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T Tursz

Publications and source records attributed to T Tursz.

At least 127 records · Page 7Linked to original sources
PubMed1989

Two distinct affinity binding sites for IL-1 on human cell lines.

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.

Adolescent↗
PubMed1989

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor.

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.

Adult↗
PubMed1989

EBV gene expression in an NPC-related tumour.

A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein-Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95-8 'prototype' viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B-lymphocytes, only clones for EBNA-1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA-1 mRNA splicing pattern in the carcinoma to differ from that observed in B-lymphocytes. Further, contrary to observations with B-cell lines, most viral transcription in the tumour is localized onto the 'rightmost' region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95-8 EBV DNA. Novel transcripts have also been identified that proceed in an anti-sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B-cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2-6, being 'switched off.' (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95-8 genome, some of which could result in amino acid alterations in proteins.

Amino Acid Sequence↗
PubMed1989

The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus.

Seven virus-encoded proteins are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid (LCL) cell lines: the EBV nuclear antigens EBNA 1 to 6 and the latent membrane protein (LMP). In nasopharyngeal carcinoma (NPC), only EBNA 1 is regularly expressed; LMP is detected in about 50% of the tumours. In Burkitt's lymphoma (BL) tumours, only EBNA 1 is expressed. Also, in BL-derived cell lines that maintain the phenotypic markers characteristic of the in vivo tumour (group I), only EBNA 1 is expressed. EBV was rescued by induction or cocultivation from one BL cell line with a restricted group I pattern, and from one NPC tumour, into normal B cells. In the resulting LCLs EBNA 1 to 6 and LMP were expressed. We assessed the level of methylation in the genes encoding ENBNA 2 and LMP by restriction fragment analysis using the methylation-sensitive enzymes SmaI and HpaII. These genes were extensively methylated in the group I BL line Rael and the nude mouse-passaged C15 NPC tumour, but were demethylated in the derived LCLs. In the LMP-expressing NPC C15 tumour the 5' flanking region of the gene was hypomethylated, whereas the coding exons were methylated [corrected]. The EBNA 1 coding exon was methylated in the Rael line and in NPC, in spite of expression. In contrast, CpG pairs in oriP were originally hypomethylated and remained so after their transfer to LCLs. The cell phenotype-dependent pattern of EBV gene methylation correlated with the phenotype-dependent pattern of EBNA and LMP expression. The specific patterns of methylation localized to controlling regions (oriP and 5' flanking sequences) also suggest a specific role for methylation in the regulation of EBNA and LMP expression.

Antigens, Viral↗
PubMed1989

Epstein-Barr virus (EBV)-containing nasopharyngeal carcinoma cells express the B-cell activation antigen blast2/CD23 and low levels of the EBV receptor CR2.

Anaplastic nasopharyngeal carcinoma (NPC) cells invariably harbor the Epstein-Barr virus (EBV) genome, an association that is unique among human virus-associated cancers. Although EBV is able to replicate in epithelial cells, results with expression of the EBV receptor (complement receptor type 2 [CR2]; also called CD21) in normal and malignant epithelial cells are conflicting. We grew five different EBV-associated NPC tumors in nude mice, and by using a sensitive transcriptional assay, we detected a very weak transcription signal of the EBV receptor CR2 gene in these cells. This suggests that low levels of EBV receptor may be expressed by malignant epithelial nasopharyngeal cells. The gene coding for Blast2/CD23, a B-cell activation molecule induced by EBV, was transcribed in three of the transplanted NPC tumors. The soluble form of the Blast2/CD23 protein was also detected in medium taken from short-term cultures of the same NPC cell lines. In contrast to the lymphoid system, in which Blast2/CD23 expression is associated with EBV nuclear antigen (EBNA2) expression, no EBNA2 protein could be detected in these NPC epithelial cells. Our study represents the first demonstration of Blast2/CD23 expression in epithelial cells. As the soluble form of the Blast2/CD23 protein possesses growth factor activity associated with EBV-induced B-cell immortalization, these results suggest a possible role for this molecule in the pathogenesis of NPC.

