The oral microbiome of a family including Papillon-Lefèvre-syndrome patients and clinically healthy members.

AIMS: The oral microbiota composition of patients diagnosed with Papillon-Lefèvre-syndrome and treated for several years were compared to those existing in the oral cavity of the clinically healthy family members and a cohort of patients having various stages of chronic periodontitis. MATERIALS AND METHODS: A family with two sisters affected with severe periodontitis and with the typical skin symptoms of Papillon-Lefèvre-syndrome, and symptomless parents and third sibling were investigated. The Patients received periodontal treatment for several years and their oral microbiome was analysed by amplicon sequencing. Data were evaluated by microbial cluster analysis. RESULTS: The microbiome of the patients with Papillon-Lefèvre-syndrome was predominated with Aggregatibacter actinomycetemcomitans and associated oral periodontopathogens. Although the clinically healthy family members showed no oral disorder, their microbiome resembled that of subjects having mild periodontitis. CONCLUSIONS: Predominance of A. actinomycetemcomitans in the subgingival microbiome of patients with Papillon-Lefèvre-syndrome suggests that specific treatment strategies directed against this pathobiont may improve the oral health status of the affected individuals. TRIAL REGISTRATION: The study was conducted in accordance with the Declaration of Helsinki and the ethical permission has been issued by the Human Investigation Review Board of the University of Szeged, Albert Szent-Györgyi Clinical Centre (Permission No. 63/2017-SZTE). September 19, 2017.  https://u-szeged.hu/klinikaikutatas/rkeb-altal-jovahagyott/rkeb-2017 .

Human tumor viruses: induction of three-dimensional alterations in the host genome structure.

Certain viruses called tumor viruses or oncoviruses are capable to change the gene expression pattern of distinct human or animal cell types in tissue culture, resulting in uncontrolled proliferation as well as a change in the social behavior of the infected cells: the oncovirus-transformed, immortalized cells are capable to form malignant neoplasms in suitable animal models. At present, seven human viruses are categorized as causative agents of distinct human malignancies. The genomes of human tumor viruses, typically encode viral oncoproteins and non- translated viral RNAs that affect the gene expression pattern of their target cells or induce genetic and epigenetic alterations contributing to oncogenesis. Recently, the application of chromatin conformation capture technologies and three-dimensional (3D) molecular imaging techniques revealed how the gene products or genomes of certain human tumor viruses interact with and induce alterations in the 3D host genome structure. This Mini Review aims to cover selected aspects of these developments. The papers, discussed briefly, describe how insertion of a novel viral binding site for the 3D genome organizer cellular protein CCCTC-binding factor (CTCF) into the DNA of T cells infected by human T-cell lymphotropic virus type 1 (HTLV-1) may contribute to lymphomagenesis, as well as how integration of high risk human papillomavirus genome into the host cell DNA may facilitate cervical carcinogenesis. Recent results regarding the interactions of cellular genomes with the episomal, chromatinized DNA genomes of oncogenic human herpesvirus, Epstein-Barr virus (EBV) will also be summarized, similarly to available data regarding contacts formed by episomal or integrated hepatitis B virus (HBV) DNA with host chromatin. Finally, a putative mechanism of hepatitis C virus (HCV) induced chromatin alterations will be presented, which may solve the riddle, how a cytoplasmic RNA virus without a viral oncogene could induce malingnant transfrormation of hepatocytes.

Integrative profiling of Epstein-Barr virus transcriptome using a multiplatform approach.

BACKGROUND: Epstein-Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. METHODS: In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other's data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. RESULTS: This study detected novel genes embedded into longer host genes containing 5'-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. CONCLUSIONS: An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.

Anaerobic bacterial communities associated with oral carcinoma: Intratumoral, surface-biofilm and salivary microbiota.

