Table of contents :

Epidemiology : in 1958, Dennis Burkitt first described a disorder associated with jaw tumours in African children^ref. In 1961, the neoplasm was identified as a form of malignant lymphoma, and what had initially emerged as a clinical syndrome became a pathological entity called Burkitt’s lymphoma (BL)^ref. It is the most frequent tumor in children aged 5 to 9 years in equatorial Sub-Saharan Africa, South America, and Papua New Guinea. The overall incidence of BL in the USA was 1.4 per million for white males and 0.4 per million for white females during 1973-1981^ref.
Aetiology :

• endemic Burkitt's lymphoma (eBL)^ref affects mostly children. The most common site of involvement of endemic BL is the kidneys. Jaw tumours are age related with an overall incidence in Uganda of 50%^ref.
• HHV-4 / EBV is contained in all eBL cells, thus implicating it as a likely etiologic factor. Viral expression is reduced essentially to small non-coding RNA, non-polyadenilates, and EBERs (10⁶ copies per cell) and EBNA1 : expression of EBNA in transgenes leads to lymphoma in mice and could play a role in the expression of the c-myc gene involved in translocations. Very early (during the first months of life) EBV infection observed in North or equatorial Africa increases the risk of BL by 20-times that in Europe. EBV is considered by some an advantage for selection^ref
• reovirus 3
• impairment of EBV-specific CTL immune responses by :
• malaria : geographic distribution of eBL appears to be related to climatic conditions and coincides with areas of holoendemic malarial infection, which is thought to facilitate this oncogenic process. Acute Plasmodium falciparum malaria infection in African children allows expansion of latent EBV infection => increase in the number of EBV-containing B-cells in the circulation, lymph nodes (in 60% of cases)^ref and cell-free EBV DNA in plasma^ref. In healthy seropositive adults, the EBV-carrying B cell is predominantly within the IgM⁺IgD⁺ circulating virgin B cells recently released from the bone marrow but not the IgG⁺ subpopulations. These B cells have an estimated life span of only 6-8 weeks suggesting that long-term EBV persistence in the body may be the result of infection of a more primitive B-cell type. In children with acute malaria EBV-carrying B cells are also found in the IgM⁺, IgG^- B-cell subpopulation. The majority of these cells are found in the low-density (large cell) Percoll fraction although in some patients a proportion was derived from the high-density (small cell) fraction. This cellular phenotype is not representative of a BL cell. Control of malaria endemicity has been associated with reduced incidence of eBL^ref
• chronic schistosomiasis is associated with elevated T_h2 cytokine expression resulting in reduced cell-mediated cytotoxicity^ref
Models :
• Klein’s model : EBV infection => malaria infection =>
• Bornkamm’s model : germinal center B-cell => malaria infection => increased c-MYC => EBV infection
• Niller-Minarovits model : binding of transcription factor and oncoprotein c-Myc to the major locus control region (LCR) of the viral genome directs us to an alternative model for the origin of Burkitt's lymphoma (BL). In this model, improved nuclear maintenance of the viral genome and the continuous expression of anti-apoptotic functions in B cells exhibiting class I EBV latency contribute to the generation of BL, without any detour through EBV nuclear antigen (EBNA) 2-driven B-cell immortalization (also called class III latency)^{ref1, ref2}.
• Niller-Minarovits ping-pong evolution hypothesis : persisting viruses served as a complement for the organismal germline like in a ping-pong game The left part of the EBV genome exhibits a strong colinearity of structural and functional elements with the Ig gene loci which is only partially reflected in nucleotide sequence homologies. This colinearity may be the result of an inter-dependent co-evolution of the immunoglobulin loci together with EBV. Our observation could help elucidating the mechanisms of somatic hypermutation, explaining the ability of EBV to accidentally cause tumors, and shedding more light on the general mechanisms of viral and organismal evolution^ref.
There are 2 main classes of Epstein-Barr virus (EBV) associated lymphomas: primarily malignant Burkitt's Lymphoma (BL) and Hodgkin's Disease (HD), on one hand, and primarily benign lymphoproliferations, e.g., post-transplant lymphoproliferative disease (PTLD) on the other hand. PTLD may start as a benign lymphoproliferation which becomes malignant if out of T cell control for too long. Our discovery of a binding site for the oncoprotein c-Myc at a central position of the EBV genome favours a distinction of pathogenetic pathways or scenarios for the proposed lymphoma classes. In the first scenario nuclear maintenance of the EBV genome and activation of viral anti-apoptotic functions with the help of c-Myc are indispensable for the origin of malignant tumours (BL, HD) from the germinal centre B-cell. In the second scenario expression of the main viral transforming protein EBNA2 is essential for immortalisation and non-malignant morphological transformation of any (germinal centre derived or non-germinal centre) B-cell in the absence of T cell control. Although EBNA2 expression is permissible, under specific circumstances, in malignant B-cells, it is not required for oncogenesis^ref.
LMP2A has been implicated in EBV related tumorigenesis. To understand the host cell dependent expression of the LMP2A gene, it is necessary to analyse the regulatory mechanisms of the LMP2A promoter (LMP2Ap). By transient transfection and in vitro binding analyses two CBF1 sites have previously been shown to be involved in the regulation of LMP2Ap. However, the promoter structure has not been examined at the nucleotide level in vivo. Therefore we undertook a comprehensive analysis of in vivo protein binding and of CpG-methylation patterns at LMP2Ap in a panel of B cell lines carrying latent EBV genomes. The presence of characteristic footprints on two CBF1 and further binding-sites, together with overall hypomethylation of CpG dinucleotides correlated well with promoter activity. In contrast, the absence of several genomic footprints, as well as the presence of patches of highly methylated CpG dinucleotides were characteristic of silent LMP2Aps^ref.
