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Papers by Teresa Frisan

Cancer Research

Several lines of evidence indicate that an impairment of EBV-specific immune responses may contri... more Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's dis ease (HD). At present, however, it is not clear whether a defective immu nity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in stand ard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the All and B44 HLA alÃ-eles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a ThO phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.

The Journal of Immunology

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from ... more We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has a...

International Journal of Cancer

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymat... more We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. ...

Cellular Microbiology

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the ... more Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth...

Nature cell biology, 2001

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal tran... more Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

infusion of EBV-specific cytotoxic T cells to develop posttransplant lymphoproliferative disease:... more infusion of EBV-specific cytotoxic T cells to develop posttransplant lymphoproliferative disease: prophylactic Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk http://bloodjournal.hematologylibrary.org/content/95/3/807.full.html Updated information and services can be found at: (3712 articles) Clinical Trials and Observations Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-celldepleted grafts showed a 4-to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10 7 EBV-specific cytotoxic Tlymphocytes (CTLs)/m 2 starting from the time of maximal virus load resulted in a 2to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBVspecific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. (Blood. 2000; 95:807-814)

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection ... more The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the natu...

Blood, Jan 15, 1995

Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-... more Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-encoded latent membrane proteins LMP1 and LMP2 that could serve as rejection targets in Hodgkin's disease (HD). To examine whether EBV-triggered reactivities can be detected in the tumor, we have compared cytokine mRNA expression, cell phenotype, and cytotoxic activity in biopsies from 8 EBV-carrying and 6 EBV-HD patients. Neither the pattern of lymphokine production nor the cell phenotype of the in vivo-activated interleukin-2-responding populations provided a clear discrimination between EBV+ and EBV- cases. HLA class I-restricted EBV-specific cytotoxicity was shown in interleukin-2-dependent cultures from 3 of 3 EBV- tumors, whereas cultures from 6 of 6 EBV+ tumors were either noncytotoxic or exerted LAK-type cytotoxicity. EBV-specific cytotoxic T lymphocyte precursors were present in the blood of 1 patient carrying an EBV+ tumor. The results suggest that a tumor-associated suppre...

Generation of Lymphoblastoid Cell Lines (LCLs)

Epstein-Barr Virus Protocols, 2001

ABSTRACT Epstein-Barr virus (EBV) is a lymphotropic γ herpes virus. Infection of human B cells wi... more ABSTRACT Epstein-Barr virus (EBV) is a lymphotropic γ herpes virus. Infection of human B cells with EBV in vitro results in their immortalization and the resulting cell lines are named lymphoblastoid cell lines (LCLs) (1). In these cells, EBV establishes mainly a latent infection, characterized by the expression of a limited number of viral proteins. LCLs express 6 EBV nuclear proteins (EBNA1 to 6), 3 membrane proteins (LMP1, LMP2A, and LMP2B) and two small untranslated nuclear RNA molecules (EBER1 and EBER2) (reviewed in refs. 2, 3). LCLs have the phenotype of highly activated B cells as assessed by expression of activation markers (CD23, CD39), high levels of expression of adhesion molecules (LFA1, LFA3, ICAM1) and MHC class I and II alleles (reviewed in ref. 4). Owing to these characteristics, these cells are highly immunogenic (reviewed in ref. 5) and provide a useful tool for reactivation of EBV-specific cytotoxic T cells (CTLs) in vitro. EBV-transformed LCLs can be obtained by explantation of blood or lymphoid tissues from EBV seropositive individuals without need for exogenous infection (6). In addition, LCLs from EBV seropositive and seronegative donors can be obtained by in vitro infection of peripheral blood mononuclear cells (PBMCs) with EBV. The most commonly used strain for laboratory work is derived from the marmoset cell line B95.8 (see Chapter 1 and ref. 7). Production of supernatant from this virus producer cell line is described in Subheading 3.4.

Limiting Dilution Assay

Epstein-Barr Virus Protocols, 2001

ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector c... more ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector cells in a population. LDA are dose-response assays that allow detection of an all-or-none (positive or negative) immunoresponse in each individual culture within replicates that vary in the number of responder cells tested (reviewed in ref. 1). The frequency of positive cultures is not informative because it is never clear whether one or more precursors in the culture well are giving the positive response. The negative response instead demonstrates that there are no precursors of a given specificity. Therefore, the evaluation of the cell frequency in the original population is possible by determining the number of cultures that are negative in the experiment. Multiple cultures are set up at different cell concentrations and the larger the number of replicates used for each cell concentration, the more precise the estimate will be. If the percentage of negative cultures is converted to its negative logarithm, the results can be plotted graphically. The fraction of negative cultures is plotted on the ordinate while the cell concentration is plotted on the abscissa to give a straight line. Suitable statistic tests are used to fit this line, including regression analysis and the least square method (2,3).