It was estimated that more than 700 bacterial species inhabit the oral cavity of healthy humans. Anaerobes comprise a significant fraction of the oral bacteriome and play an important role in the formation of multi-species biofilms attached to various anatomical sites. Bacterial biofilms are also associated with pathologic laesions of the oral cavity, including oral squamous cell carcinoma (OSCC), and distinct oral taxa could also be detected within the tumors, i.e. in deep biopsy samples. These observations suggested that certain oral bacteria or oral bacterial communities may play a causative role in oral carcinogenesis, in addition to the well characterized risk factors of oral cancer. Alternatively, it was also proposed that a subset of oral bacteria may have a growth advantage in the unique microenvironment of OSCC. Recently, a series of studies analysed the OSCC-associated bacterial communities using metataxonomic, metagenomic and metatranscriptomic approaches. This review outlines the major differences between the community structure of microbiota in tumor biopsy, surface-biofilm and salivary or oral wash samples collected from OSCC patients, compared to corresponding samples from control persons. A special emphasis is given to the anaerobic bacteria Fusobacterium nucleatum and Fusobacterium periodonticum that were characterised repeatedly as "OSCC-associated" in independent studies. Predicted microbial functions and relevant in vivo experimental models of oral carcinogenesis will also be summarized.

A case study of salivary microbiome in smokers and non-smokers in Hungary: analysis by shotgun metagenome sequencing.

OBJECTIVE: To investigate the role of cigarette smoking in disease-development through altering the composition of the oral microbial community. Periodontitis and oral cancer are highly prevalent in Hungary; therefore, the salivary microbiome of smoker and non-smoker Hungarian adults was characterized. METHODS: Shotgun metagenome sequencing of salivary DNA samples from 22 individuals (11 non-smokers and 11 current smokers) was performed using the Ion Torrent PGMTM platform. Quality-filtered reads were analysed by both alignment-based sequence similarity searches and genome-centric binning. RESULTS: Prevotella, Veillonella and Streptococcus were the predominant genera in the saliva of both groups. Although the overall composition and diversity of the microbiota were similar, Prevotella was significantly more abundant in salivary samples of current smokers compared to non-smokers. Members of the genus Prevotella were implicated in the development of inflammatory diseases and oral cancer. The abundance of the genus Megasphaera also increased in current smokers, whereas the genera Neisseria, Oribacterium, Capnocytophaga and Porphyromonas were significantly reduced. The data generated by read-based taxonomic classification and genome-centric binning mutually validated the two distinct metagenomic approaches. CONCLUSION: Smoking-associated dysbiosis of the salivary microbiome in current cigarette smokers, especially increased abundance of Prevotella and Megasphaera genera, may facilitate disease development.

Truncated oncoproteins of retroviruses and hepatitis B virus: A lesson in contrasts.

Members of the virus families Retroviridae and Hepadnaviridae use reverse transcriptase (RT) to synthesize a DNA copy of their genomic and pregenomic RNA, respectively, during the viral life cycle. A group of viruses belonging to Retroviridae ("acute transforming" retroviruses) as well as human hepatitis B virus (HBV), the prototype member of Hepadnaviridae (hepadnaviruses) are able to cause malignant neoplasms in infected hosts, due to the expression of pleiotropic "transforming proteins" encoded by the genomes of these reverse-transcribing tumor viruses. In this review we wish to compare the common and unique features of replication strategies characteristic of acute transforming retroviruses and HBV and summarize data related to the origin and evolution of their viral oncogenes either via transduction of cellular genes, or by accumulation of mutations in viral sequences that create a new open reading frame (overprinting). The exons of cellular genes (proto-onc genes or c-onc genes) incorporated into the genome of acute transforming retroviruses are regularly affected by deletions resulting in the expression of truncated viral oncoproteins which are frequently dysregulated compared to their cellular counterparts. These retroviral transforming proteins alter the behavior of their target cells (malignant transformation). HBx, a pleiotropic protein of HBV, regulates virus replication and contributes to hepatocarcinogenesis. In contrast to the v-onc genes of acute transforming retroviruses, the viral gene encoding the full-length, wild-type HBx (wtHBx) protein does not have a cellular counterpart. Mutations and deletions frequently affect, however, the HBV genome as well, resulting in the expression of truncated HBx proteins (trHBx) in liver cells. Truncated, especially C-terminal truncated variants of HBx (Ct-HBX proteins), may facilitate initiation and progression of liver carcinoma.

