Epstein-Barr Virus Infected B-cells Research Articles

Targeting latent viral infection in EBV-associated lymphomas.

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

Thermodynamic and structural characterization of an EBV infected B-cell lymphoma transcriptome.

Epstein–Barr virus (EBV) is a widely prevalent human herpes virus infecting over 95% of all adults and is associated with a variety of B-cell cancers and induction of multiple sclerosis. EBV accomplishes this in part by expression of coding and noncoding RNAs and alteration of the host cell transcriptome. To better understand the structures which are forming in the viral and host transcriptomes of infected cells, the RNA structure probing technique Structure-seq2 was applied to the BJAB-B1 cell line (an EBV infected B-cell lymphoma). This resulted in reactivity profiles and secondary structural analyses for over 10000 human mRNAs and lncRNAs, along with 19 lytic and latent EBV transcripts. We report in-depth structural analyses for the human MYC mRNA and the human lncRNA CYTOR. Additionally, we provide a new model for the EBV noncoding RNA EBER2 and provide the first reported model for the EBV tandem terminal repeat RNA. In-depth thermodynamic and structural analyses were carried out with the motif discovery tool ScanFold and RNAfold prediction tool; subsequent covariation analyses were performed on resulting models finding various levels of support. ScanFold results for all analyzed transcripts are made available for viewing and download on the user-friendly RNAStructuromeDB.

Methotrexate-associated lymphoproliferative disorders of T-cell phenotype: clinicopathological analysis of 28 cases

Methotrexate-associated lymphoproliferative disorders are categorized as “other immunodeficiency-associated lymphoproliferative disorders in the WHO classification. Methotrexate-associated lymphoproliferative disorder is mainly a B-cell lymphoproliferative disorders or Hodgkin lymphoma type, whereas T-cell lymphoproliferative disorders are relatively rare (4–8%). Only a small number of methotrexate-associated T-cell lymphoproliferative disorders have been detailed thus far. Because of the rarity, methotrexate-associated T-cell lymphoproliferative disorder has not been well studied and its clinicopathological characteristics are unknown. A total of 28 cases of methotrexate-associated T-cell lymphoproliferative disorders were retrospectively analyzed. Histologically and immunohistochemically, they were divided into three main types: angioimmunoblastic T-cell lymphoma (n = 19), peripheral T-cell lymphoma, NOS (n = 6), and CD8+ cytotoxic T-cell lymphoma (n = 3). Among the 28 cases, only one CD8+ cytotoxic T-cell lymphoma case was Epstein-Barr virus-positive. The other 27 cases were negative for Epstein-Barr virus on tumor cells, but scattered Epstein-Barr virus-infected B-cells were detected in 24 cases (89%), implying the reactivation of Epstein-Barr virus caused by immunodeficient status of the patients. After the diagnosis of methotrexate-associated T-cell lymphoproliferative disorder, methotrexate was immediately withdrawn in 26 cases. Twenty (77%) cases presented with spontaneous regression. Compared to methotrexate-associated B-cell lymphoproliferative disorder, patients with methotrexate-associated T-cell lymphoproliferative disorder had a significantly higher proportion of males (p = 0.035) and presence of B-symptoms (p = 0.036), and lower proportion of Epstein-Barr virus+ tumor cells (p < 0.001). Although the difference was not significant, the methotrexate-associated T-cell lymphoproliferative disorder also had more frequent spontaneous regression (p = 0.061). In conclusion, methotrexate-associated T-cell lymphoproliferative disorder was divided into three main types: angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma, NOS, and CD8+ cytotoxic T-cell lymphoma. Angioimmunoblastic T-cell lymphoma was the most common type. Methotrexate-associated T-cell lymphoproliferative disorder was characterized by a high rate of spontaneous regression after methotrexate cessation. Epstein-Barr virus positivity was relatively rare in methotrexate-associated T-cell lymphoproliferative disorder, significantly less frequent than methotrexate-associated B-cell lymphoproliferative disorder, suggesting different pathogenesis.