The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the EBV genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes^ref.
We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent EBV genomes using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. Transcripts for 2 latent membrane proteins, LMP1 (a transforming protein) and LMP2B, are initiated in this region in opposite directions. B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylation-mediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sis-inducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter^ref.
EBV latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity^ref.
• van den Bosch model : EBV => arbovirus (> malaria. Geographical and age distributions of eBL in Africa parallel those of certain potentially oncogenic, mosquito-borne arboviruses. Arboviruses seem to be associated with case clusters of endemic Burkitt's lymphoma, and symptoms compatible with arbovirus infection have been seen immediately before the onset of the tumour. Chikungunya virus^ref) => plant promoters (Euphorbia tirucalli, the distribution of which coincides with the boundaries of the lymphoma belt : RNA and DNA viruses, including EBV, are promoted by extracts of this commonly used plant. Extracts of E. tirucalli are tumour promoters and can induce the characteristic 8;14 translocation of endemic Burkitt's lymphoma in EBV-infected cell-lines. They also activate latent EBV in infected cells, enhance EBV-mediated cell transformation, and modulate EBV-specific immunity^ref)
• microarray models : gene signatures analyzed by cDNA microarray, and they were validated by immunohistochemistry, flow cytometry, and Western blotting
• TIMP-1 repressed expression of germinal center (GC) markers CD10, Bcl-6, PAX-5 and up-regulated plasma cell-associated antigens CD138, MUM-1/IRF-4, XBP-1, and CD44, suggesting a plasma cell differentiation. This is accompanied by activation of STAT-3 and switch to cyclin D2 expression. However, TIMP-1JD38 cells expressed an inactive form of XBP-1, lacking antibody production/secretion. This incomplete plasmacytic differentiation occurs without altering cell proliferation, and despite c-Myc deregulation, indicating an arrested plasmacytic/plasmablastic stage of differentiation. Further validation in human lymphoma cell lines and in primary B-cell tumors demonstrated a predominant TIMP-1 expression in tumors with plasmacytic/plasmablastic phenotypes, including multiple myelomas^ref
• EBNA-2 upregulates IL-16, an immunomodulatory cytokine involved in the regulation of CD4 T cells, and AML-2, a member of the Runt domain family of transcription factors. AML-2 expression is normally predominant in EBV latency III, whereas AML-1 is associated with EBV latency I or EBV^- cells^ref. EBNA2 induces expression of the 2 chains of the IL-18R in Burkitt lymphoma (BL) cell lines and in nontransformed B cells. Activation of IL-18R expression by EBNA2 is independent of its interaction with the transcriptional repressor RBPJ kappa. It occurs in the absence of any other viral protein but requires de novo synthesis of cellular proteins. IL-18R induction is a highly specific function of EBNA2, because neither other EBV latent proteins nor the cellular proteins c-myc or Notch can exert this effect. Using cDNA microarray expression profiling, we find that the IL-18 receptor expressed in EBV-infected BL cells has signaling capacity, because IL-18 significantly modified gene expression. EBNA2 expression is associated with IL-18R expression in vivo in EBV⁺ B-lymphomas from AIDS patients^ref.
• Kuppers-Dalla-Favera :
• Hodgkin and Reed-Sternberg (HRS) cells represent the malignant cells in classical Hodgkin lymphoma (HL). Because their immunophenotype cannot be attributed to any normal cell of the hematopoietic lineage, the origin of HRS cells has been controversially discussed, but molecular studies established their derivation from germinal center B cells. In this study, gene expression profiles generated by serial analysis of gene expression (SAGE) and DNA chip microarrays from HL cell lines were compared with those of normal B-cell subsets, focusing here on the expression of B-lineage markers. This analysis revealed decreased mRNA levels for nearly all established B-lineage-specific genes. For 9 of these genes, lack of protein expression was histochemically confirmed. Down-regulation of genes affected multiple components of signaling pathways active in B cells, including B-cell receptor (BCR) signaling. Because several genes down-regulated in HRS cells are positively regulated by the transcriptional activator Pax-5, which is expressed in most HRS cells, we studied HL cell lines for mutations in the Pax-5 gene. However, no mutations were found. We propose that the lost B-lineage identity in HRS cells may explain their survival without BCR expression and reflect a fundamental defect in maintaining the B-cell differentiation state in HRS cells, which is likely caused by a novel, yet unknown, pathogenic mechanism^ref.
• Hodgkin lymphoma (HL) is a malignancy of unknown pathogenesis. The malignant Hodgkin and Reed/Sternberg (HRS) cells derive from germinal center B cells (or rarely, T cells) but have a heterogeneous and largely uncharacterized phenotype. Using microarrays, we compared the gene expression profile of four HL cell lines with profiles of the main B cell subsets and B cell non-HLs to find out whether HRS cells, despite their described heterogeneity, show a distinct gene expression, to study their relationship to other normal and malignant B cells, and to identify genes aberrantly or overexpressed by HRS cells. The HL lines indeed clustered as a distinct entity, irrespective of their B or T cell derivation, and their gene expression was most similar to that of EBV-transformed B cells and cell lines derived from diffuse large cell lymphomas showing features of in vitro-activated B cells. 27 genes, most of which were previously unknown to be expressed by HRS cells, showed aberrant expression specifically in these cells, e.g., the transcription factors GATA-3, ABF1, EAR3, and Nrf3. For five genes, expression in primary HRS cells was confirmed. The newly identified HL-specific genes may play important roles in the pathogenesis of HL, potentially represent novel diagnostic markers, and can be considered for therapeutic targeting^ref.
