The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells

Epstein-Barr virus (EBV) is an exceptionally successful human viral pathogen maintained as a licensed, plasmid replicon in proliferating cells. We have measured the distributions of EBV-derived plasmids in single live cells throughout the cell cycle in the absence of selection and confirmed the measured rates of duplication and partitioning computationally and experimentally. These analyses have uncovered a striking, non-random partitioning for this minimalist plasmid replicon and revealed additional properties of it and its host cells: (1) 84% of the plasmids duplicate during each S phase; (2) all duplicated plasmids are spatially colocalized as pairs, a positioning that is coupled to their non-random partitioning; (3) each clone of cells requires a certain threshold number of plasmids per cell for its optimal growth under selection; (4) defects in plasmid synthesis and partitioning are balanced to yield wide distributions of plasmids in clonal populations of cells for which the plasmids provide a selective advantage. These properties of its plasmid replicon underlie EBV's success as a human pathogen.     

Epstein�CBarr virus-associated inflammatory pseudotumor of the spleen: report of two cases and review of the literature

We report two rare examples of Epstein–Barr virus (EBV)-associated inflammatory pseudotumor of the spleen. One patient presented with night sweats, abdominal pain, and weight loss and was found to have a splenic mass on CT scan suspected of lymphoma. The splenic mass in second patient was found incidentally at the time of work up for kidney stones. The pathologic examination of these splenectomy specimens showed similar histologic features. However, the spindle cells were composed of EBV-infected follicular dendritic cells in one case whereas the second case lacked significant follicular dendritic cell proliferation and showed only focal EBV-infected cells suggesting that these proliferations are heterogenous in nature.     

C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line,MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with ³²P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.     

NK cell lymphoma, nasal type, with massive lung involvement: a case report

Extranodal NK/T cell lymphoma, nasal type, is an Epstein–Barr virus-associated lymphoma that most commonly involves the nasal cavity and upper respiratory tract. Lung involvement by NK/T cell lymphoma is rare and seldom reported in the literature. We describe the unusual case of a 41-year-old male with NK cell lymphoma, nasal type, who presented with massive secondary lung involvement 2.5 years after the detection of a retroperitoneal mass. The diagnosis was made by open lung biopsy. Despite aggressive treatment, the patient died shortly after the initiation of therapy. Lung involvement by NK/T cell lymphoma occurs most commonly as part of widely disseminated disease and carries a poor prognosis for the patient. Novel agents and innovative therapies need to be developed for this aggressive lymphoma.     

In vitro immunization of Epstein–Barr virus-immortalized B cells augments antigen-specific antibody production

The current method for in vitro immunization (IVI) uses several antigens including toxins, food allergens, pathogenic bacteria, and self-antigen-derived peptides that induce an antigen-specific immune response in peripheral blood mononuclear cells (PBMCs). This protocol, however, requires donor blood collection and preparation of PBMCs before every IVI. In the present study, we aimed to design a more efficient system utilizing B cells immortalized with Epstein–Barr virus (EBV-B) as host cells for IVI to make antigen-specific antibodies. Results showed that previously antigen-sensitized, EBV-B cells exposed to the antigen along with IL-6, CpG oligonucleotides, and CD40 ligand signal produced antigen-specific antibodies. These results provide evidence for a novel and easy method to expand memory-type B cells and produce antigen-specific antibodies.     

EBV感染及TPA处理前后人CR2转染细胞的基因差异表达谱

采用Mouse Atlas^TM cDNA Expression Arrays对EBV感染前后及TPA处理前后的人CR2转染小鼠细胞基因表达进行分析,通过Eagle EyeⅡ图像分析系统进行密度扫描以寻找差异表达基因。结果表明已初步建立EBV和TPA的转染细胞基因差异表达谱,为进一步研究二者对转染小鼠 细胞的影响奠定了良好基础,也为进一步发现转染小鼠细胞中EBV和TPA相关的信号转导通路提供线索。     

Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture

Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. This revised version was published online in July 2006 with corrections to the Cover Date.     

T cell lymphoproliferative disorders associated with anti-tumor necrosis factor alpha antibody therapy for ulcerative colitis: literature summary

The enhanced risk of development of lymphoproliferative disorders in patients with inflammatory bowel disease has been attributed to immunosuppressive/immunomodulatory therapies. Infliximab is a chimeric monoclonal immunoglobulin G1 antibody directed against tumor necrosis factor alpha (TNF-α) that was approved by the Food and Drug Administration (FDA) in 1998 as an effective therapeutic agent against inflammatory bowel disease. Malignant lymphomas of both B and T cell lineage have been described in patients undergoing therapy involving TNF-α blockade. To date, eight cases of Epstein–Barr virus (EBV)-negative hepatosplenic T cell lymphoma associated with infliximab have been reported to the FDA’s Adverse Event Reporting System, as well as several other T cell lymphoproliferative disorders with aggressive clinical outcomes. We present the histologic, immunophenotypic, and molecular features of a T cell lymphoproliferative disorder involving the axillary lymph node of a 33-year-old male following infliximab treatment for ulcerative colitis. These EBV-negative lymphomas suggest that lymphoproliferative disorders following infliximab treatment for inflammatory bowel disease may involve EBV-independent immune dysregulation. The spectrum of lymphoproliferative disorders associated with infliximab and the potential mechanisms by which they occur are discussed.     

