Shosuke Imai

Prognostic Factors for Chronic Active Epstein-Barr Virus Infection

Chronic active Epstein-Barr virus infection (CAEBV) is a high-mortality and high-morbidity disease. To clarify the prognostic factors, a national survey was performed in Japan, and data for 82 patients who met the criteria for CAEBV were analyzed. Of these 82 patients, 47 were alive and 35 had already died. Multivariate analysis revealed that thromobocytopenia and age at disease onset were correlated with mortality. The probability of 5-year survival was 0.45 for older patients (onset age, > or = 8 years), 0.94 for younger patients (P<.001), 0.38 for patients with thrombocytopenia (platelet count < 12 x 10(4) platelets/microL at diagnosis), and 0.76 for patients without thrombocytopenia (P=.01). Furthermore, patients with T cell infection by EBV had shorter survival times than patients with natural killer cell infection (probability of 5-year survival, 0.59 vs. 0.87; P<.009). Patients with CAEBV with late onset of disease, thrombocytopenia, and T cell infection had significantly poorer outcomes.

Efficacy of Bacteriophage Therapy against Gut-Derived Sepsis Caused by Pseudomonas aeruginosa in Mice

ABSTRACT We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P < 0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-α, interleukin-1β [IL-1β], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.

Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients

Low-grade malignant lymphomas arising from mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity of B-cell non-Hodgkin lymphoma (Isaacson & Wright, 1984). MALT lymphoma tends to remain localised and the extranodal sites of involvement include the gastrointestinal tract, salivary gland, thyroid and ocular region. MALT lymphoma often occurs in relation to autoimmune disorders or chronic antigen stimulation, but the pathogenetic mechanisms of this type of lymphoma have not been well defined. Recently, Ferreri et al (2004) reported that 32 (80%) of 40 Italian patients with ocular adnexal lymphoma harboured Chlamydia psittaci DNA. The findings suggested that C. psittaci infection might contribute to the development of these lymphomas. Soon after this, two other groups independently investigated ocular adnexal lymphoma for the presence of C. psittaci DNA in 57 patients from south Florida (Rosado et al, 2005) and 11 patients from the north-eastern USA (Vargas et al, 2005) and found that none of them carried C. psittaci DNA. One explanation for this discrepancy concerns geographic differences in the incidence of C. psittaci infection. However, all of these reports are based on findings from patients in the USA and Europe, and therefore additional surveys evaluating the association between C. psittaci and ocular adnexal lymphomas including MALT lymphoma in other geographical regions of the world would be warranted. There has been no report from Asia thus far. In this study, we looked for C. psittaci DNA in tumour specimens from Japanese patients with primary ocular adnexal lymphoma. Tissues obtained by an incisional biopsy from 24 Japanese patients (12 males and 12 females, mean age 56 years; range 34–73 years) with localised lymphoproliferative conditions of the ocular adnexa, including 18 MALT lymphomas, three nonMALT lymphomas and three reactive ocular lymphoid hyperplasias, were studied. The localities of the tumours were the orbit in 14 cases, lacrimal glands in four cases, and conjunctiva in six cases. DNA was obtained from frozen or paraffinembedded tissues using the phenol/chroroform extraction technique or DNA extraction system according to the manufacturer’s instructions (Takara, Tokyo, Japan). All samples were successfully amplified by polymerase chain reaction (PCR) for human b-globin sequences, yielding a 123-bp amplicon, which indicated that amplifiable DNAs were present. Three independent PCRs were used to detect C. psittaci DNA in this study. First, we used the same method employed in the previous studies (Ferreri et al, 2004; Rosado et al, 2005; Vargas et al, 2005). The touchdown enzyme time release PCR with CPS100 and CPS101 primers produces a 111-bp amplicon (Madico et al, 2000). The primers amplify a highly conserved region encoding the 16S ribosomal RNA gene. The second method was a nested PCR to detect the same DNA region with the first-step primers (5¢-ACGGAATAATGACTTCGG-3¢ and 5¢-TACCTGGTACGCTCAATT-3¢) and the second-step primers (5¢-ATAATGACTTCGGTTGTTATT-3¢ and 5¢-TGTTTTA GATGCCTAAACAT-3¢), generating a 127-bp amplicon. The PCR primers for the third C. psittaci PCR were derived from another DNA region encoding the ompA gene, and the primer sequences were 5¢-GCCTTAAACATCTGGGATCG-3¢ and 5¢-GCACAACCACATTCCCATAAAG-3¢, giving a 248-bp fragment. All three PCRs failed to detect C. psittaci DNA in any of our samples, while positive control reactions using DNA prepared from C. psittaci strain Cal-10 yielded amplicons of expected size. In addition, our samples were negative for Chlamydia pneumoniae and Chlamydia trachomatis DNA sequences. Our results showed no correlation between C. psittaci infection and ocular adnexal lymphoma in Japan, supporting the observation from the USA (Rosado et al, 2005; Vargas et al, 2005). All these studies were based on testing C. psittaci DNA sequences. Given the serological evidence of an association between chronic chlamydia infections (C. pneumoniae and C. trachomatis) and lymphomas (Anttila et al, 1998), serological surveys for C. psittaci may also help to better understand the relationship between C. psittaci and ocular adnexal lymphoma. Although the ‘hit-and-run’ mechanism cannot be fully excluded, the present findings indicate that C. psittaci is unlikely to have played a pathogenetic role in ocular adnexal lymphoma in the Japanese population.