A Rare Case of Lymphomatoid Granulomatosis With Multisystemic Involvement

SESSION TITLE: Pulmonary Manifestations of Systemic Disease 1 SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus (EBV)-driven lymphoproliferative disease. Clinical manifestations often mimic infectious diseases, malignancy and vasculitis, frequently leading to diagnostic delays. CASE PRESENTATION: A 53-year-old Chinese man with recurrent perianal abscesses, was found to have bilateral nodular opacities on a pre-operative chest radiograph (Figure 1A). Shortly after, he developed an acute onset of diplopia. On examination, bilateral cranial nerve 6, and left cranial nerve 7 and 8 palsies were noted. His medical history was notable for polymyositis treated with mycophenolate mofetil and prednisolone for the past 4 years. Imaging of the thorax was consistent with bilateral pulmonary nodules of varying sizes, and several demonstrated cavitation (Figure 1). Magnetic resonance imaging of the brain detected T2/FLAIR hyper intense signals in the left cerebellar superior sulci, left hemi pons and right 6th cranial nerve (Figure 1). Lumbar puncture was performed, and cerebrospinal fluid cytological examination yielded atypical CD20+ B cells. Transbronchial lung biopsy revealed florid fibrinohemorrhagic exudates and polymorphous infiltrates consisting of numerous B lymphocytes, some T lymphocytes, histiocytes and scattered atypical large lymphoid cells, focally involving vessel walls. EBV by in situ hybridization showed diffuse positive reaction (Figure 2). Histology of the recurring perianal abscess reported at another institution was reviewed at our hospital, demonstrating involvement by LYG. Fungal and acid fast bacilli stains were negative. A diagnosis of LYG, grade 3 was made. DISCUSSION: LYG is a rare EBV-associated lymphoproliferative disorder. The lungs are almost invariably affected, presenting classically as bilateral pulmonary nodules1. Central nervous system (CNS) involvement occurs in one-third of patients, and portends a poor prognosis1. Our patient presented with synchronous pulmonary, CNS and perianal skin/soft tissue involvement, which is uncommon in LYG. Reported associations of LYG are immune-related (autoimmune conditions, immunodeficiency states e.g. common variable immunodeficiency)1, and it is postulated that immune dysregulation may result in inadequate control of EBV infected B-cells. Our patient was immunosuppressed with mycophenolate mofetil, which may have contributed to the development of LYG. Management involves a consideration for stopping immunosuppression if possible, and chemotherapy which should be started immediately in high grade disease or CNS involvement. CONCLUSIONS: We describe a rare case of multi-systemic LYG with bilateral cavitary nodules, cranial nerve palsies, and recurring perianal abscess. Differential diagnoses for multiple pulmonary nodules are extensive. However, the possibility of LYG should be suspected, especially in immunosuppressed patients. Reference #1: Roschewski M, Wilson WH. Lymphomatoid granulomatosis. Cancer J. 2012; 18(5):469-74. DISCLOSURE: The following authors have nothing to disclose: Ken Junyang Goh, Angela Takano, Pei Yee Tiew, Ming Hui Yong, Chee Kiang Tay No Product/Research Disclosure Information

Low-Dose Ionizing Radiation Modulates Expression of Gpam in Immortalized EBV Infected B Cells through miRNA Network

Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006).To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p<0.05).To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour).We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells.To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results.Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation.In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells. DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Integrative genomic and proteomic analyses identifies glycerol-3-phosphate acyltransferase as a target of low-dose ionizing radiation in EBV infected-B cells

Purpose: We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment.Materials and methods: The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively.Results: The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells.Conclusions: These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell’s microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.

Abstract 3006: The CCL28 oncogene in NPC pathogenesis.