Chicken or the Egg: Microbial Alterations in Biopsy Samples of Patients with Oral Potentially Malignant Disorders.

Oral carcinogenesis often leads to the alteration of the microbiota at the site of the tumor, but data are scarce regarding the microbial communities of oral potentially malignant disorders (OPMDs). Punch biopsies were taken from healthy and non-healthy mucosa of OPMD patients to analyze the microbiome using metagenome sequencing. In healthy oral mucosa biopsies the bacterial phyla Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were detected by Ion Torrent sequencing. The same phyla as well as the phyla Fibrobacteres and Spirochaetes were present in the OPMD biopsies. On the species level, there were 10 bacterial species unique to the healthy tissue and 35 species unique to the OPMD lesions whereas eight species were detected in both samples. We observed that the relative abundance of Streptococcus mitis decreased in the OPMD lesions compared to the uninvolved tissue. In contrast, the relative abundance of Fusobacterium nucleatum, implicated in carcinogenesis, was elevated in OPMD. We detected markedly increased bacterial diversity in the OPMD lesions compared to the healthy oral mucosa. The ratio of S. mitis and F. nucleatum are characteristically altered in the OPMD lesions compared to the healthy mucosa.

Prevalence and genotypes of human papillomavirus in saliva and tumor samples of head and neck cancer patients in Hungary.

In addition to traditional risk factors such as smoking, alcohol consumption and betel nut use, human papillomavirus (HPV) infection also plays a role in the development of head and neck squamous cell carcinomas (HNSCCs). Although among European countries the highest incidence and mortality rates of head and neck cancer types were recorded in Hungary, data regarding HPV prevalence in HNSCCs is scarce. We collected biopsy and saliva samples from patients diagnosed with HNSCC or oral potentially malignant disorders (OPMDs) and tested them for the presence of HPV using the PCR consensus primer set MY09/11 and the GP5+/6+ primer pair. HPV genotypes were assessed by sequencing of the amplified PCR fragments. Oral mucosa and saliva samples from tumor- and OPMD-free individuals were also analysed. HPV was detected in 11 out of 60 HNSCC samples (18%). All of the HPV positive tumors carried HPV type 16. 5 out of the 57 saliva samples collected from HNSCC patients was HPV positive (8.8%); among them, in addition to HPV16, HPV13 was also detected. Tumors located to the oropharynx had the highest HPV positivity rate with 50% (7 out of 14), which was significantly higher than the HPV prevalence in oral mucosa samples collected from controls (0 out of 20; p > 0.001) or in OPMD biopsies (0 out of 21, p > 0.001). 2 out of 57 control saliva samples (3.5%, subtype HPV13 and 11) and 3 out of 39 saliva samples from OPMD patients (7.7%, subtype HPV18, 81 and 10) were HPV positive. Our data suggested that HPV16 infection may contribute, in concert with cigarette smoking, to the development of a subset of head and neck cancers in Hungary. HPV16 infection per se does not account, however, for the high HNSCC incidence rate recorded in this country.

Pathogenic mechanisms of intracellular bacteria.

PURPOSE OF REVIEW: We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. RECENT FINDINGS: DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. SUMMARY: Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Current Trends and Alternative Scenarios in EBV Research.