Animals↗
PubMed1988

Establishment and characterization of three transplantable EBV-containing nasopharyngeal carcinomas.

Three transplantable nasopharyngeal carcinoma (NPC) tumors, designated C15, C17 and C18, have been obtained and characterized. C15, derived from a primary NPC tumor, has been propagated in nude mice for 30 passages. C17 and C18, derived from metastatic NPC tissue, have been passaged 10 times. Desmosomes, present in every case, provided confirmation of the epithelial origin of all 3 tumors. The Epstein-Barr virus (EBV) genome is contained in C15, C18 and C17 tumor cells with 30, 12 and 3 copies, respectively. The Epstein-Barr virus nuclear antigen (EBNA) was stained by the classical anti-complement immunofluorescence (ACIF) technique. Fluorescence intensity was strong in C15, moderate in C18, and hardly detectable in C17 cells. No expression of the EA and VCA antigens was detected. Flow cytometry analysis performed on monocellular suspensions showed the absence of detectable CR2 molecules (the EBV receptor on B lymphocytes) in all 3 tumors, and the constitutive expression of HLA class-II antigens in C15 and C17 cells. IL-1 activity was demonstrated in the supernatant of C15 and C17 cells cultivated in vitro for 3 days. These data confirm that the constitutive synthesis of MHC class-II molecules and the release of IL-1-like activities are frequent features of NPC cells. These characteristics could be of importance in relation with the T-cell infiltrate found in NPC primary tumors.

Adolescent↗
PubMed1988

New hematopoietic differentiation antigens detected by anti-K562 monoclonal antibodies.

Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immunoprecipitates a Mr 105,000 glycoprotein on K562 cells. (b) Two MoAbs, 14B6 and 12B1, react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-O-tetradecanoylphorbol-13-acetate treated K562 cells as a Mr 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-O-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (c) Two anti-K562 MoAbs, CF4 and HE10, recognize a myeloid differentiation antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an apparently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 recognizes the monocyte low affinity Fc receptor (Mr 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies a Mr 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of antibodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hematopoietic progenitors.

Animals↗
PubMed1988

Isolation of a normal B cell subset with a Burkitt-like phenotype and transformation in vitro with Epstein-Barr virus.

Epstein-Barr virus (EBV) is causally linked with endemic Burkitt's lymphoma (BL), a tumor whose homogeneous cell surface phenotype suggests derivation from a particular subset of activated germinal centre B cells in vivo. Endemic BL also shows an unusual form of EBV infection with down-regulation of certain of the virus latent proteins which are constitutively expressed when EBV infects and transforms normal resting B cells in vitro. Here we question whether this virus:cell interaction is unique to malignant BL cells or whether it might be reproduced by in vitro infection of those particular germinal centre cells displaying the BL-like phenotype. Firstly, we show by biochemical means that a subset of normal tonsillar B cells does indeed express the globotriaosylceramide glycolipid BLA and the common acute lymphoblastic leukaemia antigen CALLA, 2 important markers of the BL phenotype. Secondly, using 2-colour immunofluorescence labelling with anti-BLA and anti-CALLA monoclonal antibodies (MAbs), 4 subsets of low buoyant density tonsillar B cells (BLA+ CALLA+, BLA+ CALLA-, BLA- CALLA+, BLA- CALLA-) have been separated by means of a FACS and tested for their susceptibility to EBV-induced growth transformation in a limiting dilution assay. The BLA+ CALLA+ (i.e., BL-like) subset contained the highest proportion of cells already actively in cycle in vivo and gave the lowest yield of transformants, perhaps reflecting the greater efficiency with which EBV transforms resting target cells. Of the cell lines established from the BLA+ CALLA+ population, a significant number retained BLA expression but CALLA was always lost. In 2 further respects, these lines resembled conventional in vitro transformants rather than lines of BL type; thus the cells expressed cellular "activation" antigens (CD23, CD39, CD30, Ki-24) characteristic of the lymphoblastoid phenotype and contained the full spectrum of EBV latent proteins.

Antigens, Differentiation↗
PubMed1988

Expression of the c-fgr related transcripts in Epstein-Barr virus-associated malignancies.