Regression Assay

Epstein-Barr Virus Protocols, 2001

ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV... more ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV can be monitored in vitro as the capacity of T-cell lymphocytes to inhibit virusinduced proliferation of autologous B cells. In vitro infection of peripheral blood mononuclear cells (PBMCs) from EBV-seronegative and EBV-seropositive donors is accompanied by the appearance of foci of proliferating cells expressing the EBV nuclear antigens (EBNAs) within 7–14 d postinfection. These cells continue to expand giving rise to EBV-transformed lymphoblastoid cell lines (LCLs) in cultures from EBVseronegative individuals. In corresponding cultures from EBV-seropositive donors, the initial proliferation is followed by a regression of growth, seen preferentially at high cell concentration, that culminates within 1 mo in the complete degeneration of the culture (1). This phenomenon has been termed regression or outgrowth inhibition and can be monitored by the regression assay. The strength of the regression can be quantitatively expressed as the minimal number of cells required for 50% growth inhibition.

Membrane independent activation of fibroblast proMMP-2 by snake venom: novel roles for venom proteinases

Toxicon, 2004

ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mamma... more ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mammalian cell culture and a cell-free system. Zymography revealed an increment of latent and activated forms of MMP-2 in muscle homogenates 1-3 days after venom injection. To clarify if venom can induce expression and activation of MMP-2, independently of the inflammatory response, venom was added to cultured human fibroblasts, endothelial and skeletal muscle cells, which expressed proMMP-2 constitutively. Venom activated proMMP-2 without promoting its expression. Venom also activated and degraded proMMP-2 in supernatants collected from fibroblast cultures, indicating that cells are not required for this activation. Pretreatment with EDTA increased MMP-2 activation and reduced degradation. Venom serine proteinases activated proMMP-2, whereas BaP1, a P-I metalloproteinase, predominantly degraded the latent and active forms of MMP-2. Moreover, pretreatment of conditioned medium with serine proteinase inhibitors greatly reduced the venom-induced activation, suggesting that venom proteinases activate MMP-2 via a serine proteinase secreted by fibroblasts. Venom also directly activated and degraded purified proMMP-2, albeit requiring a high concentration. Thus, B. asper venom proteinases activate and degrade proMMP-2 without inducing its synthesis. Serine proteinases play a dominant role in the activation, whereas metalloproteinases predominantly degrade MMP-2. Activation of proMMP-2 by snake venom proteinases, independently of the MT1-MMP/TIMP-2 pathway, extracellular matrix degradation or apoptosis, represents a novel mechanism in human fibroblasts.

Toxins, 2011

The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacter... more The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B 2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?

FEBS Journal, 2011

Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted... more Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.

The FASEB Journal, 2012

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that ... more Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120catenin, ␤-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.-Frisan, T., Coppotelli, G., Dryselius, R., Masucci, M. G. Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth. FASEB J. 26, 000 -000 (2012). www.fasebj.org

Proceedings of the National Academy of Sciences, 2002

have strong antigenpresenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are ... more have strong antigenpresenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are highly allostimulatory in mixed lymphocyte culture. By contrast, EBV-positive Burkitt lymphoma (BL) cells are poor antigen presenters, are not recognized by EBV-specific cytotoxic T cells, and are poorly allostimulatory, which raises the question of whether immunological pressure exerted during BL pathogenesis in vivo has selected for a 'nonimmunogenic' tumor phenotype. The present work addresses this question by examining the immuno-genicity͞antigenicity of cell lines, generated by conversion of a conditionally immortalized lymphoblastoid cell line to permanent growth independent of EBV-latent proteins by introduction of a constitutively active or tetracycline-regulated c-myc gene (A1 and P493-6 cells, respectively). Compared with its parental lymphoblastoid cell line, A1 cells showed many of the features of the nonimmunogenic BL phenotype, namely poor allostimulatory activity, poor antigen-presenting function associated with impaired proteasomal activity, down-regulation of peptide transporter, reduced HLA class I expression, and an inability to present endogenously expressed EBV-latent proteins to cytotoxic T cells. P493-6 cells, when grown in the presence of estrogen with the exogenous c-myc gene switched off, were strongly immunogenic. The cells had lost their immunogenic potential, however, when grown on a c-myc-driven proliferation program in the absence of estrogen. Deregulation of c-myc, a step central to the development of uncontrolled BL cell growth in vivo, can thus impose a nonimmunogenic phenotype on proliferating human B cells in the absence of any immune pressure.

PLoS ONE, 2011

Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced ... more Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).