Epstein-Barr virus (EBV) infection is associated with several distinct hematological and epithelial malignancies, e.g., Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and others. The association with several malignant tumors of local and worldwide distribution makes EBV one of the most important tumor viruses. Furthermore, because EBV can cause posttransplant lymphoproliferative disease, transplant medicine has to deal with EBV as a major pathogenic virus second only to cytomegalovirus. In this review, we summarize briefly the natural history of EBV infection and outline some of the recent advances in the pathogenesis of the major EBV-associated neoplasms. We present alternative scenarios and discuss them in the light of most recent experimental data. Emerging research areas including EBV-induced patho-epigenetic alterations in host cells and the putative role of exosome-mediated information transfer in disease development are also within the scope of this review. This book contains an in-depth description of a series of modern methodologies used in EBV research. In this introductory chapter, we thoroughly refer to the applications of these methods and demonstrate how they contributed to the understanding of EBV-host cell interactions. The data gathered using recent technological advancements in molecular biology and immunology as well as the application of sophisticated in vitro and in vivo experimental models certainly provided deep and novel insights into the pathogenetic mechanisms of EBV infection and EBV-associated tumorigenesis. Furthermore, the development of adoptive T cell immunotherapy has provided a novel approach to the therapy of viral disease in transplant medicine and hematology.

Detection and Phylogenetic Analysis of Torque Teno Virus in Salivary and Tumor Biopsy Samples from Head and Neck Carcinoma Patients.

OBJECTIVES: Because torque teno virus (TTV) has been implicated in tumorigenesis as a cocarcinogen, we studied TTV prevalence in saliva and biopsy samples from head and neck cancer (HNCC) patients, patients with premalignant lesions of oral cancer, and controls. We also wished to determine the TTV genotypes in HNCC patients. METHODS: A seminested polymerase chain reaction (PCR) amplifying the N22 region of the TTV genome, as well as direct sequencing of PCR fragments, was used. RESULTS: TTV prevalence was higher in HNCC patients (saliva: 27/71, 38%; tumor biopsy: 22/74, 30%) than in controls (saliva: 8/56, 14%; oral mucosa: 1/19, 5%). TTV prevalence was also high in patients with premalignant lesions of oral carcinoma (saliva: 9/18, 50%; biopsy: 5/21, 24%). By phylogenetic analysis, TTV belonging mostly to genotypes 1 and 2 was found in HNCC patients. In most of the cases, identical TTV strains were present in the biopsy and salivary sample of the same HNCC patient. In addition, the same TTV strain was detected in 2 laryngeal carcinoma biopsies obtained from 2 independent patients. CONCLUSIONS: Our data are compatible with the idea that TTV might act as a cocarcinogen in certain cases of HNCC. Alternatively, HNCC may facilitate either TTV replication or TTV entry into the saliva.

Infectious Agents Associated with Head and Neck Carcinomas.

In addition to traditional risk factors such as smoking habits and alcohol consumption, certain microbes also play an important role in the generation of head and neck carcinomas. Infection with high-risk human papillomavirus types is strongly associated with the development of oropharyngeal carcinoma, and Epstein-Barr virus appears to be indispensable for the development of non-keratinizing squamous cell carcinoma of the nasopharynx. Other viruses including torque teno virus and hepatitis C virus may act as co-carcinogens, increasing the risk of malignant transformation. A shift in the composition of the oral microbiome was associated with the development of oral squamous cell carcinoma, although the causal or casual role of oral bacteria remains to be clarified. Conversion of ethanol to acetaldehyde, a mutagenic compound, by members of the oral microflora as well as by fungi including Candida albicans and others is a potential mechanism that may increase oral cancer risk. In addition, distinct Candida spp. also produce NBMA (N-nitrosobenzylmethylamine), a potent carcinogen. Inflammatory processes elicited by microbes may also facilitate tumorigenesis in the head and neck region.

Wild type HBx and truncated HBx: Pleiotropic regulators driving sequential genetic and epigenetic steps of hepatocarcinogenesis and progression of HBV-associated neoplasms.

Hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma. The molecular mechanisms of tumorigenesis are complex. One of the host factors involved is apparently the long-lasting inflammatory reaction which accompanies chronic HBV infection. Although HBV lacks a typical viral oncogene, the HBx gene encoding a pleiotropic regulatory protein emerged as a major player in liver carcinogenesis. Here we review the tumorigenic functions of HBx with an emphasis on wild type and truncated HBx variants, and their role in the transcriptional dysregulation and epigenetic reprogramming of the host cell genome. We suggest that HBx acquired by the HBV genome during evolution acts like a cellular proto-onc gene that is activated by deletion during hepatocarcinogenesis. The resulting viral oncogene (v-onc gene) codes for a truncated HBx protein that facilitates tumor progression. Copyright © 2015 John Wiley & Sons, Ltd.

Epigenetic Alterations in Epstein-Barr Virus-Associated Diseases.

Latent Epstein-Bar virus genomes undergo epigenetic modifications which are dependent on the respective tissue type and cellular phenotype. These define distinct viral epigenotypes corresponding with latent viral gene expression profiles. Viral Latent Membrane Proteins 1 and 2A can induce cellular DNA methyltransferases, thereby influencing the methylation status of the viral and cellular genomes. Therefore, not only the viral genomes carry epigenetic modifications, but also the cellular genomes adopt major epigenetic alterations upon EBV infection. The distinct cellular epigenotypes of EBV-infected cells differ from the epigenotypes of their normal counterparts. In Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) significant changes in the host cell methylome with a strong tendency towards CpG island hypermethylation are observed. Hypermethylated genes unique for EBVaGC suggest the existence of an EBV-specific "epigenetic signature". Contrary to the primary malignancies carrying latent EBV genomes, lymphoblastoid cells (LCs) established by EBV infection of peripheral B cells in vitro are characterized by a massive genome-wide demethylation and a significant decrease and redistribution of heterochromatic histone marks. Establishing complete epigenomes of the diverse EBV-associated malignancies shall clarify their similarities and differences and further clarify the contribution of EBV to the pathogenesis, especially for the epithelial malignancies, NPC and EBVaGC.

Epigenetic Dysregulation in Virus-Associated Neoplasms.

The oncoproteins of human tumor viruses regularly interact with the cellular epigenetic machinery. Such interactions alter the epigenome of the host cell and reprogram its gene expression pattern. Altered levels or redistribution of (cytosine-5)-DNA methyltransferases and changes in the cellular methylome were observed in Kaposi sarcoma-associated herpesvirus (KSHV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), and human papillomavirus (HPV) associated neoplasms and cell lines. Methylation-mediated silencing of cellular promoters was also noted in Merkel cell polyomavirus (MCPyV) positive Merkel cell carcinomas, and, as discussed elsewhere, in EBV-associated malignancies and adenovirus-induced rodent tumors as well. Promoter activation also occurred, either associated with DNA hypomethylation or with the induction of euchromatic histone modifications by viral oncoproteins. It is worthy to notice that HCV infection induced large, hypomethylated blocks of cellular chromatin, although the exact molecular mechanism remains to be elucidated. In hepatoma cells expressing HBx, the oncoprotein encoded by the HBV genome, demethylation of the repetitive satellite 2 sequences was observed, due to downregulation of the de novo DNA methyltransferase DNMT3B. Tax and HBZ, the oncoproteins of human T-cell lymphotropic virus type I (HTLV-I), can both activate and silence distinct cellular promoters by interacting with cellular enzymes involved in histone modification.

Patho-epigenetics of Infectious Diseases Caused by Intracellular Bacteria.

