Epstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial ...cells. Therefore, cellular transcription factors, such as BLIMP1, that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV-positive (EBV(+)) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nucleotide (nt) -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately early genes, BZLF1 and BRLF1.
This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.
Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue ...lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.
Ikaros is a zinc finger DNA-binding protein that regulates chromatin remodeling and the expression of genes involved in the cell cycle, apoptosis, and Notch signaling. It is a master regulator of ...lymphocyte differentiation and functions as a tumor suppressor in acute lymphoblastic leukemia. Nevertheless, no previous reports described effects of Ikaros on the life cycle of any human lymphotropic virus. Here, we demonstrate that full-length Ikaros (IK-1) functions as a major factor in the maintenance of viral latency in Epstein-Barr virus (EBV)-positive Burkitt's lymphoma Sal and MutuI cell lines. Either silencing of Ikaros expression by small hairpin RNA (shRNA) knockdown or ectopic expression of a non-DNA-binding isoform induced lytic gene expression. These effects synergized with other lytic inducers of EBV, including transforming growth factor β (TGF-β) and the hypoxia mimic desferrioxamine. Data from chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) and ChIP-sequencing (ChIP-seq) analyses indicated that Ikaros did not bind to either of the EBV immediate early genes BZLF1 and BRLF1. Rather, Ikaros affected the expression of Oct-2 and Bcl-6, other transcription factors that directly inhibit EBV reactivation and plasma cell differentiation, respectively. IK-1 also complexed with the EBV immediate early R protein in coimmunoprecipitation assays and partially colocalized with R within cells. The presence of R alleviated IK-1-mediated transcriptional repression, with IK-1 then cooperating with Z and R to enhance lytic gene expression. Thus, we conclude that Ikaros plays distinct roles at different stages of EBV's life cycle: it contributes to maintaining latency via indirect mechanisms, and it may also synergize with Z and R to enhance lytic replication through direct association with R and/or R-induced alterations in Ikaros' functional activities via cellular signaling pathways.
This is the first report showing that the cellular protein Ikaros, a known master regulator of hematopoiesis and critical tumor suppressor in acute lymphoblastic leukemia, also plays important roles in the life cycle of Epstein-Barr virus in B cells.
•The transcriptional elongation factor ELL3 is up-regulated in activated B cells.•Two ELL family members are non-concurrently expressed at specific B cell stages.•The expression of ELL3 precedes ...expression of ELL2 during B cell differentiation.•ELL3 plays a critical role in proliferation and survival of B cell models.•PRDM1 (Blimp-1) silences expression of ELL3 in plasma cells.
B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt’s lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma’s with a germinal center origin.
The data presented here are related to the research article entitled “Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival” ...(Alexander et al., 2017) 1. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) 2. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.
Habilidades sociais (HS) podem ser compreendidas como classes de comportamentos no repertório do indivíduo para lidar de maneira adequada com as situações interpessoais, sendo um construto teórico ...vinculado ao modelo comportamental. Este estudo tem por objetivo relatar a experiência de um estágio curricular em Psicologia que teve foco no desenvolvimento de habilidades sociais em 32 estudantes, sendo 12 alunos de Ensino Médio e 20 de Educação de Jovens e Adultos (EJA). O intuito dos grupos foi promover habilidades comunicacionais mais eficazes e prevenir conflitos. Foram realizados seis encontros, um por semana com cada turma, no turno da noite, com a duração média de 45 minutos cada encontro. As intervenções foram por meio de diálogos, utilização de recursos audiovisuais, dinâmicas e dramatizações. A partir das intervenções propostas, buscou-se possibilitar o desenvolvimento de comportamentos mais assertivos dos alunos em sala de aula, por meio da expressão de seus pensamentos e sentimentos de forma clara, preservando os direitos dos outros e sem prejudicar aos seus próprios direitos, a fim de diminuir os conflitos e melhorar as relações interpessoais. Os resultados apontaram para a melhoria das relações interpessoais no ambiente escolar, conforme observação do comportamento e relato dos próprios alunos e professores.
Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue ...lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.
