BCL-6 expression during B-cell activation

Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5' untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL- 6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti- IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL- 6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34- fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression.     

Bcl-x rather than Bcl-2 mediates CD40-dependent centrocyte survival in the germinal center

Both rapid B-cell proliferation and programmed cell death (PCD) occur during the differentiation and selection of B cells within the germinal center. To help elucidate the role of Bcl-x in B-cell antigen selection and PCD within the germinal center, we examined its expression in defined B-cell populations and by immunochemistry of tonsil tissue. Purified B-cell fractions enriched for centrocytes express high amounts of Bcl-x and relatively low amounts of Bcl-2, whereas fractions enriched for centroblasts lack significant levels of both proteins. Consistent with this observation, immunocytochemistry localized Bcl-x within cells scattered throughout the germinal center. Stimulation of tonsil B cells with either CD40 or Staphylococcus aureus Cowan increase bcl-x mRNA and protein levels. Treatment of a cell line with a germinal center phenotype (RAMOS) or the tonsillar B-cell centroblast fraction with CD40 rapidly increased Bcl-x levels and partially rescued B cells from PCD. These data suggest that Bcl-x rather than Bcl-2 may rescue centrocytes during selection in the germinal center.     

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with In vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log 10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.     

APOBEC3G expression is restricted to neurons in the brains of pigtailed macaques

The Vif protein of human immunodeficiency virus-1 (HIV-1) has been shown to interact with members of the APOBEC family of cytidine deaminases, particularly APOBEC3G/F. In this study, we isolated RNA from 12 regions of the brain from two pigtailed macaques that were exsanguinated and perfused with saline. Our results indicate that APOBEC3G was detected in all regions of the brain analyzed. Immunoblot analysis using lysates prepared from these same regions of the brain and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on whole brain sections. Our results clearly show that the pyramidal neurons within the gray matter of cerebral and cerebellar cortices express APOBEC3G. However, APOBEC3G expression in the pyramidal neurons appeared to be nuclear or associated with nuclei. In contrast to our findings in the cerebral cortex, immunohistochemical analysis of the spleen and kidney tissues revealed that APOBEC3G expression in the cells of these tissues was predominantly cytoplasmic. We further investigated the expression of APOBEC3G in astrocytes. Immunohistochemical staining of serial sections was performed using antibodies to glial fibrillary acidic protein (GFAP) and APOBEC3G. As expected, the cortical and cerebellar white matter showed extensive immunostaining of astrocytes with the antibody against GFAP but a lack of reactivity to the antibody to APOBEC3G. Additionally, Immunoblot analysis of lysates prepared from primary human fetal astrocytes revealed a lack of APOBEC3G expression. Taken together, these results indicate that APOBEC3G expression is restricted to neurons in the brain and that astrocytes and microglia probably do not express this protein or express it at levels undetectable by immunohistochemistry. These finding have implications for the brain as a potential reservoir for Vif-defective viruses.     

Immunological aspects in chronic lymphocytic leukemia (CLL) development

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens-including apoptotic bodies-in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.     

Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas

Activation of telomerase seems to be a prerequisite for immortalization and is found in permanent cell lines and most malignant tumors. Normal somatic cells are generally telomerase negative, except for bone marrow stem cells. Weak activity is also present in peripheral blood cells. In the present study strong telomerase activity was demonstrated in vivo in normal mature cells of the immune system, as well as in malignant lymphomas. Benign lymph nodes had lower telomerase activity than benign tonsils, which exhibited intermediate to high activity comparable with findings in malignant lymphomas. In benign tonsils the activity seemed to be restricted to germinal center B cells. In benign lymphoid tissues telomerase activity correlated with B-cell numbers and cell proliferation, but this was not observed in the lymphoma group. High- grade lymphomas exhibited higher levels of telomerase compared with low- grade cases. The data showed that in vivo activation of telomerase is a characteristic feature of germinal center B cells. Different signals for activation of telomerase are likely to exist, one of them being immune stimulation. The data suggest that telomerase activity in malignant lymphomas can be explained by an "induction and retention" model, ie, transformation occurs in a normal, mature B cell with reactivated telomerase, which is retained in the neoplastic clone.     