• reciprocal chromosomal translocations involving the Ig loci are a hallmark of most mature B cell lymphomas and usually result in dysregulated expression of oncogenes brought under the control of the Ig enhancers. Although the precise mechanisms involved in the development of these translocations remains essentially unknown, a clear relationship has been established with the mechanisms that lead to Ig gene remodeling, including V(D)J recombination, isotype switching and somatic hypermutation. The common denominator of these three processes in the formation of Ig-associated translocations is probably represented by the fact that each of these processes intrinsically generates double-strand DNA breaks. Since isotype switching and somatic hypermutation occur in GC B cells, the origin of a large number of B cell lymphomas from GC B cells is likely closely related to aberrant hypermutation and isotype switching activity in these B cells^ref.
• genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis^ref.
• Thorley-Lawson model^ref : LMP1 and LMP2A--allow EBV to exploit the normal pathways of B-cell differentiation so that the EBV-infected B blast can become a resting memory cell^ref. EBV is a human herpesvirus that infects over 90% of the world's adult population. Although EBV infection is usually benign, it is associated with several neoplasias and is the causative agent of acute infectious mononucleosis (AIM)^ref. As with other herpesviruses, primary infection by EBV is followed by lifelong persistence. EBV persists in resting, recirculating memory B lymphocytes^{ref1, ref2, ref3}. We have previously presented evidence^ref in support of a model^{ref1, ref2, ref3} whereby EBV gains access to memory B cells by using different transcription programs to first activate latently infected cells and then allow them to differentiate into resting memory B cells. One major unresolved issue with EBV in vivo is the nature of the cells responsible for replicating the virus and the signals necessary to drive a latently infected cell into viral replication. Lytic replication of EBV occurs regularly in healthy carriers, since virus particles are found in their saliva^ref. It is assumed that the latently infected memory B cells circulating in the body return periodically to Waldeyer's ring (tonsils and adenoids)^ref, where they undergo periodic reactivation to produce infectious virus to be shed into saliva. The signal that initiates viral replication in vivo is unknown; however, we assume that it is not provided by the virus, since viral proteins are not expressed in the latently infected memory cells^ref. The process of activating normal memory cells in lymph nodes usually causes them to differentiate into antibody-producing plasma cells^ref. These cells are essentially biochemical factories, so they would be ideal for the efficient production of virions. Also, they are located in the tonsil epithelium^{ref1, ref2}, which would allow them to shed virus into the saliva. The first suggestion that EBV replication may be associated with terminal differentiation came from an early study that detected expression of a poorly characterized, plasma cell-associated surface marker (PC1) on cells replicating the virus in tissue culture cell lines^ref. It has also been reported^{ref1, ref2} that very rare cells, in tonsils from AIM patients, express proteins from the viral lytic cycle. The cells were detected by immunohistochemistry and resembled plasma cells morphologically. These results need to be interpreted with care, however, since immunohistochemical studies are prone to artifacts, especially when detecting rare positive cells for which no independent verification of infection status or cell phenotype is established. These concerns are highlighted because those authors also observed rare infected cells bearing T-cell markers. Since infection of T cells is not normally associated with EBV, this raises concerns that the rare cells replicating EBV in AIM tonsils are artifacts or aberrations of the very high levels of infection found in AIM. It was also unresolved whether plasma cell differentiation is the signal for reactivation or whether reactivation results in plasma cell differentiation. EBV reactivation has been extensively studied in vitro with latently infected cell lines. Lytic replication begins through expression of the immediate-early transcription factor BZLF1^ref. BZLF1 then initiates a cascade of gene expression beginning with the early genes, some of which are involved in viral DNA replication, and late genes, which are expressed after viral DNA synthesis and include components of the virion. Various signals have been shown to trigger rapid induction of the promoter for BZLF1^ref and to induce viral replication in culture^{ref1, ref2}. Although these in vitro systems employ potential differentiation signals^{ref1, ref2} to initiate viral replication, terminal differentiation of cells replicating the virus into plasma cells is not observed. Thus, the in vitro studies stand in contrast to the histochemical studies, and it is unclear what, if any, relationship exists between plasma cell differentiation and induction of viral replication. In this paper we address the question of which cell type initiates viral replication in the tonsils of healthy carriers by separating subsets of tonsil cells and identifying which ones express the BZLF1 gene. We identify the plasma cell as the cell type in which EBV undergoes reactivation in vivo in human tonsils and provide evidence that the terminal differentiation of B cells into plasma cells may provide the signal that triggers the switch from latency into the lytic cycle. The cells that