Ken-Ichi Imadome

A New Humanized Mouse Model of Epstein-Barr Virus Infection That Reproduces Persistent Infection, Lymphoproliferative Disorder, and Cell-Mediated and Humoral Immune Responses

The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (<or=1 x 10(1) TD(50)), in contrast, resulted in apparently asymptomatic persistent infection. Levels of activated CD8(+) T cells increased dramatically in the peripheral blood of infected mice, and enzyme-linked immunospot assay and flow cytometry demonstrated an EBV-specific T cell response. Immunoglobulin M antibody specific to the EBV-encoded protein BFRF3 was detected in serum from infected mice. The NOG mouse is the most comprehensive small-animal model of EBV infection described to date and should facilitate studies of the pathogenesis, prevention, and treatment of EBV infection.

T Cell-Mediated Control of Epstein-Barr Virus Infection in Humanized Mice

Humanized NOD/Shi-scid/interleukin-2Rgamma(null) (NOG) mice with full T cell development had significantly longer life span after Epstein-Barr virus (EBV) infection, compared with those with minimal T cell development. Removing CD3(+) or CD8(+) T cells from EBV-infected humanized mice by administration of anti-CD3 or anti-CD8 antibodies reduced their life span. CD8(+) T cells obtained from EBV-infected mice suppressed the outgrowth of autologous B cells isolated from uninfected mice and inoculated with EBV in vitro. These results indicate that humanized NOG mice are capable of T cell-mediated control of EBV infection and imply their usefulness as a tool to evaluate immunotherapeutic and prophylactic strategies for EBV infection.

Epstein-Barr Virus Induces Erosive Arthritis in Humanized Mice

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of rheumatoid arthritis (RA) on the basis of indirect evidence, such as its presence in affected joint tissues, antigenic cross reactions between EBV and human proteins, and elevated humoral and cellular anti-EBV immune responses in patients. Here we report development of erosive arthritis closely resembling RA in humanized mice inoculated with EBV. Human immune system components were reconstituted in mice of the NOD/Shi-scid/IL-2Rγnull (NOG) strain by transplantation with CD34+ hematopoietic stem cells isolated from cord blood. These humanized mice were then inoculated with EBV and examined pathologically for the signs of arthritis. Erosive arthritis accompanied by synovial membrane proliferation, pannus formation, and bone marrow edema developed in fifteen of twenty-three NOG mice transplanted with human HSC and inoculated with EBV, but not in the nine NOG mice that were transplanted with HSC but not inoculated with EBV. This is the first report of an animal model of EBV-induced arthritis and strongly suggest a causative role of the virus in RA.

CD40 ligand is a critical effector of Epstein–Barr virus in host cell survival and transformation

Epstein–Barr virus (EBV), implicated in numerous human diseases, including lymphoid malignancies, persistently infects peripheral B cells and transforms them into lymphoblastoid cell lines. Here we found that EBV equally infected B cells from patients with X-linked hyper IgM syndrome and those from healthy donors; however, it hardly transformed X-linked hyper IgM syndrome B cells, because of the dysfunctional gene of CD40 ligand (CD40L) of the patients. Unlike CD40, CD40L is not usually expressed on B cells. However, we found that EBV infection of normal B cells induced CD40L expression as a critical effector in host cell transformation and survival. Moreover, chronic active EBV infection of peripheral T cells, implicated in T cell malignancies, was associated with ectopic expression of CD40, and, in Jurkat T cells, EBV infection induced CD40 expression. These results suggest that EBV infection induces CD40L/CD40 signaling in host cells, which appears to play an essential role in its persistent infection and malignancies of lymphocytes.

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.

Phenotypic variations between affected siblings with ataxia-telangiectasia: ataxia-telangiectasia in Japan.

A nationwide survey was conducted for identifying ataxia-telangiectasia (AT) patients in Japan. Eighty-nine patients were diagnosed between 1971 and 2006. Detailed clinical and laboratory data of 64 patients including affected siblings were collected. Analyses focused on malignancy, therapy-related toxicity, infection, and hematological/immunological parameters. The phenotypic variability of AT was assessed by comparing 26 affected siblings from 13 families. Malignancy developed in 22% of the cases and was associated with a high rate of severe therapy-related complications: chemotherapy-related cardiac toxicity in 2 children, and severe hemorrhagic cystitis requiring surgery in 2 patients. The frequency of serious viral infections correlated with the T cell count. Hypogammaglobulinemia with hyper-IgM (HIGM) was recorded in 5 patients, and 3 patients developed panhypogammaglobulinemia. Differences in immunological parameters were noted in siblings. Four patients showed an HIGM phenotype, in contrast to their siblings with normal IgG and IgM levels. The patients with HIGM phenotype showed reduced levels of TRECs and CD27+CD20+ memory B cells. The findings suggest that hitherto unidentified modifier genes or exogenous environmental factors can influence the overall immune responses. Our data along with future prospective study will lead to better understanding of the hematological/immunological phenotypes and to better care of the patients.