Abstract CCL28 Gene plays a role as an oncogene in the pathogenesis of Lymphoepitheliomatous Carcinoma of Nasopharynx Nasopharyngeal Carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. The molecular mechanisms involved in the pathogenesis of NPC still are not yet well defined. Although it was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis, but recently many studies have reported that EBV behaves more likely as a progression factor but not an initiation factor. The purpose of this research was to find out the genes associated with NPC pathogenesis. Using cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells, SOX5 gene expression was found significantly increased in NPC cell lines. In our previous studies of the function of SOX-5 gene in NPC, we found that chemokine (CC motif) of ligand 28 (CCL28) was also significantly increased in NPC cell lines, especially in NPC-TW03 line which is a lymphoepitheliomatous carcinoma of nasopharynx (LE-NPC). In 50 cases of Type III NPC biopsy specimens it was found that about 46% of cases revealed high expression of CCL28 protein in their tumor cells. To further identify the relationship between SOX5 and CCL28, we found that SOX5 and CCL28 gene expressions could be reciprocally regulated in NPC cells and suggested that CCL28 may be a critical factor for the formation of LE-NPC. To study the role of CCL28 in the molecular pathogenesis of LE-NPC and its functions, we constructed a stable pBIG2i-CCL28 transfected NPC cell line. We found that tumor cells could be up-regulated to express CCL28 mRNA and protein remarkably in those transfectants. This gene could promote tumor cell migration, proliferation, invasion, and attract lymphocytes especially B cells in vitro. In addition, EBV-immortalized B cells can also be attracted by CCL28. On the other hand, EBV could infect the CCL28 transfectants; furthermore, CCL28 could enhance IgA anti EBV-VCA production from the EBV infected-B cells. However, in SCID mice bearing CCL28 transfected NPC xenograft, the tumor growth and metastatic activity were slightly to moderately increased by this gene, but it has failed to attract mouse lymphocytic infiltration, probably due to the dissimilarity of CCL28 molecular structure between human and mouse species. Also, cDNA microarray analysis of CCL28 overexpressed NPC cells showed a group of oncogenes, such as IFI6 (Interferon L-inducible Protein 6, a survival protein) up-regulated and some oncosuppressor genes such as GPR68 (a G-protein-couple receptor 68) (a tumor metastasis suppressor gene) down-regulated. It is concluded that CCL28 gene may play a role to promote the formation of LE-NPC and as an oncogene in NPC pathogenesis to promote LE-NPC migration, proliferation and invasion in addition to its chemotactic property to attract B cell migration and to enhance IgA anti EBV-VCA production from the EBV+ B cells. Citation Format: Chin-Tarng Lin, Yi-Ying Chen, Cheng-Der Wu, Dah-yeou Huang, Yuan-Sung Kuo. The CCL28 oncogene in NPC pathogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2013-3006

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.

THIS ARTICLE HAS BEEN RETRACTED Epstein–Barr virus‐encoded latent membrane protein 1 activates β‐catenin signaling in B lymphocytes

The Epstein-Barr virus-encoded latent membrane protein 1 is considered the Epstein-Barr virus oncogene based on its importance in Epstein-Barr virus-induced B-lymphocyte transformation. Beta-catenin is a potential oncogene, and its accumulation has been implicated in a variety of human cancers. Here, we found that beta-catenin protein was highly expressed in Epstein-Barr virus-immortalized B-cell lines compared with peripheral blood mononuclear cells from healthy donors. Beta-catenin expression in Epstein-Barr virus-immortalized B-cell line decreased following treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase. Treatment with LY294002 or knockdown of beta-catenin by small interfering RNA reduced the growth of Epstein-Barr virus-immortalized B-cell line. Transient transfection of latent membrane protein 1 expression plasmid increased beta-catenin protein expression and beta-catenin-dependent transcription. Latent membrane protein 1 deletions mutants lacking the carboxyl-terminal activating region 1 domain failed to enhance beta-catenin protein expression and beta-catenin-dependent transcriptional activity. They also failed to increase phosphorylated AKT expression. Dominant-negative AKT suppressed latent membrane protein 1-induced beta-catenin-dependent transcriptional activity. These results suggest that latent membrane protein 1 activates beta-catenin through the phosphatidylinositol 3-kinase/AKT signaling pathway. Activation of the beta-catenin pathway by Epstein-Barr virus may contribute to the lymphoproliferation characteristic of Epstein-Barr virus-infected B-cells.

Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency

In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5'-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.

Immune suppression in healthy carriers of adult T-cell leukemia retrovirus (HTLV-I): impairment of T-cell control of Epstein-Barr virus-infected B-cells.

We have examined the activities of Epstein-Barr virus (EBV)-specific memory T-cells of carriers of adult T-cell leukemia (ATL) associated retrovirus (HTLV-I) and non-carriers in an ATL-endemic area, all of whom were EBV-seropositive, by assay of EBV-induced B-cell focus regression. The result showed that immune suppression, as represented by lowering of the regression, was present in 18 (29%) out of 63 healthy HTLV-I carriers, in contrast to none of 63 matched control persons (healthy non-carriers). This finding suggests that a suppression of cell-mediated immunity is induced by a persistent infection with ATL retrovirus.

Cyclosporin A-Induced Lipid and Protein Oxidation in Human B-Cells and in Epstein-Barr Virus-Infected B-Cells is Prevented by Antioxidants

The incidence of post-transplant lymphoproliferative disorder (PTLD) has increased since cyclosporin A (CsA) became the mainstay of transplant immunosuppression. We have previously shown that, in addition to its potent immunosuppressive property, CsA-induced oxidative stress plays an important role in Epstein-Barr virus (EBV)-related PTLD. Using lipid hydroperoxide and malondialdehyde as markers of lipid oxidation, and protein carbonyls as markers of protein oxidation, we further investigated the in vitro effect of CsA on human B cells and EBV-infected human B cells. We found that CsA at 500 ng/ml, a relatively safe and effective blood concentration in organ transplant recipients, induced the highest lipid hydroperoxide and malondialdehyde after 10 min of treatment in time- and concentration-related kinetic studies. We also found that treatment with CsA at 500 ng/ml for 10 min increased the EBV-infected B cell protein carbonyl formation as assayed by immunoblot method. CsA-induced lipid and protein oxidation could be inhibited by vitamin E, N-acetyl cysteine, and pyrollidine dithiocarbamate. CsA significantly promoted the EBV-B cell transformation as assayed by colony counting, cell counting, and 3H-thymidine incorporation. Our recent study provides further evidence to support the hypothesis that CsA exerts direct oxidative stress in EBV-infected as well as non-EBV-infected human B cells. A greater understanding of these cellular and molecular mechanisms may benefit the clinical practice and prevention of PTLD.

EBV+ B-cell Lymphoproliferative Disorder Associated with Subsequent Development of Burkitt Lymphoma in a Patient with Idiopathic CD4+ T-lymphocytopenia

We report here a case of idiopathic CD4(+) T-lymphocytopenia (ICL) associated with Epstein-Barr virus (EBV)(+) lymphoproliferative disorder (LPD) terminating in Burkitt lymphoma (BL). A 33-year-old Japanese male was admitted to the hospital showing severe CD4(+) lymphocytopenia and neutropenia that was diagnosed as ICL in 1993. Twenty months after the onset of disease, right cervical lymphadenopathy was detected. Biopsy of the specimen showed reactive lymph node hyperplasia and interfollicular B-cell hyperplasia. Ninety-one months later, polypoid tumors were resected from the bilateral nasal cavities and were diagnosed as BL. Immunohistological studies suggested the reactive nature of the initial lymph node biopsy specimen. Polymerase chain reaction (PCR) analyses of immunoglobulin heavy-chain gene (IgH) demonstrated a polyclonal pattern in the initial lymph node lesion. However, the subsequent BL demonstrated a clonal band in the PCR assay for the IgH gene. As demonstrated in human immunodeficiency virus (HIV)-patients, clonal expansion of EBV infected B-cells in the initial lymph node lesion may progress to BL in this patient. The present case did not associate with severe opportunistic infections during the course of disease. EBV(+) BL may be the first manifestation of severe immunodeficiency of the ICL in this patient.