PLoS ONE, 2010

Background: The MYC protein controls cellular functions such as differentiation, proliferation, a... more Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

PLoS ONE, 2008

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal d... more Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Cancer Research

Several lines of evidence indicate that an impairment of EBV-specific immune responses may contri... more Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's dis ease (HD). At present, however, it is not clear whether a defective immu nity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in stand ard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the All and B44 HLA alÃ-eles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a ThO phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.

The Journal of Immunology

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from ... more We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has a...

International Journal of Cancer

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymat... more We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. ...

Cellular Microbiology

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the ... more Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth...

Nature cell biology, 2001

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal tran... more Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

infusion of EBV-specific cytotoxic T cells to develop posttransplant lymphoproliferative disease:... more infusion of EBV-specific cytotoxic T cells to develop posttransplant lymphoproliferative disease: prophylactic Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk http://bloodjournal.hematologylibrary.org/content/95/3/807.full.html Updated information and services can be found at: (3712 articles) Clinical Trials and Observations Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-celldepleted grafts showed a 4-to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10 7 EBV-specific cytotoxic Tlymphocytes (CTLs)/m 2 starting from the time of maximal virus load resulted in a 2to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBVspecific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease. (Blood. 2000; 95:807-814)

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection ... more The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the natu...

Blood, Jan 15, 1995

Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-... more Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-encoded latent membrane proteins LMP1 and LMP2 that could serve as rejection targets in Hodgkin's disease (HD). To examine whether EBV-triggered reactivities can be detected in the tumor, we have compared cytokine mRNA expression, cell phenotype, and cytotoxic activity in biopsies from 8 EBV-carrying and 6 EBV-HD patients. Neither the pattern of lymphokine production nor the cell phenotype of the in vivo-activated interleukin-2-responding populations provided a clear discrimination between EBV+ and EBV- cases. HLA class I-restricted EBV-specific cytotoxicity was shown in interleukin-2-dependent cultures from 3 of 3 EBV- tumors, whereas cultures from 6 of 6 EBV+ tumors were either noncytotoxic or exerted LAK-type cytotoxicity. EBV-specific cytotoxic T lymphocyte precursors were present in the blood of 1 patient carrying an EBV+ tumor. The results suggest that a tumor-associated suppre...

Generation of Lymphoblastoid Cell Lines (LCLs)

Epstein-Barr Virus Protocols, 2001

ABSTRACT Epstein-Barr virus (EBV) is a lymphotropic γ herpes virus. Infection of human B cells wi... more ABSTRACT Epstein-Barr virus (EBV) is a lymphotropic γ herpes virus. Infection of human B cells with EBV in vitro results in their immortalization and the resulting cell lines are named lymphoblastoid cell lines (LCLs) (1). In these cells, EBV establishes mainly a latent infection, characterized by the expression of a limited number of viral proteins. LCLs express 6 EBV nuclear proteins (EBNA1 to 6), 3 membrane proteins (LMP1, LMP2A, and LMP2B) and two small untranslated nuclear RNA molecules (EBER1 and EBER2) (reviewed in refs. 2, 3). LCLs have the phenotype of highly activated B cells as assessed by expression of activation markers (CD23, CD39), high levels of expression of adhesion molecules (LFA1, LFA3, ICAM1) and MHC class I and II alleles (reviewed in ref. 4). Owing to these characteristics, these cells are highly immunogenic (reviewed in ref. 5) and provide a useful tool for reactivation of EBV-specific cytotoxic T cells (CTLs) in vitro. EBV-transformed LCLs can be obtained by explantation of blood or lymphoid tissues from EBV seropositive individuals without need for exogenous infection (6). In addition, LCLs from EBV seropositive and seronegative donors can be obtained by in vitro infection of peripheral blood mononuclear cells (PBMCs) with EBV. The most commonly used strain for laboratory work is derived from the marmoset cell line B95.8 (see Chapter 1 and ref. 7). Production of supernatant from this virus producer cell line is described in Subheading 3.4.

Limiting Dilution Assay

Epstein-Barr Virus Protocols, 2001

ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector c... more ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector cells in a population. LDA are dose-response assays that allow detection of an all-or-none (positive or negative) immunoresponse in each individual culture within replicates that vary in the number of responder cells tested (reviewed in ref. 1). The frequency of positive cultures is not informative because it is never clear whether one or more precursors in the culture well are giving the positive response. The negative response instead demonstrates that there are no precursors of a given specificity. Therefore, the evaluation of the cell frequency in the original population is possible by determining the number of cultures that are negative in the experiment. Multiple cultures are set up at different cell concentrations and the larger the number of replicates used for each cell concentration, the more precise the estimate will be. If the percentage of negative cultures is converted to its negative logarithm, the results can be plotted graphically. The fraction of negative cultures is plotted on the ordinate while the cell concentration is plotted on the abscissa to give a straight line. Suitable statistic tests are used to fit this line, including regression analysis and the least square method (2,3).

