相关论文
1995 - Journal of immunology
Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13.

Production by endothelial cells of the regulated on activation normal T expressed and secreted chemokine (RANTES) has recently been evidenced during delayed-type hypersensitivity (DTH) reactions and may contribute to the local accumulation of macrophages and CD4+ memory T lymphocytes. To document the mechanism inducing RANTES production in this condition, we analyzed the effect of cytokines known to influence the formation of DTH granulomas. Little or no RANTES was produced after stimulation of HUVEC with IFN-gamma, IL-1 beta, or TNF-alpha. However, the combination TNF-alpha+IFN-gamma induced a strong RANTES production. In situ hybridization experiments with a RANTES probe showed that this synergy was also observed at the mRNA level and that the effect of the combination was mainly to increase the amount of RANTES mRNA per cell. The expression of the luciferase gene under the control of the RANTES gene regulatory elements was analyzed; TNF-alpha and the combination TNF-alpha+IFN-gamma activated the regulatory elements. Sequential treatment of HUVEC with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized HUVEC to the stimulating effect of TNF-alpha. The production of RANTES induced by TNF-alpha+IFN-gamma was partly but significantly inhibited by the Th2-type cytokines IL-4 and IL-13. In contrast, IL-10 had no effect. These results indicate that the microenvironment of DTH granulomas, containing high levels of both TNF-alpha and IFN-gamma, may be responsible for RANTES production by perigranulomatous endothelial cells. Inhibition of this production by Th2-type cytokines may be a mechanism by which these cytokines interfere with the formation of DTH granulomas.

2003 - Cancer research
Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis.

Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-homologue 1 (B7-H1), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H1 in human glioma cells in vitro and in vivo. Although lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-gamma, interleukin-2, and interleukin-10) and expression levels of the T-cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T-cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction.

2012 - Gastroenterology
Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells.

BACKGROUND & AIMS Wnt signaling regulates multiple aspects of intestinal physiology, including stem cell maintenance. Paneth cells support stem cells by secreting Wnt, but little is known about the exact sources and primary functions of individual Wnt family members. METHODS We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice, which lack secretory cells. RESULTS Unexpectedly, Wnt3 was dispensable for maintenance of intestinal stem cells in mice, indicating a redundancy of Wnt signals. By contrast, cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt, or coculture with mesenchymal cells, restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. CONCLUSIONS Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures, whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt.

2011 - Proteomics
Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery

Exosomes are membranous vesicles released by cells in extracellular fluids: they have been found and analyzed in blood, urine, amniotic fluid, breast milk, seminal fluid, saliva and malignant effusions, besides conditioned media from different cell lines. Several recent papers show that exosome proteomes of different origin include both a common set of membrane and cytosolic proteins, and specific subsets of proteins, likely correlated to cell‐type associated functions. This is particularly interesting in relation to their possible involvement in human diseases. The knowledge of exosome proteomics can help not only in understanding their biological roles but also in supplying new biomarkers to be searched for in patients' fluids. This review offers an overview of technical and analytical issues in exosome proteomics, and it highlights the significance of proteomic studies in terms of biological and clinical usefulness.

2002 - Cancer research
Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.

1998 - Blood
Prognostic significance of T-cell phenotype in aggressive non-Hodgkin's lymphomas. Groupe d'Etudes des Lymphomes de l'Adulte (GELA).

Peripheral T-cell lymphomas (PTCL) have been generally reported to have a worse prognosis than B-cell lymphomas (BCL). Because of their heterogeneity and scarcity, the outcomes of the different histological subtypes have not been compared. From October 1987 to March 1993, 1,883 patients with diffuse aggressive non-Hodgkin's lymphomas (NHL) included in the LNH87 protocol could be assessed for both morphology and immunophenotyping. Among them, 288 (15%) had PTCL and 1,595 (85%) had BCL. According to the Kiel classification, most PTCL were classified as angioimmunoblastic (AIL; 23%), pleomorphic medium and large T-cell (PML; 49%), or anaplastic large cell (ALCL; 20%) lymphomas. Comparing PTCL with BCL patients, the former had more disseminated disease (78% v 58%), B symptoms (57% v 40%), bone marrow involvement (31% v 17%), skin involvement (21% v 4%), and increased beta2-microglobulin (50% v 34%), whereas BCL patients had more bulky disease (41% v 26%). According to the International Prognostic Index (IPI), PTCL and BCL scores were, respectively: 0 factors, 13% and 15%; 1 factor, 17% and 22%; 2 factors, 24% and 25%; >/=3 factors, 45% and 37% (P = .09). For BCL and PTCL, respectively, complete remission rates were 63% and 54% (P = .004); the 5-year overall survival (OS) rates were 53% and 41% (P = .0004) and event-free survival (EFS) rates were 42% and 33% (P < . 0001). Comparison of the different histological subtypes of lymphoma showed that the 5-year OS rate for T-ALCL (64%) was superior to those of other PTCL (35%) as well as diffuse large B-cell (53%) NHL. When multivariate analysis was applied using the IPI score as one factor, nonanaplastic PTCL remained an independent parameter (P = . 0004). Although the poor prognosis of non-ALCL PTCL could be due in part to the presence of adverse prognostic factors at diagnosis, this study shows that the T-cell phenotype is an independent significant factor, which should be incorporated into the definition of prognostic groups.