MORC4, a novel member of the MORC family, is highly expressed in a subset of diffuse large B-cell lymphomas

The OX-TES-4 antigen originally elicited an antibody response in 50% of diffuse large B-cell lymphoma (DLBCL) patients but not in control subjects. OX-TES-4 is encoded by a novel gene, MORC4, located at chromosome Xq22.3. The MORC4 protein contains a HATPase-c domain, CW zinc finger motif, nuclear localisation signals and a nuclear matrix-binding domain, together with a coiled-coil region. MORC4 mRNA is widely expressed at low levels in normal tissues, showing highest expression levels in placenta and testis. mRNA levels were increased in non-germinal centre-derived DLBCL and Hodgkin lymphoma cell lines, compared with germinal centre-derived DLBCL cell lines and normal B cells. Nineteen DLBCL patients (66%) expressed significantly higher levels of MORC4 mRNA than normal B cells (P = 0.0031). The differential expression of MORC4 identifies this molecule as a potential lymphoma biomarker, whose overexpression may contribute to the immunological recognition of MORC4 by a subgroup of DLBCL patients.     

Oct-2、Bob.1在肺结核组织B细胞中的表达及其意义

目的探讨肺结核组织B细胞中Oct-2、Bob.1的表达与机体免疫的关系。方法采用免疫组织化学检测20例肺结核组织B细胞中Oct-2、Bob.1的表达情况。结果 (1)在肺结核组织生发中心的B细胞中Oct-2和Bob.1主要表达在细胞核;在无形成生发中心聚集的B细胞中,细胞核和细胞浆均呈阳性;(2)肺结核组织B细胞中Oct-2阳性的细胞数明显少于Bob.1阳性的细胞数,Oct-2和Bob.1阳性率分别为:10.8%、65.6%;(3)形成生发中心的肺结核组织B细胞中Oct-2和Bob.1阳性的细胞数明显高于无生发中心形成的肺结核组织,差异有统计学意义(P<0.05)。结论肺结核组织B细胞Oct-2表达低下和/或Oct-2、Bob.1表达不一致可能导致B细胞在发育过程中未能形成生发中心。     

HGAL is a novel interleukin-4-inducible gene that strongly predicts survival in diffuse large B-cell lymphoma

We have cloned and characterized a novel human gene, HGAL (human germinal center-associated lymphoma), which predicts outcome in patients with diffuse large B-cell lymphoma (DLBCL). The HGAL gene comprises 6 exons and encodes a cytoplasmic protein of 178 amino acids that contains an immunoreceptor tyrosine-based activation motif (ITAM). It is highly expressed in germinal center (GC) lymphocytes and GC-derived lymphomas and is homologous to the mouse GC-specific gene M17. Expression of the HGAL gene is specifically induced in B cells by interleukin-4 (IL-4). Patients with DLBCL expressing high levels of HGAL mRNA demonstrate significantly longer overall survival than do patients with low HGAL expression. This association was independent of the clinical international prognostic index. High HGAL mRNA expression should be used as a prognostic factor in DLBCL.     

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser(502) that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.     

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.     

Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia

We examined isolated leukemia B cells of patients with chronic lymphocytic leukemia (CLL) for expression of zeta-associated protein 70 (ZAP-70). CLL B cells that have nonmutated immunoglobulin variable region genes (V genes) expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells. In contrast, CLL B cells that had mutated immunoglobulin variable V genes, or that had low-level expression of CD38, generally did not express detectable amounts of ZAP-70 protein. Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined. Ligation of the B-cell receptor (BCR) complex on CLL cells that expressed ZAP-70 induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72(Syk), than did similar stimulation of CLL cells that did not express ZAP-70. Also, exceptional cases of CLL cells that expressed mutated immunoglobulin V genes and ZAP-70 also experienced higher levels tyrosine phosphorylation of such cytosolic proteins following BCR ligation. Following BCR ligation, ZAP-70 underwent tyrosine phosphorylation and became associated with surface immunoglobulin and CD79b, arguing for the involvement of ZAP-70 in BCR signaling. These data indicate that expression of ZAP-70 is associated with enhanced signal transduction via the BCR complex, which may contribute to the more aggressive clinical course associated with CLL cells that express nonmutated immunoglobulin receptors.     

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.     