Interferon-α/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006

BackgroundOligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported.ResultsThis study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip®. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-κB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-α/β receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN.ConclusionThis study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-α/β receptor-mediated paracrine pathway.

Induction of Lytic Epstein-Barr Virus (EBV) Infection by Synergistic Action of Rituximab and Dexamethasone Renders EBV-Positive Lymphoma Cells More Susceptible to Ganciclovir Cytotoxicity In Vitro and In Vivo

ABSTRACT The purposeful induction of the lytic form of Epstein-Barr virus (EBV) infection combined with ganciclovir (GCV) treatment has been advocated as a novel strategy for EBV-positive B-cell lymphoma. We demonstrated that rituximab had a synergistic effect with dexamethasone on induction of the lytic EBV infection in CD20-positive lymphoma cells. Addition of GCV to the dexamethasone/rituximab-treated cells was more effective than dexamethasone/rituximab alone in killing EBV-positive lymphoma cells in vitro and in lymphoma-bearing nude mice but not in EBV-negative cells. These data suggest that induction of the lytic EBV infection with dexamethasone/rituximab in combination with GCV could be a potential virally targeted therapy for EBV-associated B-cell lymphoma.

Epstein–Barr virus (EBV)-positive pyothorax-associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2-positive PAL B-cell line

Summary.  Pyothorax‐associated lymphoma (PAL) is a clinico‐pathological entity arising in the pleural cavity of patients with long‐standing inflammatory pyothorax. PAL is closely associated with Epstein–Barr virus (EBV), but how this virus contributes to the development of the lymphoma is unknown. We have successfully obtained a novel EBV‐infected PAL cell line, designated Pal‐1. The cell line and its source coexpressed CD2 and CD20 molecules, but other representative B‐ and T‐cell markers such as CD1, CD3, CD5, CD7, CD10 and CD19 were not found. The B‐cell origin of Pal‐1 cells was proven by rearrangement of the immunoglobulin heavy‐ and light‐chain genes without rearranged T‐cell receptor genes. Both the cell line and primary tumour cells carried monoclonal EBV genome. Although EBV genome is known to be maintained as circular extrachromosomal DNA, neither circular nor linear extrachromosomal EBV DNA was detectable in Pal‐1 cells by in situ lysis gel analysis. Fluorescence in situ hybridization demonstrated viral integration at a marker chromosome mostly consisting of the centromere region of chromosome 1. The viral integration event may enhance a chromosomal instability at the insertion site. This cell line represents the first example of EBV integration in PAL and could enable the study of the potential role of integrated viral infection in the development of PAL as well as mechanism of the aberrant phenotype expression.