1999 - Journal of clinical oncology : official journal of the American Society of Clinical Oncology
Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy.

PURPOSE To determine the safety and efficacy of the combination of the chimeric anti-CD20 antibody, Rituxan (Rituximab, IDEC-C2B8; IDEC Pharmaceuticals Corporation, San Diego, CA), and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy. PATIENTS AND METHODS Forty patients with low-grade or follicular B-cell non-Hodgkin's lymphoma received six infusions of Rituxan (375 mg/m2 per dose) in combination with six doses of CHOP chemotherapy. RESULTS The overall response rate was 95% (38 of 40 patients). Twenty-two patients experienced a complete response (55%), 16 patients had a partial response (40%), and two patients, who received no treatment, were classified as nonresponders. Medians for duration of response and time to progression had not been reached after a median observation time of 29 + months. Twenty-eight of 38 assessable patients (74%) continued in remission during this median follow-up period. The most frequent adverse events attributable to CHOP were alopecia (38 patients), neutropenia (31 patients), and fever (23 patients). The most frequent events attributed to Rituxan were fever and chills, observed primarily with the first infusion. No quantifiable immune response to the chimeric antibody was detected. In a subset of 18 patients, the bcl-2 [t(14;18)] translocation was positive in eight patients; seven of these patients had complete remissions and converted to polymerase chain reaction (PCR) negativity by completion of therapy. CONCLUSION This is the first report demonstrating the safety and efficacy of Rituxan anti-CD20 chimeric antibody in combination with standard-dose systemic chemotherapy in the treatment of indolent B-cell lymphoma. The clinical responses suggest an additive therapeutic benefit for the combination with no significant added toxicity. The conversion of bcl-2 from positive to negative by PCR in blood and/or marrow suggests possible clearing of minimal residual disease not previously demonstrated by CHOP chemotherapy alone.

2000 - The Journal of Immunology
Up-Regulation of IL-17 Is Associated with Bioactive IL-8 Expression in Helicobacter pylori-Infected Human Gastric Mucosa1

Helicobacter pylori (Hp)-associated gastritis is characterized by an increased number of acute and chronic inflammatory cells secreting cytokines that contribute to maintain and expand the local inflammation. Locally induced IL-8 is believed to play a major role in the Hp-associated acute inflammatory response. Factors/mechanisms that regulate IL-8 induction are, however, not fully understood. In the present study we investigated whether Hp infection is associated with an increased production of IL-17, a T cell-derived cytokine capable of modulating IL-8 gene expression. We showed that both IL-17 RNA transcripts and protein were expressed at a higher level in the whole gastric mucosal and lamina propria mononuclear cell samples from Hp-infected patients than in those from uninfected subjects. Hp eradication was associated with a marked down-regulation of IL-17 expression. The addition of a neutralizing anti-IL-17 Ab to the gastric lamina propria mononuclear cell cultures resulted in a significant inhibition of IL-8 secretion, indicating that IL-17 contributes to enhance IL-8 in the Hp-colonized gastric mucosa. Consistently, stimulation of MKN 28 cells, a gastric epithelial cell line, with IL-17 increased IL-8 secretion. Finally, conditioned medium from the IL-17-stimulated MKN 28 cell cultures promoted the in vitro polymorphonuclear leukocyte migration. This effect was inhibitable by a neutralizing IL-8 but not IL-17 Ab. Together, these data indicate that biologically active IL-17 production is increased during Hp infection, suggesting the possibility that this cytokine may play an important role in the inflammatory response to the Hp colonization.

1981 - Proceedings of the National Academy of Sciences of the United States of America
Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera.