慢性乙肝患者外周血单个核细胞APOBEC3G mRNA表达的水平及意义

目的探讨外周血单个核细胞（PBMC）APOBEC3GmRNA表达水平与乙肝病毒（HBV）慢性感染的关系。方法用实时荧光相对定量RT．PCR的方法检测27例慢性乙肝患者以及16名健康人PBMC中APOBEC3GmRNA的水平，同时检测乙肝患者外周血HBV病毒载量及肝功能ALT。结果慢性乙肝患者PBMC中APOBEC3GmRNA的表达水平高于健康对照组，差异具有统计学意义（P〈0．01），且与外周血HBV病毒载量成正相关（r=0．73，P〈0．01），与ALT水平无明显相关性（P〉0．05）。结论APOBEC3G在人体内对HBV复制的影响可能不是直接的抑制作用。     

Human Th2 cells selectively express the orexigenic peptide, pro-melanin-concentrating hormone

Th1 and Th2 cells represent the two main functional subsets of CD4(+) T helper cell, and are defined by their cytokine expression. Human Th1 cells express IFNgamma, whilst Th2 cells express IL-4, IL-5, and IL-13. Th1 and Th2 cells have distinct immunological functions, and can drive different immunopathologies. Here, we show that in vitro-differentiated human Th2 cells highly selectively express the gene for pro-melanin-concentrating hormone (PMCH), using real-time RT-PCR, enzyme immunoassay, and Western blot analysis. PMCH encodes the prohormone, promelanin-concentrating hormone (PMCH), which is proteolytically processed to produce several peptides, including the orexigenic hormone melanin-concentrating hormone (MCH). PMCH expression by Th2 cells was activation responsive and increased throughout the 28-day differentiation in parallel with the expression of the Th2 cytokine genes. MCH immunoreactivity was detected in the differentiated Th2 but not Th1 cell culture supernatants after activation, and contained the entire PMCH protein, in addition to several smaller peptides. Human Th1 and Th2 cells were isolated by their expression of IFNgamma and CRTH2, respectively, and the ex vivo Th2 cells expressed PMCH upon activation, in contrast to the Th1 cells. Because Th2 cells are central to the pathogenesis of allergic diseases including asthma, expression of PMCH by activated Th2 cells in vivo may directly link allergic inflammation to energy homeostasis and may contribute to the association between asthma and obesity.     

Ovarian aromatase activity in the domestic fowl (Gallus domesticus)

The effect of excessive tri-iodothyronine (T3) in vivo was assessed using normal human lymphocytes. Cells from normal subjects were frozen in liquid nitrogen before and after oral administration of T3 for 1 week to permit a direct comparison under identical culture conditions. Within the group of individuals studied, some subjects did show changes in B or T cell function but hypertri-iodothyroninaemia produced no consistent effect for the whole group on circulating T cell subsets or T and B cell activation measured by short-term culture or stimulation of lymphocyte cultures with phytohaemagglutinin or pokeweed mitogen. Tri-iodothyronine supplementation of cultures in vitro did not affect pokeweed mitogen stimulation. These findings suggest that the immunological abnormalities in Graves' disease are not the result of increased circulating thyroid hormone levels and that remission following medical treatment is due to an immuno-suppressive effect of the drug rather than the restoration of euthyroidism.     

Expression of the AID protein in normal and neoplastic B cells

Somatic hypermutation (SHM) targets primarily the immunoglobulin variable region (IgV) genes in germinal center (GC) B cells, thereby allowing antibody affinity maturation. A malfunction of SHM, termed aberrant somatic hypermutation (ASHM), was found in about 50% of diffuse large B-cell lymphomas (DLBCLs), leading to mutations in the 5' sequences of multiple genes, including oncogenes. Although the SHM mechanism is largely unknown, it was shown to require the activation-induced cytidine deaminase (AID) gene. AID mRNA is expressed in GC B cells and GC-derived lymphomas, but the pattern of expression of the AID protein is not known. Using 2 specific antibodies, here we show that the AID protein can be detected in GC centroblasts and their transformed counterpart (Burkitt lymphoma) but not in pre-GC B cells and post-GC neoplasms, including B-cell chronic lymphocytic leukemia and multiple myeloma. DLBCLs displayed variable levels of AID expression, which did not correlate with IgV ongoing hypermutation, ASHM, or disease subtype. Finally, both in normal and malignant B cells the AID protein appeared predominantly localized in the cytoplasm. These results indicate that the AID protein is specifically expressed in normal and transformed GC B cells; nonetheless, its predominantly cytoplasmic localization suggests that additional mechanisms may regulate its function and may be altered during lymphomagenesis.