Current research on chronic active Epstein–Barr virus infection in Japan

Epstein–Barr virus (EBV) infection is usually asymptomatic and persists lifelong. Although EBV‐infected B cells have the potential for unlimited proliferation, they are effectively removed by the virus‐specific cytotoxic T cells, and EBV‐associated lymphoproliferative disease develops only in immunocompromised hosts. Rarely, however, individuals without apparent immunodeficiency develop chronic EBV infection with persistent infectious mononucleosis‐like symptoms. These patients have high EBV‐DNA load in the peripheral blood and systemic clonal expansion of EBV‐infected T cells or natural killer (NK) cells. Their prognosis is poor with life‐threatening complications including hemophagocytic lymphohistiocytosis, organ failure, and malignant lymphomas. The term “chronic active EBV infection” (CAEBV) is now generally used for this disease. The geographical distribution of CAEBV is markedly uneven and most cases have been reported from Japan and other East Asian countries. Here we summarize the current understanding of CAEBV and describe the recent progress of CAEBV research in Japan.

Coexpression of CD40 and CD40 Ligand in Epstein-Barr Virus–Infected T and NK Cells and Their Role in Cell Survival

We investigated the role that CD40-CD40 ligand (CD40L) signaling plays in survival of Epstein-Barr virus (EBV)-infected T and NK cells. EBV-infected T and NK cell lines derived from patients with either chronic active EBV infection (CAEBV) or nasal T/NK cell lymphoma, as well as virus-infected peripheral T cells freshly isolated from a patient with CAEBV, were shown to express both CD40 and CD40L on their surface. Apoptosis of these cells was enhanced by blockade of CD40-CD40L signaling by a fusion protein of CD40 and immunoglobulin G (CD40Ig). Expression of CD40 was induced in human CD40L-positive Jurkat T cells after experimental EBV infection, and apoptosis of infected cells was enhanced by CD40Ig. These results suggest that CD40-CD40L signaling promotes survival of EBV-infected T and NK cells and, thus, plays an important role in the pathogenesis of T/NK lymphoproliferative disorders associated with the virus.

Novel Immediate-Early Protein IE19 of Human Cytomegalovirus Activates the Origin Recognition Complex I Promoter in a Cooperative Manner with IE72

ABSTRACT The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) expresses IE86, IE72, IE55, and IE18 mRNA by differential splicing. Reverse transcription-PCR with IE72-specific primers generated an 0.65-kb cDNA from HCMV-infected fibroblast RNA, which does not correspond to any known MIE cDNA. Nucleotide sequencing revealed that the 0.65-kb cDNA is from exons 1, 2, and 3 and part of exon 4, indicating that it is derived from a novel alternatively spliced mRNA of the MIE gene. The cDNA encodes a 172-amino-acid polypeptide, termed IE19, which corresponds to an IE72 variant with an internal deletion from Val86 to Pro404 and appears as a band at 38 kDa on a sodium dodecyl sulfate-polyacrylamide gel. IE19 mRNA was expressed at a low level in the immediate-early, early, and late period of viral infection. IE19 was localized in nuclei, and a transient-expression assay revealed that IE19 enhances IE72-dependent activation of the HsOrc1 promoter, which is identified here as an IE72 target promoter. Another MIE protein, IE86, activated the same promoter but only weakly compared to IE72, and coexpression of IE19 did not alter the IE86-mediated transcriptional activation. In addition, IE19 did not enhance the IE72-dependent activation of the HCMV UL54 promoter. These results suggest that IE19 is a transcriptional coactivator that works with IE72.

Effective control of Epstein–Barr virus infection following pediatric liver transplantation by monitoring of viral DNA load and lymphocyte surface markers

Imadome K‐I, Fukuda A, Kawano F, Imai Y, Ichikawa S, Mochizuki M, Shigeta T, Kakiuchi T, Sakamoto S, Kasahara M, Fujiwara S. Effective control of Epstein–Barr virus infection following pediatric liver transplantation by monitoring of viral DNA load and lymphocyte surface markers.