Promoter Hypermethylation of the Bone Morphogenetic Protein-6 Gene in Malignant Lymphoma

Purpose: Bone morphogenetic proteins (BMP), belonging to the transforming growth factor-β superfamily, are important regulators of cell growth, differentiation, and apoptosis. The biological effects of BMPs on malignant lymphoma, however, remain unknown. Promoter methylation of the BMP-6 gene in lymphomas was investigated. Experimental Design: We investigated BMP-6 promoter methylation and its gene expression in various histologic types of 90 primary lymphomas and 30 lymphoma cell lines. The effect of BMP-6 promoter hypermethylation on clinical outcome was also evaluated. Results:BMP-6 was epigenetically inactivated in subsets of lymphomas. The silencing occurred with high frequency in diffuse large B-cell lymphoma (DLBCL) and Burkitts lymphoma in association with aberrant BMP-6 promoter methylation. The methylation was observed in 60% (21 of 35) of DLBCL cases and 100% (7 of 7) of DLBCL cell lines, and in 83% (5 of 6) of Burkitts lymphoma cases and 86% (12 of 14) of Burkitts lymphoma cell lines. In contrast, other histologic types of primary lymphomas studied had little or no detectable methylation (1 of 49; 2%). The presence of BMP-6 promoter hypermethylation in DLBCL statistically correlated with a decrease in disease-free survival (P = 0.014) and overall survival (P = 0.038). Multivariate analysis showed that the methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.022) and overall survival (P = 0. 046). Conclusion:BMP-6 promoter was hypermethylated more often in aggressive types of lymphomas, and the hypermethylation is likely to be related to the histologic type of lymphomas. BMP-6 promoter methylation may be a potential new biomarker of risk prediction in DLBCL.

Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus- and Activation-Regulated Chemokine in B Cells

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.

Promoter methylation of the bone morphogenetic protein-6 gene in association with adult T-cell leukemia.

Bone morphogenetic proteins (BMP), belonging to the transforming growth factor‐β superfamily, are multifunctional regulators of cell proliferation, differentiation and apoptosis in various types of malignant cells. In this study, we investigated BMP‐6 promoter methylation in patients with various types of leukemias. The BMP‐6 methylation was found preferentially in adult T‐cell leukemia (ATL) (49 of 60, 82%) compared with other types of leukemias studied including acute myeloid leukemia (3 of 67, 5%), acute lymphoblastic leukemia (6 of 38, 16%) and chronic lymphocytic leukemia (1 of 21, 5%). Among subtypes of ATL, the BMP‐6 gene was more frequently methylated in aggressive ATL forms of acute (96%) and lymphoma (94%) types than less malignant chronic ATL (44%) and smoldering ATL (20%). We also analyzed the methylation status of peripheral blood mononuclear cells from healthy donors and nonmalignant lymph nodes with reactive lymphadenopathy, none of which showed detectable BMP‐6 methylation in this study. The BMP‐6 methyaltion was correlated with decreased mRNA transcript and protein expression. Expression of BMP‐6 was restored by the demethylating agent 5‐aza‐2′‐deoxycytidine, suggesting that methylation was associated with the transcriptional silencing. Serial analysis demonstrated an increasing methylation of CpG sites in the BMP‐6 promoter and the resultant suppression of BMP‐6 expression as ATL progressed. These findings suggested that BMP‐6 promoter methylation is likely to be a common epigenetic event at later stages of ATL and that the methylation profiles may be useful for the staging of ATL as well as for evaluation of the individual risk of developing the disease.

Secretory IgA, Salivary Peroxidase, and Catalase-Mediated Microbicidal Activity during Hydrogen Peroxide Catabolism in Viridans Streptococci: Pathogen Coaggregation

Viridans streptococci can kill methicillin-resistant Staphylococcus aureus (MRSA) through the production of hydrogen peroxide (H2O2). However, several hundred viridans streptococci cells are necessary to kill 1 cfu of MRSA. We analyzed the potency of bactericidal and fungicidal effector molecules induced by catabolism of H2O2 in the oral cavity. Secretory IgA (SIgA) and an unidentified salivary component bound Streptococcus sanguinis, a viridans streprococcus, and MRSA into coaggregates. In these coaggregates, salivary peroxidase and the MRSA catalase produced singlet molecular oxygen (1O2) from H2O2 produced by viridans streptococci. SIgA converted 1O2 into ozone, which has potent bactericidal and fungicidal activity. We calculated that <10 cfu of Streptococcus sanguinis were necessary to kill 1 cfu of MRSA in the coaggregate. SIgA, Aspergillus niger catalase, and H2O2 in saliva killed Candida albicans, which is highly resistant to reagent H2O2. Together with indigenous bacteria and innate immunity, SIgA potentially constitutes a novel system that may sustain oral homeostasis.