Indirect immunofluorescence of certain human sera demonstrated an antigen(s) in the cytoplasm of 1--5% of the cells of a T-cell line, MT-1, from a patient with adult T-cell leukemia (ATL), which is endemic in southwestern Japan. The antigen was not detected in other human lymphoid cell lines, including six T-cell lines, seven B-cell lines, and four non-T non-B cell lines. The antigen did not show cross antigenicity with that of herpesviruses, including Epstein--Barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, herpesvirus saimiri, and Marek disease virus. The proportion of antigen-bearing cells was increased by a factor of approximately 5 on culture in the presence of 5-iodo-2'-deoxyuridine. Antibodies against the antigen in MT-1 cells were found in all 44 patients with ATL examined and in 32 of 40 patients with malignant T-cell lymphomas (most of them had diseases similar to ATL except that leukemic cells were not found in the peripheral blood). The antibodies were also detected in 26% of the healthy adults examined from ATL-endemic areas but in only a few of those examined from ATL-non-endemic areas. On electron microscopy, extracellular type C virus particles were detected in pelleted MT-1 cells cultured in the presence of 5-iodo-2'-deoxyuridine.

2019 - Science Signaling
Specificity, versatility, and control of TGF-β family signaling

Molecular function and pathway cross-talk in TGF-β signaling reveal its complexity and open questions. Gloss The transforming growth factor–β (TGF-β) family of secreted proteins is implicated in the control of widely diverse physiologies and its dysregulation with disease. In this review, which contains 7 figures and 343 references, we describe the exquisite nature of TGF-β family signaling in its roles in diverse and context-specific cellular behaviors. These pathways are far from linear and still far from being fully illuminated. Encoded in mammalian cells by 33 genes, the transforming growth factor–β (TGF-β) family of secreted, homodimeric and heterodimeric proteins controls the differentiation of most, if not all, cell lineages and many aspects of cell and tissue physiology in multicellular eukaryotes. Deregulation of TGF-β family signaling leads to developmental anomalies and disease, whereas enhanced TGF-β signaling contributes to cancer and fibrosis. Here, we review the fundamentals of the signaling mechanisms that are initiated upon TGF-β ligand binding to its cell surface receptors and the dependence of the signaling responses on input from and cooperation with other signaling pathways. We discuss how cells exquisitely control the functional presentation and activation of heteromeric receptor complexes of transmembrane, dual-specificity kinases and, thus, define their context-dependent responsiveness to ligands. We also introduce the mechanisms through which proteins called Smads act as intracellular effectors of ligand-induced gene expression responses and show that the specificity and impressive versatility of Smad signaling depend on cross-talk from other pathways. Last, we discuss how non-Smad signaling mechanisms, initiated by distinct ligand-activated receptor complexes, complement Smad signaling and thus contribute to cellular responses.

2012 - The Journal of clinical investigation
The molecular basis of T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemias (T-ALLs) arise from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process involving constitutive activation of NOTCH signaling and genetic alterations in transcription factors, signaling oncogenes, and tumor suppressors. Notably, these genetic alterations define distinct molecular groups of T-ALL with specific gene expression signatures and clinicobiological features. This review summarizes recent advances in our understanding of the molecular genetics of T-ALL.

1986 - Science
Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance.

Cachectin (tumor necrosis factor) is a macrophage hormone strongly implicated in the pathogenesis of endotoxin-induced shock. The availability of a DNA probe complementary to the cachectin messenger RNA (mRNA), as well as a specific antibody capable of recognizing the cachectin gene product, has made it possible to analyze the regulation of cachectin gene expression under a variety of conditions. Thioglycollate-elicited peritoneal macrophages obtained from mice contain a pool of cachectin mRNA that is not expressed as protein. When the cells are stimulated with endotoxin, large quantity of additional cachectin mRNA is produced, and immunoreactive cachectin is secreted. Macrophages from mice of the C3H/HeJ strain do not produce cachectin in response to endotoxin. A dual defect appears to prevent cachectin expression. First, a diminished quantity of cachectin mRNA is expressed in response to low concentrations of endotoxin. Second, a post-transcriptional defect prevents the production of cachectin protein. Macrophages from endotoxin-sensitive mice do not produce cachectin if they are first treated with dexamethasone, apparently for similar reasons. These findings give new insight into the nature of the C3H/HeJ mutation and suggest an important mechanism by which glucocorticoids may act to suppress inflammation.

2007 - Science's STKE
How Do MicroRNAs Regulate Gene Expression?