initiate EBV replication, i.e., express the immediate-early gene BZLF1, in the tonsils have the phenotype of plasma cells (CD38^hi, CD10^–, CD19⁺, CD20^lo, IgD^–, surface Ig^–, and cytoplasmic Ig⁺). Unfortunately, cells expressing viral late proteins were extremely rare, and we have no way to test whether infectious virus is actually produced by this population. These technical difficulties make it difficult to conclude with certainty that plasma cells actually produce infectious virus, although they clearly initiate replication. The possibility that the replication is abortive cannot be excluded at this time. The main arguments against this conclusion are that we did detect single BcLF1⁺ cells in some experiments, we showed that memory cells have to differentiate into plasma cells before the BZLF1 promoter is activated, and plasma cells were the only population where a significant fraction of the infected cells initiate replication based on BZLF1 expression. Therefore, if virus is being produced by any B-cell subset, it must be in the plasma cells. Viral replication was not detected in naive (IgD⁺ CD38^– CD20⁺) or germinal center (CD10⁺ CD38⁺ CD20⁺) cells. Rare cells expressing BZLF1 or early or late antigens were found in the CD38⁺ fraction, but these probably represent contamination by a small number of CD38^hi cells. For example, 25% of the infected CD38hi cells were BZLF1 positive for tonsil 1, whereas only 0.1% of the infected CD38+ cells were BZLF1 positive. Since there are similar frequencies of virus-infected cells in both populations, it would take only a 0.5% contamination of CD38hi cells in the CD38+ population to account for this result. Most likely, therefore, the BZLF1 signals in the CD38⁺ population are caused by small numbers of contaminating CD38hi cells. Even if there was no contamination and the CD38⁺ signals were real, the ratio of CD38^hi to CD38+ to CD38– B cells in the tonsil is 1:10:30^ref. Therefore, in absolute terms, 25 out of every 26 (95%) BZLF1-expressing cells reside in the CD38^hi population for tonsil 1. Similarly, 85% can be estimated to reside in the CD38^hi population for tonsil 2. Plasma cells are not thought to be self-renewing, and therefore it is unlikely that they are in themselves a site of persistent infection. This is because the infected cells would be rapidly depleted when they die producing infectious virus. EBV could persist through chronic reinfection and replication in plasma cells; however, this is unlikely because they do not express a known viral receptor and there is no evidence that plasma cells can be directly infected. It is most likely that plasma cells replicating the virus are produced through terminal differentiation of a small number of cells from the pool of latently infected circulating resting memory cells that are generally believed to be the site of long-term persistent infection for EBV^{ref1, ref2}. It follows that these cells must circulate back to Waldeyer's ring^ref, where they occasionally undergo viral replication to release infectious virus into the saliva^ref. There are 2 possible mechanisms by which EBV in a tonsil memory cell could be induced to replicate:
• viral replication is initiated in memory cells that subsequently differentiate into plasma cells
• latently infected memory cells differentiate into plasma cells and then initiate viral replication.
We favor the second mechanism. The observation that BZLF1 is predominantly expressed in the plasma cell fraction together with our quantitation showing that only about a quarter of the infected plasma cells express BZLF1 implies that infected memory cells differentiate into plasma cells while remaining latently infected and then initiate viral replication. This conclusion is supported by our in vitro studies demonstrating that the BZLF1 promoter functions in memory cells only after they have been driven in culture to become plasma cells. Since this process takes 7 days, it is apparent that lytic replication is induced by plasma cell differentiation and not by the acute signals that drive the differentiation. Plasma cell differentiation is the signal for initiating viral replication rather than that viral replication allows or drives cellular differentiation. Since the circulating, latently infected memory cells express no viral proteins^ref, the signal that causes them to undergo terminal differentiation in vivo is probably host derived. An explanation for how this could work is offered by elegant studies demonstrating that immunological B-cell memory does not require antigen but may be sustained by polyclonal stimuli provided by bystander T-cell help^ref. This mechanism provides for continuous renewal of memory cells through sporadic division, which also produces plasma cells and antibody. The result is maintenance of the memory population while stable levels of antibody are produced over time in the absence of antigen. Applied to EBV, this would explain how the population of latently infected memory cells can be maintained at stable levels for years^ref while, through the continuous generation of plasma cells, virus can also be continuously produced^ref. A model of how EBV may persist in the circulating memory B-cell compartment while being continuously shed into saliva, based on the work of Bernasconi et al^ref. They have suggested that memory B cells exposed to bystander T-cell help become activated and divide, generating new memory cells and plasma cells. This process maintains the level of memory cells and antibody-secreting plasma cells in an antigen-independent way. If the memory B cell contained latent EBV, the process would produce another latently infected memory cell, maintaining the levels of latently infected cells in the peripheral circulation, while also producing a plasma cell, thus also ensuring the continuous release of infectious virus. Since this process occurs in the tonsils, the plasma cell would migrate into the lymphoepithelium and produce the virus. Whether this virus is released directly into saliva or is first amplified through lytic infection of epithelial cells is a controversial and, as yet, unresolved issue.