The proto-oncogene c-fgr is expressed at high levels in cell lines derived from lymphomas which are infected with Epstein-Barr virus (EBV) (Cheah et al., 1986). mRNA extracted directly from biopsies of EBV-infected tissues was analyzed on Northern blots to determine if c-fgr is expressed during lympho-proliferations induced in vivo by EBV and in nasopharyngeal carcinoma (NPC), the epithelial malignancy associated with the virus. Elevated levels of c-fgr expression were detected in all EBV-positive lympho-proliferations in vivo but not in cell lines established by EBV infection in vitro. This indicates that the induction of the c-fgr proto-oncogene is not an essential component of EBV-induced transformation. Although no c-fgr expression was detected in EBV-positive or -negative epithelial cell lines, the 3.0-kb c-fgr mRNA was detected at low levels in mRNA obtained from NPC biopsy specimens. However, NPC tissue, after passage in nude mice (which eliminates infiltrating lymphoid and myeloid cells) did not contain the 3.0-kb c-fgr mRNA. The absence of expression of c-fgr in the malignant epithelial cells infected with EBV contrasts with the elevated level of the proto-oncogene in EBV-infected lymphoma tissue and cell lines established from lymphomas. This suggests differences in the expression of cellular functions in EBV-induced malignancies of these 2 distinct cell types.

Genes, Viral↗
PubMed1988

Kinetic analysis of choriocarcinoma cell intoxication induced by ricin and ricin A chain immunotoxin.

The kinetics of protein synthesis inhibition was studied in the choriocarcinoma-derived BeWo cell line treated with ricin and an immunotoxin (IT) constructed by linking a specific antibody to the A chain of ricin. The IT was specifically cytotoxic to BeWo and other choriocarcinoma cells. The multistep process underlying this kinetics was explored using two mathematical models where the protein synthesis-inactivation step is preceded by one or two processing rate-limiting steps. Theoretical curves were computed for different concentrations of toxic reagents. Two processing steps were found necessary to predict the duration of the observed latent period before initiation of protein synthesis inhibition. With this model, a satisfactory fit was obtained. A mathematical modeling of the intoxication induced by ricin or ricin A chain IT thus requires two processing steps to account for the data observed. In addition, the data suggest that the cytoplasmic internalization of ricin is a slow process compensated by an extremely fast enzymatic inactivation of ribosomal activity. This implies that in this and similar systems, endocytosis might not be involved in the cytoplasmic internalization of the few molecules of ricin A chain responsible for cell intoxication.

Antibodies, Monoclonal↗
PubMed1988

Comparison of interleukin 2 and 12-O-tetradecanoylphorbol 13-acetate as signals for protein kinase C activation in purified human T lymphocytes.

Interleukin 2 (IL2) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti-CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes. TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti-CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti-CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti-CD3 mAb-treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation. In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high-affinity IL2 receptors.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Comparison of signals delivered through CD3 and CD2 for T-cell activation: the role of calcium influx and interleukin 1.

In the absence of monocytes, resting T lymphocytes extensively purified from human peripheral blood failed to proliferate when stimulated with a mixture of calcium ionophore, which elevates intracellular calcium levels, and TPA, which activated protein kinase C. A third signal, i.e., the triggering via CD3 or CD2 molecules, was necessary in order to observe proliferation. These highly purified T cells required the presence of monocytes in both CD3 and CD2 systems for their proliferation. Exogenous interleukin 1 clearly substituted for monocytes in CD2- but not in CD3- triggered T-cell proliferation. In contrast, the effect of CD2 and CD3 antibodies on Ca++ influx was apparently not dependent on the presence of monocytes. In the presence or absence of the monocytes, CD3, as well as certain combinations of CD2 monoclonal antibodies including the D66 monoclonal antibody, were able to increase the intracellular calcium concentration as measured by Quin 2 fluorescence. EGTA, a Ca++ chelator, completely inhibited CD2- and CD3- mediated T-cell proliferation, indicating that calcium uptake is necessary during the T-cell proliferation. The addition of TPA abrogated the inhibitory effect of EGTA and completely restored the response of the T cells stimulated by CD3, but not by CD2, monoclonal antibodies. In the CD2 pathway, EGTA-inhibited proliferation of T cells could be completely restored by addition of exogenous interleukin 2 as well as exogenous recombinant interleukin 1. Our results indicate that EGTA inhibits the production of interleukin 1 but has no direct effect on either interleukin 2 production or on Tac antigen expression. In this system, recombinant interleukin 1 alpha demonstrated a more potent ability for restoring the T-cell response than did recombinant interleukin 1 beta. These results suggest that interleukin 1 could act as a potent costimulatory factor in the non-antigen-specific T-cell activation.