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the ~21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3′-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression. The human genome encodes several hundred short (~21 residue) microRNAs (miRNAs), which are predicted to collectively regulate several thousand distinct genes, amounting to about one-third of all human genes. MicroRNAs generally interact with their target mRNAs through imperfect or incomplete complementary base-pairing to sites in the 3′-untranslated region of the messenger RNA (mRNA), which are usually present in multiple copies. These interactions result in a decrease in synthesis of the protein encoded by the mRNA, but until very recently, little was known about the mechanism(s) of this repression. An miRNA can decrease the intracellular concentration of its target mRNAs by accelerating the normal process of mRNA degradation and can inhibit the translation or decoding of the target mRNA, with the relative importance of these two mechanisms differing between different miRNA-mRNA pairs. The mechanism of inhibition of translation remains controversial, with some data pointing to inhibition of the translation initiation step, and other results indicative of inhibition at some later stage during the actual decoding of the mRNA sequence. This article reviews these recent data, discusses the controversies that remain unresolved, and makes suggestions for future research.

2000 - The Journal of experimental medicine
Natural Killer T Cell Activation Inhibits Hepatitis B Virus Replication in Vivo

We have previously reported that hepatitis B virus (HBV)–specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-γ when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of α-galactosylceramide (α-GalCer), a glycolipid antigen presented to Vα14+NK1.1+ T cells by the nonclassical major histocompatibility complex class I–like molecule CD1d. Within 24 h of α-GalCer injection, IFN-γ and IFN-α/β were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4+ and CD8+ T cells from the mice did not diminish the ability of α-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of α-GalCer was inhibited in mice that are genetically deficient for either IFN-γ or the IFN-α/β receptor, indicating that most of the antiviral activity of α-GalCer is mediated by these cytokines. Based on these results, we conclude that α-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.

2009 - Analytical and Bioanalytical Chemistry
MicroRNA detection by microarray

MicroRNAs (miRNAs) are a class of small noncoding RNAs ∼22 nt in length that regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as disease processes. The study of miRNA has thus become a rapidly emerging field in life science. The detection of miRNA expression is a very important first step in miRNA exploration. Several methodologies, including cloning, northern blotting, real-time RT-PCR, microRNA arrays and ISH (in situ hybridization), have been developed and applied successfully in miRNA profiling. This review discusses the main existing microRNA detection technologies, while emphasizing microRNA arrays.

1994 - Journal of immunology
Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity.

Activation of the Fas cell surface molecule, either by specific antibody or by its as yet unidentified ligand, has been shown to induce apoptosis. Because apoptosis is also evoked in target cells by cytolytic T cells, we investigated whether the Fas pathway is involved in CD4+ T cell-mediated cytotoxicity. Analysis of Fas expression in APC, such as the B lymphoma A20.2J and MHC class II-transfected fibroblasts RT2.3, revealed a correlation between the degree of expression and sensitivity to cytotoxic attack, high level of Fas expression in A20.2J being associated with efficient lysis. To examine whether increased Fas expression in RT2.3 would render these cells more susceptible to CD4+ CTL lysis, they were transfected with a Fas gene expression vector. Indeed, Fas- but not mock-transfected RT2.3 proved to be more sensitive to lysis by either Ag specifically or nonspecifically activated CD4+ CTL. Similarly, MHC class II-negative, Fas-transfected L1210 leukemia cells were lysed with nonspecifically activated CD4+ CTL. The importance of the Fas engagement in CD4+ CTL-mediated cytotoxicity is further substantiated by the failure of both cloned and normal CD4+ CTL to lyse B cell blasts from Ipr mice. These mice are known to have a defect in functional Fas expression. Although the bulk of CD4+ T cell-mediated lysis appears to be Fas induced, the fact that the effector phase of A20.2J lysis is only partially Ca2+ independent indicates that other pathways also contribute to target cell death.

2004 - Science
Nuclear Export of MicroRNA Precursors

MicroRNAs (miRNAs), which function as regulators of gene expression in eukaryotes, are processed from larger transcripts by sequential action of nuclear and cytoplasmic ribonuclease III–like endonucleases. We show that Exportin-5 (Exp5) mediates efficient nuclear export of short miRNA precursors (pre-miRNAs) and that its depletion by RNA interference results in reduced miRNA levels. Exp5 binds correctly processed pre-miRNAs directly and specifically, in a Ran guanosine triphosphate–dependent manner, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed by Dicer in vitro. Thus, Exp5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.