FAT10/Diubiquitin-Like Protein-Deficient Mice Exhibit Minimal Phenotypic Differences

The FAT10 gene encodes a diubiquitin-like protein containing two tandem head-to-tail ubiquitin-like domains. There is a high degree of similarity between murine and human FAT10 sequences at both the mRNA and protein levels. In various cell lines, FAT10 expression was shown to be induced by gamma interferon or by tumor necrosis factor alpha. In addition, FAT10 expression was found to be up-regulated in some Epstein-Barr virus-infected B-cell lines, in activated dendritic cells, and in several epithelial tumors. However, forced expression of FAT10 in cultured cells was also found to produce apoptotic cell death. Overall, these findings suggest that FAT10 may modulate cellular growth or cellular viability. Here we describe the steps to generate, by genetic targeting, a FAT10 gene knockout mouse model. The FAT10 knockout homozygous mice are viable and fertile. No gross lesions or obvious histological differences were found in these mutated mice. Examination of lymphocyte populations from spleen, thymus, and bone marrow did not reveal any abnormalities. However, flow cytometry analysis demonstrated that the lymphocytes of FAT10 knockout mice were, on average, more prone to spontaneous apoptotic death. Physiologically, these mice demonstrated a high level of sensitivity toward endotoxin challenge. These findings indicate that FAT10 may function as a survival factor.

Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder.

Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.

Small bowel transplantation: selection criteria, operative techniques, advances in specific immunosuppresion, prognosis

Intestinal transplantation is now an accepted therapy for intestinal failure when parenteral nutrition therapy cannot be tolerated. During the past year, evidence has been provided indicating that neither stomach nor colon need to be included in the transplant, even if a primary motility disorder is the indication for surgery. The liver should be included in the composite allograft when there are clinical indications of portal hypertension resulting from parenteral nutrition associated cholestasis. When liver disease develops, operations intended to improve gut function should be avoided in preference of early listing for transplantation. During the past year, initial attempts at adult to child intestinal transplantation were carried out with some success; reduction in the diameter of the adult donor bowel may not be uniformly necessary. New immunosuppressive therapies have been employed recently, but few have been subjected to peer review. Experimental models have clarified the pathology, if not the immunobiology, of chronic intestinal allograft rejection and the ability of the liver to promote tolerance of a cotransplanted intestinal allograft. Treatment of posttransplant lymphoproliferative disease has been augmented by the use of anti-CD 20 antibody that targets Epstein-Barr virus infected B-cells for destruction with high specificity.

Evidence for a Proliferation of Epstein-Barr Virus Infected B-Lymphocytes with a Prominent T-Cell Component and Vasculitis

Similarities have been noted in the histologic patterns of lymphomatoid granulomatosis and Epstein-Barr virus associated lymphoproliferative disease involving the lung. Epstein-Barr virus has also been identified by polymerase chain reaction in most cases of lymphomatoid granulomatosis; however, the precise cellular localization of Epstein-Barr virus sequences has not been extensively studied. We analyzed 10 cases of lymphomatoid granulomatosis involving the lung by immunohistochemistry and combined immunohistochemistry with in situ hybridization for Epstein-Barr virus, CD20, and CD45RO. All cases were selected from the files of the Armed Forces Institute of Pathology and met the clinical and histologic criteria for the diagnosis of lymphomatoid granulomatosis, grades 1 through 3. In all 10 cases, immunohistochemistry showed that most of the cells--small to medium-sized lymphocytes--were T cells (CD45RO+); however, a much smaller population of medium-sized to large atypical cells were B cells (CD20+). In each case, combined immunohistochemistry and in situ hybridization confirmed the presence of Epstein-Barr virus sequences within B (CD20+) cells and the absence of Epstein-Barr within T-cells (CD45RO+). Polymerase chain reaction analysis for immunoglobulin heavy-chain gene rearrangement identified a monoclonal pattern in six of nine cases tested, whereas analysis for T-cell receptor gamma-chain gene rearrangements was negative in three cases tested. On the basis of these findings, we hypothesize that most cases of lymphomatoid granulomatosis involving the lung represent a proliferation of Epstein-Barr virus infected B-cells with a prominent T-cell reaction and vasculitis, distinguishing these cases from angiocentric "T-cell lymphomas" in other sites, such as the head and neck.