An alternate hypothesis is that plasma cell generation is stimulated by cognate antigen and T-cell help. This would mean that EBV becomes reactivated when the latently infected memory cell undergoes an antigen-specific secondary response. This hypothesis has the attractive feature that latency is established in antigen-specific memory cells in the tonsil. These cells would then enter the peripheral circulation, where they would maintain persistent infection. As these cells reenter secondary lymphoid tissue, the site where they would most likely reencounter cognate antigen would be the tonsil. This would provide a mechanism for preferential homing and reactivation of the latently infected memory cells in the tonsil compared to other lymph nodes. This hypothesis is made uncertain by the fact that EBV encodes latent proteins, LMP1 and LMP2, which, respectively, can mimic CD40 and antigen receptor-like signals^{ref1, ref2}. This makes cells latently infected with EBV potentially independent of T-cell help and/or antigen. The situation is further complicated by the observation from in vitro studies that signaling through LMP1 and/or LMP2 can block reactivation initiated by antigen receptor signaling^{ref1, ref2}, implying that the virus has encoded latent proteins with the specific intent of blocking antigen-driven activation. This means that a cell destined to replicate the virus cannot be expressing LMP1 and LMP2. This strongly implicates the circulating resting memory cell, the only latently infected cells type in which expression of these proteins is absent. Taken together, this information leads to a model whereby reception of host-derived signals by a resting latently infected memory cell in the tonsil leads to activation and then terminal differentiation of the cell into a plasma cell without turning on expression of the latent genes. Once the cell becomes a full-blown plasma cell, viral replication begins. This model predicts that the BZLF1 promoter should be highly and specifically responsive to a plasma cell-specific transcription factor(s). In vitro studies with the Akata cell line appear at first glance to support the idea that antigen drives viral replication. Cross-linking of the antigen receptor (BCR) on these cells leads to their synchronous entry into the lytic cycle, and BCR signaling is known to be important in plasma cell differentiation^ref. However, a number of properties of this system suggest that it may not be representative of in vivo replication. First and most striking is that activation of the BZLF1 promoter in Akata cells occurs within minutes of the antigen receptor signal, whereas differentiation into plasma cells takes days. The response in Akata cells is too acute to be the same as the in vivo mechanism, and not surprisingly, the Akata cells do not undergo plasma cell differentiation prior to viral replication. The Akata system may be more closely related to the spontaneous reactivation that occurs when latently infected peripheral memory cells are placed into culture^ref. This is an acute reactivation, presumably in response to the stress induced upon being placed in culture, and also occurs too rapidly to be associated with terminal differentiation. It is also important to note that all of the signals that induce viral replication in vitro in cell lines, including surface Ig cross-linking of Akata cells, are associated with the induction of apoptosis in the cells^ref. However, expression of lytic EBV genes protects these cells from apoptosis^ref, suggesting that EBV has acquired specific genes to save a stressed cell from death while the virus replicates. This discussion raises the possibility that there may be two mechanisms for reactivating EBV in vivo. One may be an acute reactivation in resting memory cells in response to stress that allows the virus to escape quickly before the cell dies. The other allows replication of EBV in plasma cells located in the epithelium of the tonsil. This allows for high-level production of virions that may be shed directly into saliva. For the tonsils that we fractionated and studied in detail, 10 to 20% of the infected plasma cells initiated viral replication. We measured the frequencies of virus-infected cells in the unfractionated tonsil lymphocytes and in the subsets isolated on the basis of CD38 expression. Since we also know the relative abundance of plasma cells based on CD38 staining, we can use this information to estimate that 0.1 to 0.5% of all of the infected cells in the tonsil are replicating the virus. Assuming approximately 10¹⁰ B lymphocytes in Waldeyer's ring and a range of infected cells from 1 to 1,000/10^7ref, we may calculate that anywhere from 5 to 1,000 cells are replicating EBV in Waldeyer's ring at any one time. However, we found sequentially fewer cells expressing the immediate-early, early, and then late lytic antigens, such that it appears that only 10% of the cells complete the replicative cycle. This suggests that < 100 cells are actually releasing virus in Waldeyer's ring at any given time. This may account for the relatively low titers of EBV observed in the saliva of most healthy carriers^ref. The sequential diminution in the numbers of cells replicating the virus as they proceed through the cycle does not appear to be a technical artifact; therefore, some physiological process is impeding the progress of the cells. One possibility, as discussed above, is that replication in the majority of cells is abortive and simply never reaches completion. An alternative explanation for the diminution is that immunosurveillance by CTL kills the cells as they progress through the lytic cycle, leaving fewer and fewer cells as the cycle progresses. This is consistent with our finding that the cells expressing BZLF1, a major target of CTL, do not down regulate class I MHC and are therefore liable to CTL attack. It is possible that MHC down regulation could occur at later times, since we analyzed only immediate-early (i.e., BZLF1) expression; however, this presumably would be too late, since the CTL would already have detected the cells expressing the immediate-early genes. This distinguishes EBV from other herpesviruses, such as HSV, which encode immediate-early proteins that specifically blunt CTL responses by down regulating MHC class I^ref. The failure of EBV to do so may reflect different strategies for these viruses. For HSV the strategy of the virus is to