1988 - The Journal of experimental medicine
Downregulation of cell adhesion molecules LFA-3 and ICAM-1 in Epstein- Barr virus-positive Burkitt's lymphoma underlies tumor cell escape from virus-specific T cell surveillance

Some EBV+ BL cell lines continue to grow as single cells on in vitro passage, show an unusually restricted expression of EBV-latent genes and retain a BL biopsy-like cell surface phenotype (group I/II lines); others change to growth in aggregates, show a broader pattern of virus latent gene expression, and develop a cell surface phenotype more characteristic of EBV-transformed LCL (group III lines). Here we show that the cell surface adhesion molecules LFA-1, ICAM-1, and LFA-3 are expressed at very low levels, if at all, on group I/II lines and are coordinately upregulated as BL lines move towards group III. The change to growth in aggregates reflects the increasing availability of LFA-1 and ICAM-1, the two ligands whose mutual interaction underlies homotypic BL cell adhesion in vitro. The low levels of ICAM-1 and LFA-3 on group I/II BL cell lines are also associated with an impaired ability to interact with EBV-specific CTL in the antigen-independent phase of effector/target conjugation. mAb blocking studies show that the small number of conjugates that are formed with group I/II BL targets involve the LFA-1/ICAM-1 adhesion pathway but not the LFA-3 pathway; in contrast, both pathways contribute to the efficient conjugate formation shown by group III BL or LCL targets. Earlier work identified one group III line, WW1 BL, as unusual since is expressed the full spectrum of EBV-latent proteins yet remained insensitive to lysis by EBV-specific CTL. Here we show that this line has an anomalous pattern of adhesion molecule expression with high levels of LFA-1 and ICAM-1 in the absence of detectable LFA-3. The WW1 BL cells form conjugates with EBV-specific CTL through the LFA-1/ICAM-1 pathway, but in the absence of a target LFA-3/effector CD2 interaction these conjugates do not achieve target cell lysis. This may reflect an important role for target LFA-3 molecules in activating EBV-specific CTL function. From these in vitro studies, we postulate that downregulation of the adhesion molecules LFA-3 and ICAM-1 on EBV+ BL underlies the ability of the malignant clone to evade EBV-specific T cell surveillance in vivo.

1990 - The Journal of general virology
Different Epstein-Barr virus-B cell interactions in phenotypically distinct clones of a Burkitt's lymphoma cell line.

Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) biopsy cells and early passage BL cell lines have been reported as showing an unusual type of virus-cell interaction; at least two EBV latent proteins appear not to be expressed. Serial passage of such lines is often accompanied by a broadening of virus latent gene expression and a corresponding change in the cell surface/growth phenotype towards that shown by in vitro transformed lymphoblastoid cell lines (LCLs). The sequence of events, both viral and cellular, involved in this transition needs to be defined properly. In the present work, phenotypically distinct cell clones have been derived from early passage cultures of a BL cell line in phenotypic transition, thereby giving access to relatively stable cell populations through which the different EBV-B cell interactions within the parental line can be studied. Clones retaining the original BL biopsy cell phenotype (CD10/CD77-positive, activation antigen/adhesion molecule-negative) expressed the virus-encoded nuclear antigen EBNA 1 but not any of the other known latent proteins, EBNAs 2, 3a, 3b, 3c, -LP and latent membrane protein (LMP). Other clones which had developed an LCL-like phenotype (CD10/CD77-negative, activation antigen/adhesion molecule-positive) now expressed all the above latent proteins and also contained significant numbers of cells in lytic cycle. Phenotypic change occurring within the parental BL cell line itself was initiated in a small subpopulation of cells in which the virus-encoded proteins EBNA 2 and LMP were transiently induced to an unusually high level of expression; this was accompanied by the first detectable changes in cell surface phenotype, namely the increase of cellular adhesion molecules. Some control over EBNA 2/LMP expression then appeared to be re-imposed since the presumed clonal descendents of these cells stably expressed EBNA 2 and LMP at much reduced levels typical of those seen in conventional LCLs.

1987 - The EMBO journal
Differences in B cell growth phenotype reflect novel patterns of Epstein‐Barr virus latent gene expression in Burkitt's lymphoma cells.

Recently established Epstein‐Barr virus (EBV)‐positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more ‘lymphoblastoid’ cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus‐transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other ‘transforming’ proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL‐like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.

Functional delivery of viral miRNAs via exosomes

T. D. de Gruijl
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Katherine Cosmopoulos
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Monique A J van Eijndhoven
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E. Hopmans
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T. Würdinger
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Jaap M Middeldorp
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D. Pegtel
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D. Thorley-Lawson
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Jelle L Lindenberg
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M. V. van Eijndhoven
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Monique A. J. van Eijndhoven

Abstract:Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called “exosomes” that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human γ-herpesvirus EBV.

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