Antibodies, Monoclonal↗
PubMed1988

Epstein-Barr virus gene expression in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV), an agent with growth transforming potential for human B cells, is associated with certain B cell lymphomas in man and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma (NPC). Since B cell growth transformation is associated with the constitutive expression of a small number of EBV-coded latent proteins, the nuclear antigens EBNA 1, EBNA 2, EBNA 3 and EBNA-LP and the latent membrane protein (LMP), the present work sought to determine whether this same pattern of virus gene expression occurred in NPC. Tumour biopsies were taken from NPC patients from three areas of differing tumour incidence (Kenya, Algeria, Britain) and immediately snap-frozen, as were biopsies of non-EBV-related carcinomas for controls. Immunoblotting of PAGE-separated proteins with selected human sera identified 24 NPC biopsies clearly expressing EBNA 1. When the analysis was extended using selected human sera with antibodies against the other EBNAs, there was no detectable expression of EBNA 2, EBNA 3 or EBNA-LP in any of these 24 biopsies; their EBNA 2-negative status was confirmed using a monoclonal antibody (MAb) PE2 which was reactive in immunoblotting and in immunoprecipitation with EBNA 2A and EBNA 2B proteins. Similar experiments with two different LMP-specific MAbs, CS1 to 4 and S12, revealed heterogeneity between NPC biopsies; 9/24 biopsies were demonstrably LMP-positive, the degree of expression varying considerably between individual tumours in a manner which was not related to the level of EBNA 1 expression. None of the 24 NPC biopsies expressed detectable amounts of EBV lytic cycle antigens. A nude mouse-passaged NPC cell line, C15, likewise expressed EBNA 1 and LMP but none of the other EBV latent proteins nor lytic cycle antigens. This work identifies a novel type of EBV-cell interaction in NPC cells which is distinct from that seen in in vitro transformed B cell lines and from that seen to date in EBV-positive B cell lymphomas.

Antibodies, Monoclonal↗
PubMed1988

Use of a monoclonal antibody (GA3) to demonstrate lineage restricted O-glycosylation on leukosialin during terminal erythroid differentiation.

A murine monoclonal antibody (GA3) obtained by immunizing mice with cells of the human erythroleukemic cell line K562 is shown to define a 105 kilodalton (kd) membrane antigen on K562 cells that is restricted within the hematopoietic system to the erythroid lineage and to a minor population of CD3, CD4 positive T lymphocytes. Cocapping studies and immunoprecipitation experiments performed with GA3 and L10, an anti-sialophorin monoclonal antibody reacting with leukosialin (Gp 105) on K562 cells, demonstrate that the antigen detected by GA3 on K562 cells is identical to leukosialin. Neuraminidase treatment but not tunicamycin treatment of K562 cells abolishes the expression of the GA3-epitope without affecting the L10-epitope thus providing evidence that terminal sialic acid present on O-linked oligosaccharide chains on Gp 105 is essential for the expression of the GA3-epitope. Further analysis by flow cytometry and immune panning experiments performed on bone marrow cells with GA3 or L10 demonstrate that, in contrast to L10, which reacts with all types of hematopoietic progenitors, the epitope recognized by GA3 is restricted to the erythroid lineage, and appears during erythroid differentiation before glycophorin A on the earliest morphologically recognizable erythroid precursor, the proerythroblast. Our results therefore suggest that O-linked oligosaccharides on leukosialin express lineage restricted and even maturation restricted antigenic structures that might serve as cell lineage specific markers.

Animals↗