In multicellular eukaryotes including plants, animals and humans, epigenetic reprogramming may play a role in the pathogenesis of a wide variety of diseases. Recent studies revealed that in addition to viruses, pathogenic bacteria are also capable to dysregulate the epigenetic machinery of their target cells. In this chapter we focus on epigenetic alterations induced by bacteria infecting humans. Most of them are obligate or facultative intracellular bacteria that produce either bacterial toxins and surface proteins targeting the host cell membrane, or synthesise effector proteins entering the host cell nucleus. These bacterial products typically elicit histone modifications, i.e. alter the "histone code". Bacterial pathogens are capable to induce alterations of host cell DNA methylation patterns, too. Such changes in the host cell epigenotype and gene expression pattern may hinder the antibacterial immune response and create favourable conditions for bacterial colonization, growth, or spread. Epigenetic dysregulation mediated by bacterial products may also facilitate the production of inflammatory cytokines and other inflammatory mediators affecting the epigenotype of their target cells. Such indirect epigenetic changes as well as direct interference with the epigenetic machinery of the host cells may contribute to the initiation and progression of malignant tumors associated with distinct bacterial infections.

Epstein-Barr virus-host cell interactions: an epigenetic dialog?

Here, we wish to highlight the genetic exchange and epigenetic interactions between Epstein-Barr virus (EBV) and its host. EBV is associated with diverse lymphoid and epithelial malignancies. Their molecular pathogenesis is accompanied by epigenetic alterations which are distinct for each of them. While lymphoblastoid cell lines derived from B cells transformed by EBV in vitro are characterized by a massive demethylation and euchromatinization of the viral and cellular genomes, the primarily malignant lymphoid tumor Burkitt's lymphoma and the epithelial tumors nasopharyngeal carcinoma and EBV-associated gastric carcinoma are characterized by hypermethylation of a multitude of cellular tumor suppressor gene loci and of the viral genomes. In some cases, the viral latency and oncoproteins including the latent membrane proteins LMP1 and LMP2A and several nuclear antigens affect the level of cellular DNA methyltransferases or interact with the histone modifying machinery. Specific molecular mechanisms of the epigenetic dialog between virus and host cell remain to be elucidated.

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

Role of epigenetics in EBV regulation and pathogenesis.

Epigenetic modifications of the viral and host cell genomes regularly occur in EBV-associated lymphomas and carcinomas. The cell type-dependent usage of latent EBV promoters is determined by the cellular epigenetic machinery. Viral oncoproteins interact with the very same epigenetic regulators and alter the cellular epigenotype and gene-expression pattern: there are common gene sets hypermethylated in both EBV-positive and EBV-negative neoplasms of different histological types. A group of hypermethylated promoters may represent, however, a unique EBV-associated epigenetic signature in EBV-positive gastric carcinomas. By contrast, EBV-immortalized B-lymphoblastoid cell lines are characterized by genome-wide demethylation and loss and rearrangement of heterochromatic histone marks. Early steps of EBV infection may also contribute to reprogramming of the cellular epigenome.

The role of DNA hypomethylation, histone acetylation and in vivo protein-DNA binding in Epstein-Barr virus-induced CD23 upregulation.

We analyzed epigenetic marks at the CD23 regulatory regions in well characterized Epstein-Barr virus (EBV)-carrying cell lines covering the major latency types. Bisulfite sequencing showed that DNA methylation is not a major regulator of EBV-induced CD23 transcription, although a wide hypomethylated DNA sequence in the regulatory regions is always present in the cell lines with high CD23 expression. Acetylated histone H3 levels at the CD23b promoter showed strong correlation with CD23b expression, while a weaker correlation could be observed at the CD23a core promoter. DMS in vivo footprinting at the intronic EBV-responsive enhancer and the intermediate-affinity CBF1 site at the CD23a core promoter did not reveal any significant sign of in vivo protein-DNA interactions, despite the presence of strong, characteristic footprints in the same DMS-treated DNA samples at the two CBF1 sites of the LMP2A-promoter. Our in vivo results suggest a minor role for DNA methylation, while a more important role for histone acetylation in the regulation of EBV-induced CD23 expression. Furthermore, our in vivo footprinting results support the complex model of CD23 induction by EBV, rather than a simple model with direct transactivation of CD23 by EBNA-2.