Epstein-Barr virus (EBV) is a human DNA tumor virus that infects the vast majority of the world's population. Initially discovered in association with endemic Burkitt Lymphoma, latent EBV infection ...has since been found to be associated with numerous additional malignancies, including nasopharyngeal carcinoma (NPC) and a subset of gastric carcinomas. EBV establishes a life-long latent infection in the host's memory B cells, with sporadic lytic infection only observed in plasma cells and more differentiated epithelial cells. However, the cause of the switch from latent to lytic replication during cellular differentiation is not yet fully understood. The purpose of this Thesis was to further elucidate the role of the cellular differentiation factor B-lymphocyte-induced maturation protein-1 (BLIMP1; also called PRDI-BF1 and PRDM1) in EBV lytic reactivation. Here, I demonstrated that BLIMP1 induces EBV lytic replication in a variety of EBV-positive NPC, gastric cancer, and telomerase-immortalized normal oral keratinocyte (NOK) epithelial cell lines as well as in a subset of EBV-positive B cell lines. BLIMP1 does so by strongly activating transcription from two viral immediate-early promoters, Zp and Rp, with Rp being activated to a greater extent than Zp in epithelial cells. Rp was found to contain at least three synergistically acting BLIMP1-responsive elements (BREs); one required nt -660 and/or -659 relative to the transcription initiation site while two others mapped within nt -267 to -106 and nt -106 to +38. Zp contained at least one BRE mapping within nt -82 to +15. BLIMP1 associated with Rp in vivo, but did not associate with Zp. Loss of BLIMP1 during cellular differentiation due to siRNA knock-down led to decreased expression of EBV lytic proteins in NOK-Akata cells which are reactivated by R but not Z. Thus, I conclude that BLIMP1 is a key player in the initiation of EBV lytic reactivation during epithelial and B-cell differentiation; it most likely does so by activating Zp and Rp through indirect mechanisms.
Coastal oceans are particularly affected by rapid and extreme environmental changes with dramatic consequences for the entire ecosystem. Seagrasses are key ecosystem engineering or foundation species ...supporting diverse and productive ecosystems along the coastline that are particularly susceptible to fast environmental changes. In this context, the analysis of phenotypic plasticity could reveal important insights into seagrasses persistence, as it represents an individual property that allows species’ phenotypes to accommodate and react to fast environmental changes and stress. Many studies have provided different definitions of plasticity and related processes (acclimation and adaptation) resulting in a variety of associated terminology. Here, we review different ways to define phenotypic plasticity with particular reference to seagrass responses to single and multiple stressors. We relate plasticity to the shape of reaction norms, resulting from genotype by environment interactions, and examine its role in the presence of environmental shifts. The potential role of genetic and epigenetic changes in underlying seagrasses plasticity in face of environmental changes is also discussed. Different approaches aimed to assess local acclimation and adaptation in seagrasses are explored, explaining strengths and weaknesses based on the main results obtained from the most recent literature. We conclude that the implemented experimental approaches, whether performed with controlled or field experiments, provide new insights to explore the basis of plasticity in seagrasses. However, an improvement of molecular analysis and the application of multi‐factorial experiments are required to better explore genetic and epigenetic adjustments to rapid environmental shifts. These considerations revealed the potential for selecting the best phenotypes to promote assisted evolution with fundamental implications on restoration and preservation efforts.
•Exploratory statistical analysis of data from 54 manufactured batches of an IgG1 therapeutic antibody (mAb1) suggests that ADCC activity is not only modulated by afucose but also by galactose.•We ...enzymatically modulate the galactose levels of four different batches of mAb1 and confirm that the presence of terminal galactose enhances ADCC activity.•The ADCC enhancing effect of galactose is observed in 4 additional monoclonal antibodies derived using standard CHO-based manufacturing processes.•In contrast, the ADCC activity of IgG1 monoclonal antibody glycoengineered to contain a high degree of afucosylation remains unchanged by the addition of galactose, suggesting that while the levels of galactose have some influence on ADCC activity, the impact of afucose is still predominant.
The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity.
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