sporadically reactivate and produce infectious virus for a brief period of time before viral replication is shut down by the immune response. EBV, on the other hand, is chronically shed into the saliva, so transient delay of the immune response would not be useful. Rather, it appears that EBV is able to continue generating replicative cells, a few of which complete the cycle despite the cellular immune response. This suggests that EBV may have developed a mechanism to constitutively reduce the local effectiveness of the CTL response, perhaps through the production of viral IL-10 during the lytic cycle^{ref1, ref2}. In a previous study on viral replication, we used Gardella gels to detect the linear replicating form of viral DNA^ref. In that study we failed to detect linear DNA in the IgD^– fraction, which would appear to be inconsistent with the present results. However, the present study predicts that only very small numbers of plasma cells reach the late stages of replication, and it would not be possible to load sufficient cells on a Gardella gel to detect this very low level. Conversely, in that study we did detect linear viral DNA in the CD19^– fraction. In follow-up studies we were unable to associate this linear DNA with a particular cell type. It is possible that it could be due to epithelial cells with replicating virus or cell debris-associated virion DNA from dead cells. One confusion that resulted from this study was the observed surface phenotype (CD20^lo) of tonsil plasma cells. This arose in part because plasma cells from the bone marrow are CD20^{–ref1, ref2} and in part because some reviews state that tonsil plasma cells are also CD20–^ref. However, our study and several previous studies show clearly that tonsil plasma cells, unlike the bone marrow, continue to express CD20, albeit at a reduced level^{ref1, ref2, ref3}. In conclusion, we have shown that differentiation to plasma cells is associated with induction of the EBV lytic cycle in vivo. It now remains to be discovered what the signals are that drive this differentiation^ref. During acute infection with EBV, the peripheral blood fills up with latently infected, resting memory B cells to the point where up to 50% of all the memory cells may carry EBV. Despite this massive invasion of the memory compartment, the virus remains tightly restricted to memory cells, such that, in one donor, < 1 in 10⁴ infected cells were found in the naive compartment. Even during acute infection, EBV persistence is tightly regulated. This result confirms the prediction that during the early phase of infection, before cellular immunity is effective, there is nothing to prevent amplification of the viral cycle of infection, differentiation, and reactivation, causing the peripheral memory compartment to fill up with latently infected cells. Subsequently, there is a rapid decline in infected cells for the first few weeks that approximates the decay in the CTL responses to viral replicative antigens. This phase is followed by a slower decline that, even by 1 year, had not reached a steady state. Therefore, EBV may approach but never reach a stable equilibrium^ref. EBNA1 is required to maintain the viral genome but is not recognized by cytotoxic T cells. Consequently, it was proposed that this expression pattern was used by latently infected B cells in vivo. This would be the site of long-term, persistent infection by the virus and, by implication, the progenitor of BL. EBV persists in memory B cells in the peripheral blood and that BL is a tumor of memory cells. However, a normal B cell expressing EBNA1 alone has been elusive. Most infected cells in the blood express no detectable latent mRNA or proteins. The exception is that when infected cells divide they express EBNA1 only. This is the first detection of the BL viral phenotype in a normal, infected B cell in vivo. It suggests that BL may be a tumor of a latently infected memory B cell that is stuck proliferating because it is a tumor and, therefore, constitutively expressing only EBNA1^ref.
• sporadic BL affects both children and adults. Only < 20% of BL cases occurring in Western countries are HHV-4 / EBV-associated; HHV-6 ? ^{O’Conor GT, 411-17, ref2}. The terminal ileum and lymph nodes are the more commonly involved sites in sporadic BL^{ref1, ref2}.
• AIDS-related BL^{Pathology and genetics: tumors of haematopoietic and lymphoid tissues. The World Health Organisation classification of tumors. Jaffe ES, Harris NL, Stein H, et al, eds. Lyon: IARC Press, 2001} : BL has frequently been reported as a common neoplasm in HIV infected patients^ref, although it is not known why BL is so common in HIV and not in other forms of immunodepression. These lymphomas usually display an activation of c-myc by chromosome translocations that show structural similarities to those found in patients with sporadic BL^ref. Nonetheless, most AIDS related BLs in Western countries are EBV negative^ref, whereas in Africa they are strongly associated with EBV^ref
• Burkitt-like (BL-like) lymphoma has been commonly applied to those tumours that have morphological features intermediate between large cell lymphoma with centroblastic or immunoblastic features and typical BL. The REAL classification gives BL-like lymphoma provisional status, leaving the differential diagnosis between BL and diffuse large B cell lymphoma (DLBCL) unresolved^ref. The oncologists recommended that the category of BL-like lymphoma be reserved for tumours to be treated "like Burkitt lymphoma". A recent study by the southwest oncology group (SWOG) concluded that BL-like lymphoma can be recognised by its combined morphology and phenotypical features and that it represents a high grade lymphoma much closer to BL than DLBCL^ref. In the World Health Organisation (WHO) classification, BL-like lymphoma is listed as a morphological variant of BL (atypical BL), in addition to the 3 subcategories—endemic, sporadic, and immunodeficiency associated—proposed to reflect the major clinical and genetic subtypes of this disease^{Pathology and genetics: tumors of haematopoietic and lymphoid tissues. The World Health Organisation classification of tumors. Jaffe ES, Harris NL, Stein H, et al, eds. Lyon: IARC Press, 2001}. In the WHO classification, the definition of atypical BL is a lymphoma that morphologically resembles BL, but has more pleiomorphism or larger cells than classic BL, and has a proliferation fraction of > 90%.