Epstein-Barr virus infection rapidly progressing to monoclonal lymphoproliferative disease in a child with selective immunodeficiency

We report on a 30-month-old previously healthy Turkish boy who presented with fever, hepatosplenomegaly and generalized lymphadenopathy. He died 4 months after admission in spite of treatment with steroids, acycloguanosine and cyclophosphamide. Epstein-Barr virus (EBV) DNA was detected in the patient's bone marrow and in a lymph node biopsy. Cells from the lymph node biopsy showed monoclonal rearrangements of immunoglobulin heavy chain genes but no rearrangements of T-cell receptor beta-chain genes or immunoglobulin kappa chain genes. Serological data indicated chronic active EBV infection. There was a slight increase of CD8 positive cells in peripheral blood and a normal response to T-cell mitogens. However, T-cell lines established with interleukin 2 from lymph node biopsy completely failed to kill autologous EBV-transformed B-cells and K 562 target cells. Moreover, in regression tests the patient's peripheral blood mononuclear cells completely failed to limit outgrowth of autologous EBV infected B-cells. We conclude that the patient's selective immuno-deficiency had led to the rapid development of EBV-associated monoclonal lymphoproliferation.

Epstein-Barr virus and human autoimmune diseases: possibilities and pitfalls

Epidemiologie studies suggest that human autoimmune disease involves both genetic and environmental components. Although a great deal has been learned about genetic components (i.e. histocompatibility antigens), little is known about the environmental factors. Because of its ubiquitous nature and ability to stimulate lymphoid responses, Epstein-Barr virus (EBV) has been examined as a potential candidate, particularly in rheumatoid arthritis and systemic lupus erythematosus. No convincing evidence has been produced that it plays a primary etiologic role in these disorders. Nevertheless, EBV or a related herpesvirus may play an indirect role in perpetuating the disorder or in the development of extra-articular manifestations such as Sjögren's syndrome (an autoimmune disorder involving the salivary glands). Current data indicates increased EBV reactivation in some patients with autoimmune diseases, as evidenced by increased viral DNA in their saliva and increased number of circulating B-cells containing EBV in their blood. These patients also have modestly elevated anti-EBV antibody titers and altered antiviral T-cell responses, as measured by the ability to prevent outgrowth of autologous EBV infected B-cells. However, the relevance of these ‘abnormalities’ to pathogenesis remains unknown. It is hoped that new techniques such as polymerase chain reaction will provide new insights into these questions by allowing detection of viral DNA from tissue biopsies obtained early in the course of disease and by providing a method to identify viral isolates that do not grow well in vitro.

Chemiluminescence response of human B-cell lines.

Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cells share certain properties with monocytes: they are capable of presenting protein antigens to antigen-specific T-lymphocytes and of releasing an Interleukin 1-like factor. It was our interest to study whether transformed B-cells resemble monocytes by generating toxic oxygen radicals. Human B-cell lines were developed from human peripheral blood lymphocytes by EBV-transformation. The induction of the respiratory burst in the B-cells was assessed by chemiluminescence (CL) in the presence of lucigenin. B-cells were stimulated with phorbol-myristate-acetate (PMA), zymosan particles, the chemotactic peptide f-met-phe, the complement split product C5a and with a recently described granulocyte activating cytokine (GRAM). Stimulation with PMA elicited a distinct CL-response in the tested B-cell lines. The CL-signal was significantly reduced by superoxide dismutase, but not by D-mannitol and catalase. No significant response to any of the other stimuli was detected. Furthermore, none of the stimuli induced a luminol-enhanced CL signal, which, in contrast to lucigenin, is dependent on the presence of peroxidase. Our results indicate that EBV infected B-cells were able to generate significant amounts of reactive oxygen species, particularly superoxide. It appears that virus transformation uncovers genetic information which is usually not expressed in non-transformed B-cells.