Regression Assay

Epstein-Barr Virus Protocols, 2001

ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV... more ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV can be monitored in vitro as the capacity of T-cell lymphocytes to inhibit virusinduced proliferation of autologous B cells. In vitro infection of peripheral blood mononuclear cells (PBMCs) from EBV-seronegative and EBV-seropositive donors is accompanied by the appearance of foci of proliferating cells expressing the EBV nuclear antigens (EBNAs) within 7–14 d postinfection. These cells continue to expand giving rise to EBV-transformed lymphoblastoid cell lines (LCLs) in cultures from EBVseronegative individuals. In corresponding cultures from EBV-seropositive donors, the initial proliferation is followed by a regression of growth, seen preferentially at high cell concentration, that culminates within 1 mo in the complete degeneration of the culture (1). This phenomenon has been termed regression or outgrowth inhibition and can be monitored by the regression assay. The strength of the regression can be quantitatively expressed as the minimal number of cells required for 50% growth inhibition.

Membrane independent activation of fibroblast proMMP-2 by snake venom: novel roles for venom proteinases

Toxicon, 2004

ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mamma... more ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mammalian cell culture and a cell-free system. Zymography revealed an increment of latent and activated forms of MMP-2 in muscle homogenates 1-3 days after venom injection. To clarify if venom can induce expression and activation of MMP-2, independently of the inflammatory response, venom was added to cultured human fibroblasts, endothelial and skeletal muscle cells, which expressed proMMP-2 constitutively. Venom activated proMMP-2 without promoting its expression. Venom also activated and degraded proMMP-2 in supernatants collected from fibroblast cultures, indicating that cells are not required for this activation. Pretreatment with EDTA increased MMP-2 activation and reduced degradation. Venom serine proteinases activated proMMP-2, whereas BaP1, a P-I metalloproteinase, predominantly degraded the latent and active forms of MMP-2. Moreover, pretreatment of conditioned medium with serine proteinase inhibitors greatly reduced the venom-induced activation, suggesting that venom proteinases activate MMP-2 via a serine proteinase secreted by fibroblasts. Venom also directly activated and degraded purified proMMP-2, albeit requiring a high concentration. Thus, B. asper venom proteinases activate and degrade proMMP-2 without inducing its synthesis. Serine proteinases play a dominant role in the activation, whereas metalloproteinases predominantly degrade MMP-2. Activation of proMMP-2 by snake venom proteinases, independently of the MT1-MMP/TIMP-2 pathway, extracellular matrix degradation or apoptosis, represents a novel mechanism in human fibroblasts.

Toxins, 2011

The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacter... more The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B 2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?

FEBS Journal, 2011

Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted... more Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.

The FASEB Journal, 2012

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that ... more Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120catenin, ␤-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.-Frisan, T., Coppotelli, G., Dryselius, R., Masucci, M. G. Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth. FASEB J. 26, 000 -000 (2012). www.fasebj.org

Proceedings of the National Academy of Sciences, 2002

have strong antigenpresenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are ... more have strong antigenpresenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are highly allostimulatory in mixed lymphocyte culture. By contrast, EBV-positive Burkitt lymphoma (BL) cells are poor antigen presenters, are not recognized by EBV-specific cytotoxic T cells, and are poorly allostimulatory, which raises the question of whether immunological pressure exerted during BL pathogenesis in vivo has selected for a 'nonimmunogenic' tumor phenotype. The present work addresses this question by examining the immuno-genicity͞antigenicity of cell lines, generated by conversion of a conditionally immortalized lymphoblastoid cell line to permanent growth independent of EBV-latent proteins by introduction of a constitutively active or tetracycline-regulated c-myc gene (A1 and P493-6 cells, respectively). Compared with its parental lymphoblastoid cell line, A1 cells showed many of the features of the nonimmunogenic BL phenotype, namely poor allostimulatory activity, poor antigen-presenting function associated with impaired proteasomal activity, down-regulation of peptide transporter, reduced HLA class I expression, and an inability to present endogenously expressed EBV-latent proteins to cytotoxic T cells. P493-6 cells, when grown in the presence of estrogen with the exogenous c-myc gene switched off, were strongly immunogenic. The cells had lost their immunogenic potential, however, when grown on a c-myc-driven proliferation program in the absence of estrogen. Deregulation of c-myc, a step central to the development of uncontrolled BL cell growth in vivo, can thus impose a nonimmunogenic phenotype on proliferating human B cells in the absence of any immune pressure.

PLoS ONE, 2011

Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced ... more Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).

PLoS ONE, 2010

Background: The MYC protein controls cellular functions such as differentiation, proliferation, a... more Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

PLoS ONE, 2008

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal d... more Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.