a chromosomal translocation in a single B cell which leads c-myc protooncogene under the control of ...

Ig H chains promoter : t(8;14)(q32;q21) (80%)

l chain promoter : t(8;22)(q21;q11)

k chain promoter : t(2;8)(p11-12;q21)

=> overexpression have been identified in endemic and sporadic BL. However, some not very strict associations between J_H and D_H recombination have been identified at 14q32, with distant 5‘ c-myc recombination in endemic cases of BL, whereas in sporadic cases Sµ and S recombination was identified^ref at 14q32, with near 5‘ or intronic c-myc recombination at 8q24. These differences between endemic and sporadic BL do not mean that each of these subtypes of lymphoma represents a perfectly homogeneous entity^ref. Instead, it seems probable that BL is composed of a mixture of molecular types and that the incidence of each subtype might depend upon environmental factors^{ref1, ref2}. Yet, the sporadic form of BL can also occur in endemic areas^ref, as reported in the table below, which summarises the distribution of BL collected from endemic areas of Kenya according to the age of patients, the clinical pathological characteristics, and the EBV and HIV status. These observations emphasise the importance of precise disease definition for biological and epidemiological studies.

		BL age under 16 years (range 2–16; median 6)	BL age over 16 years (range, 17–58; median 29)
Total cases		23	16
Male/Female		14/9	10/6
Site	jaw	16	2
	nodal	3	7
	ileum	2	5
	other	2	2
HIV		0	8
EBV		21	12

The necessary cytogenetics for the diagnosis of BL should be the presence of the t(8;14) (q24;q32) translocation and its variants, or c-myc rearrangement. If cytogenetic or Southern blot cannot be applied to solid tumours the most reasonable surrogate for c-myc rearrangement is probably the proliferation fraction. Therefore, cases in which cytogenetic analysis is not available should not be diagnosed as BL or BL-like without a Ki-67 fraction close to 100%^{Pathology and genetics: tumors of haematopoietic and lymphoid tissues. The World Health Organisation classification of tumors. Jaffe ES, Harris NL, Stein H, et al, eds. Lyon: IARC Press, 2001}. The morphological similarity between BLs probably results from the fact that they all have a common translocation, t(8;14), and consequent c-myc rearrangement and overexpression. However, it is also true that BLs account for only 30% of lymphomas bearing a c-myc translocation at presentation^{Gaulard Ph, Delsol G, Callat MP, et al. Cytogenetic and clinicopathologic features of B-cell lymphomas associated with the Burkitt translocation t(8;14) (q24;q32) or its variants. J Clin Pathol 2002; 55(suppl 1):A22}. Furthermore, the c-myc translocation is not the only genetic lesion found in BL^ref, and myc transformed cells are usually characterised by the loss of expression of several genes^ref. Cooperating alterations of cell cycle associated genes probably contribute to the pathogenesis of BL^{ref1, ref2}. The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis : 2 common mutant MYC alleles derived from human BL uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the Bcl2 family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis^ref.

p53 mutations have been found in 30–40% of BL samples^ref, and most lymphomas with wild-type p53 might have lesions in other growth suppressor genes^ref