Screening,Isolation, and Some Properties of Microbial Cell Flocculants

Nineteen out of 214 strains of microorganisms including moulds, bacteria, actinomycetes and yeasts, which were selected from the culture collection of our laboratory were found to produce the substances which flocculate Saccharomyces cerevisiae AJ4005 (baker’s yeast). Among them are Aspergillus sojae, Anixiella reticulata, Geotrichum candidum, Eupenicillium crustaceus, Circinella sydowi, Monascus anka, Sordaria fimicola, Pseudomonas fluorescens, Staphylococcus aureus, Corynebacterium brevicale, Brevibacterium insectiphilum, Streptomyces vinaceus.

The flocculants in the culture broth of these microorganisms precipitated readily by addition of acetone, and flocculated various microorganisms nonspecifically. The flocculant produced by Asp. sojae AJ7002 sedimented activated sludge well.     

Epstein-Barr病毒诱导永生化人上皮细胞恶性转化

为了使ＥＢ病毒能直接感染人上皮细胞,将ｐＳＧ－ＣＲ２－Ｈｙｇ载体转入永生化人上皮细胞（２９３细胞）中。通过间接免疫荧光法测定发现,转化的细胞表达ＥＢ病毒受体。用ＥＢ病毒感染ＣＲ２－２９３细胞后,２３％的细胞可表达ＥＢ病毒抗原。用ＴＰＡ作用于这些细胞后,表达病毒抗原的细胞数增加。用ＰＣＲ法从ＥＢ病毒感染细胞ＤＮＡ中扩增出ＥＢ病毒ＤＮＡＷ片段。在ＴＰＡ持续作用下,ＥＢ病毒感染细胞的形态特征发生改变。把ＥＢ病毒感染细胞接种在裸鼠皮下,每周注射ＴＰＡ,可诱导细胞在裸鼠体内形成肿瘤。经组织病理检查确诊,肿瘤为低分化上皮细胞癌。杂交试验证明,肿瘤组织细胞中有ＥＢ病毒ＥＢＥＲｓ存在。上述结果表明,ＥＢ病毒在ＴＰＡ协同作用下,可诱导永生化上皮细胞恶性转化。     

Epstein—Barr病毒相关疾病的免疫治疗与疫苗发展策略

简要介绍了EB病毒的感染类型及机体对不同类型EB病毒感染的免疫反应,并对EB病毒相关疾病的免疫治疗手段和疫苗发展策略进行了评述。     

Characterization of natural killer cell activity in Macaca nemestrina

Natural killer (NK) cell activity was evaluated in three groups of Macaca nemestrina that varied with respect to SAIDS D retrovirus serotype 2 (SRV-2/W) and viremic status. Target cells used were Raji and K562 cells. No significant differences (ANOVA) in mean NK activity were detected among the three groups of animals studied. Using Raji targets, mean LU₃₀/10⁶ ± SEM was 6.3 ± 1.6 for seronegative (V-Ab−) animals, 7.3 ± 1.5 for seropositive (V-Ab+) animals, and 10.2 ± 3.5 for persistently viremic (V + Ab−) animals. Using K562 targets, mean LU₃₀/10⁶ was 7.6 ± 1.7 for seronegative (V-Ab−) animals, 6.5 ± 2.5 for seropositive (V-Ab+) animals, and 5.1 ± 1.9 for persistently viremic (V+Ab−) animals. Percentage blood CD16⁺ and CD8⁺cells also were not different in the three groups of animals. NK activity did not always correlate with percentage of CD16⁺ or CD8⁺ cells in peripheral blood at the time the assays were done. In persistently viremic animals, there was a strong positive correlation between percent CD16⁺ and CD8⁺ cells and NK activity using K562 cells but not Raji cells. Depletion experiments indicated that lysis was mediated by both CD8⁺ and CD16⁺ cells with both Raji and K562 cells. However, Raji targets were a better indicator of killing mediated by CD16⁺ cells. Our studies indicate that M. nemestrina may be classified as high or low responders with regard to NK activity, and there was no correlation with SRV-2/W viral or antibody status. Additionally, our results suggested that group housing of M. nemestrina was usually associated with increased NK activity. In conclusion, studies of NK activity in M. nemestrina should consider target cells used, phenotype of effectors, endogenous (high or low) levels of NK activity in individual animals, and housing conditions. © 1996 Wiley-Liss, Inc.