer of the tonsils was found. The other rabbit with a transient detection of EBV-DNA in PBMCs had no EBER-1-positive lymphocytes in the tissues examined. Few EBER-1-positive lymphocytes were detected in some rabbits without detection of EBV-DNA in PBMCs. CONCLUSIONS: P3HR-1-EBV showed less efficient infection in rabbits than EBV from the B95-8 cell line. However, a P3HR-1-EBV-inoculated animal model is meaningful because this is the first study of EBNA-2 function on in vivo EBV infection and it demonstrated the in vivo infectivity with lytic-type infection by EBNA-2-deleted EBV.

The influence of Epstein-Barr virus reactivation in patients with Graves' disease.

In Graves' disease, the IgG class autoantibody against thyrotropin receptor (TRAb) is produced excessively and induces hyperthyroidism. Epstein-Barr virus (EBV) is one of the human herpesviruses that persists for life, mainly in B lymphocytes, and is occasionally reactivated. Therefore, EBV may affect the antibody production of B lymphocytes that would normally produce TRAb. The purpose of the present study was to evaluate the association of EBV reactivation with the etiology of Graves' disease. Serum levels of EBV antibodies and IgE were determined by ELISA. TRAb levels were determined by radioreceptor assay. We performed in-situ hybridization (ISH) of EBV-encoded small RNA (EBER)1 on the thyroid tissue of one of our patients. In Graves' disease patients with TRAb levels ≥ 10%, EA antibody levels, which indicate EBV reactivation, were moderately but significantly correlated with the levels of TRAb, and weakly but significantly correlated with IgE. EBER1-ISH revealed that one of our patients had EBV-infected lymphocytes infiltrating the thyroid gland. EBV reactivation may stimulate antibody-producing B lymphocytes predisposed to make TRAb, and this may contribute to or exacerbate the disease.

In vitro Epstein-Barr virus infection model of rabbit lymphocytes from peripheral blood or spleen.

Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently, we reported that EBV can infect rabbits by intravenous, intranasal and/or peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Some rabbits showed chronic and lifelong EBV infection with hemophagocytosis. In this study, to reveal detailed mechanisms in rabbit EBV infection, an in vitro investigation was performed. We elucidated that: (1) EBV can infect rabbit peripheral blood mononuclear cells and splenic lymphocytes in vitro, because EBV gene expressions were confirmed. (2) It is highly likely that the B cell is the main target cell of rabbit EBV infection and is immortalized similar to humans. (3) CD8+ T cells increased in the rabbit in vivo model after EBV inoculation, whereas an increase of B cells occurred after their transient decrease. These data suggest that EBV-infected B cells were proliferated, while CD8+ T cells increased to recognize and kill them. This system may explain the paths of rabbit EBV infection and host reaction, simulating human EBV infection. In vitro studies will be helpful to reveal the pathogenesis of rabbit EBV infection and EBV-associated diseases.

Lifelong persistent EBV infection of rabbits with EBER1-positive lymphocyte infiltration and mild sublethal hemophagocytosis.

Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes in one to two-third of cases infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently we reported that EBV can infect rabbits frequently by intravenous, intranasal or/and peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Here we presented follow up data that of six primary EBV-infected rabbits out of seven inoculated intravenously with EBV, two out of six EBV-infected rabbits showed lifetime EBV infection. (1) EBV-DNA were detected in blood through life. (2) High antibody titers against EA-D were maintained over 1000 days. (3) A focal mass lesion was transiently observed by ultrasonography in the spleen of one rabbit. (4) Two lifelong EBV-detected rabbits died on day 1522 or 1400, and autopsy revealed proliferation of lymphocytes expressing EBER1 or LMP1 accompanied with mild hemophagocytosis in the spleen or lymph nodes. We hypothesized some EBV-infected rabbits could not eliminate EBV for life and showed somewhat similar features to persistent EBV infection, mild CAEBV and/or mild sublethal hemophagocytosis. These lifelong EBV-infected rabbits might be a new useful animal model for studying lifelong persistent EBV infection, taking place in almost all adults.

Epstein-Barr virus can infect rabbits by the intranasal or peroral route: an animal model for natural primary EBV infection in humans.

Epstein-Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV-associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV-infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV-DNA or mRNA expression and anti-EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV-DNA or EBV-related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV-related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV-DNA and/or mRNA of EBV-related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV-DNA and/or mRNA were detected for more than 200 days after viral inoculation. The level of EA-IgG increased and its level was maintained in all infected rabbits, whereas those of VCA-IgM and VCA-IgG increased transiently, and EBNA-IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans.

A new animal model for primary and persistent Epstein-Barr virus infection: human EBV-infected rabbit characteristics determined using sequential imaging and pathological analysis.

Most adults have persistent Epstein-Barr virus (EBV)-infection. Adolescents and young adults with primary EBV-infection frequently develop infectious mononucleosis. Latent EBV-infection is associated with various diseases, neoplasms, and hematological disorders. In vivo animal models of human EBV infection, such as non-human primates, have had limited success. A new rabbit model for primary human EBV-infection is described in this study. Seven male rabbits inoculated intravenously with EBV were sequentially imaged by ultrasonography and computed tomography, and examined for anti-EBV-VCA titer and EBV-DNA levels in blood. Six rabbits demonstrated transient splenomegaly, increased anti-EBV-VCA titers and/or EBV-DNA in blood. Transient infiltration of some EBER1-positive lymphocytes was observed in biopsied liver tissues. After splenomegaly, two rabbits tested continuously negative, two alternatively positive and negative, and one consistently positive EBV detection in blood for 470 days. One tested negative for both EBV DNA and splenomegaly. On the 14th day, mild to moderate numbers of EBER1-positive lymphocytes expressing LMP1, EBNA2, or ZEBRA infiltrated mainly in enlarged white pulps of two splenectomized materials. These cells included both B and T cells. EBV clonality analysis revealed an oligoclonal pattern. These indicate that EBV-inoculated rabbits exhibiting heterogenous host reactions are a good model for primary and persistent human EBV infection.