recently, mutations of the nuclear localisation signal of the RBL2 / p130 tumour suppressor gene have also been detected in BL cell lines and primary tumours^ref. The pRb2/p130 tumour suppressor gene belongs to the retinoblastoma (Rb) gene family, along with Rb and p107. Although they have similar functional properties, Rb family members are not functionally redundant and each protein has a different temporal profile of interaction with different E2F members^{ref1, ref2, ref3}. Whereas Rb is found in both quiescent and proliferating cells, the expression of Rb2/p130 and p107 is related to the cell cycle^ref. In an ideally controlled proliferating cell population with identical cell cycle and cycle phase times, during which a given protein can be detected in the nucleus by the corresponding antibody, the percentages of cells expressing pRb2/p130 and p107 should be inversely correlated^ref. Although such a pattern of Rb2/p130 and p107 expression has been demonstrated in different types of non-Hodgkin’s lymphomas, this is not the case in BLs^ref. The Rb2/p130 gene is mutated in most cases of endemic BL and to a lesser extent in sporadic BL. In contrast, in AIDS related BL, the wild-type pRb2/p130 gene is highly expressed^{ref1, ref2}. In those tumours in which the Rb2/p130 gene is mutated, the interaction between individual proteins and E2F family members and the timing of formation of particular protein–E2F complexes during the cell cycle can be deregulated.25 In addition to mutations of the gene, interaction with viral oncoproteins is another important mechanism of pRb2/p130 inactivation^{ref1, ref2}. pRb2/p130 shares, with all members of the retinoblastoma gene family, the ability to interact physically with certain DNA virus oncoproteins; by this mechanism, pRb2/p130 is inactivated yet maintained in its underphosphorylated form^{ref1, ref2, ref3, ref4, ref5}. Thus, the absence of mutations in the Rb2/p130 gene and the unusually high expression of pRb2/p130 in tumours with high proliferative activity, such as AIDS related BL, may suggest a physical interaction of pRb2/p130 with viral products. Although HIV-1 has long been recognised as the aetiological agent of AIDS, the role of HIV-1 as an oncogenic virus has not yet been well established. Data exist that clearly suggest that the HIV gene product Tat can contribute to the growth and oncogenesis of human and animal cells^ref. Soluble Tat can function as a biologically active extracellular protein released by infected cells and readily taken up by uninfected cells.36,37 This has also been shown to occur in AIDS related B cell lymphomas, by the diffuse and nuclear staining seen in tissue sections immunostained with anti-Tat monoclonal antibody^ref. In addition, there is experimental evidence that extracellular Tat acts directly on B cells^ref. In particular, germinal centre B cell proliferation was enhanced by the addition of Tat at the initiation of the culture, suggesting that Tat acts on the early stage of B cell activation, probably before the G₁ to S phase transition^ref. This is in line with new data indicating that the well established function of Rb2/p130 in the control of the G₀/G₁ transition can be inactivated by physical interaction with the Tat protein of HIV-1. In fact, the results of an in vitro and in vivo binding assay suggest that the Tat protein of HIV-1 is one of those viral oncoproteins that interact with the Rb family. In particular, they revealed that the Tat protein of HIV-1 interacts specifically with the pocket region of the Rb2/p130 protein^ref. This can result in the inactivation of Rb2/p130 oncosuppressive properties and the induction of genes needed to proceed through the cell cycle including p107, cyclin A, and cyclin B^ref. Consequently, some B cell clones among a virus induced B cell proliferation might have an uninterrupted cell cycle and a growth advantage, thus favouring the incidence of B cell malignancies in lymphoid organs of HIV-1 positive patients. Increased cell proliferation has been shown to account almost entirely for tumour prevalence in immunocompromised patients^ref. Spontaneous regression of HIV-1 associated lymphoproliferative disorders has been reported after highly active antiretroviral therapy^{ref1, ref2, ref3}.  In quiescent G₀ cells, the nuclear E2F–pRb2/p130 complex is responsible for the active repression of several cellular promoters. After its release into the cell cycle, pRb2/p130 is phosphorylated by G1 cyclin dependent kinases (cdks) and subsequently degraded through a proteosome dependent mechanism, resulting in the derepression of a variety of genes, including p107. The accumulated p107 protein is then able to interact with E2F4 and E2F5, which have been released from pRb2/p130, and associate with cyclin A (cyc A)/cdk2.

immunophenotype : CD5^-10 / CALLA⁺19⁺20⁺22⁺23^-45⁺79a⁺, sIgM⁺

cytomorphology : Burkitt’s tumours are composed of monomorphic, medium sized cells with round nuclei, multiple nucleoli, and relatively abundant basophilic cytoplasm, which may give the cells a "cohesive appearance". Typical morphological pattern of Burkitt’s lymphoma (Giemsa stained; original magnification, x375).

These tumours have an extremely high rate of proliferation, in addition to a high rate of cell death (apoptosis). A "starry sky" pattern is usually present, imparted by numerous benign macrophages that have ingested apoptotic tumour cells. Based on immunoglobulin gene rearrangement studies, the cell of origin of BL is currently thought to be a germinal centre B cell^ref, although several studies of IgHV genes in BLs suggest that they may derive from memory B cells rather than germinal centre B cells^ref. However, most of these studies rely on cell lines or a small series of cases. Endemic and AIDS-related forms showed a considerably higher V_H mutation rate than the sporadic form (5.1%, 5.4% and 1.5% respectively). The mutations in eBL and AIDS-related BL also showed signs of antigen selection, while no signs of antigen selection were found in sBL. Finally, after subcloning the amplificates, sequence analysis revealed no signs of ongoing mutations in any of the cases analyzed. Since one of the main differences between eBL and AIDS related BL on the one hand and sBL on the other hand is the association with EBV, we compared EBV-positive and EBV-negative BL, independently of their geographical origin and HIV status. The differences in the number of somatic mutations and antigen selection were even more evident when this approach was used. According to our molecular results, it appears that EBV⁺ and EBV^- BL may originate from two distinct subsets of B cells, pointing to a particular role for the germinal-centre reaction in the pathogenesis of these tumors. The different types of C-MYC translocation reported in BL may also be related to the different stages of B-cell maturation^ref.

Copyright © 2001-2005 Daniele Focosi. All rights reserved  |  Terms of use  | Legal notices
About this site  |  Site map  |  Acknowledgements | Open forum  |  Tell a friend about this site  |  Current link partners
 Abbreviations and acronyms  |  Medical terminology  |  Add a link  |  Translate this site into your favourite language  |  Softwares

Rate Us:10 - The Best987654321 - The Poorest

---

---

Search EntrezNCBI Web Site-------------PubMedGeneLocusLinkTaxonomyProteinNucleotideStructureGenomeBooksCancerChromosomesConserved Domains3D DomainsGEO ProfilesGEO DatasetsHomoloGeneJournalsMeSHNLM CatalogOMIMPMCPopSetPubChem BioAssayPubChem CompoundPubChem SubstanceSNPUniGeneUniSTS for

Search AboutAltaVistaAsk JeevesDictionaryDirectHitExciteFindWhatGoogleGoogle GroupsHotBotLookSmartLycosMetacrawlerMetaLocateNorthern LightOpen DirectorySnapThesaurusWebcrawlerYahoo for

Search Medical Dictionary  for