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T Triglia

Publications and source records attributed to T Triglia.

At least 37 records · Page 2Linked to original sources
PubMed1992

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

Animals↗
PubMed1991

Large fragments of Plasmodium falciparum DNA can be stable when cloned in yeast artificial chromosomes.

A major problem in cloning large segments of Plasmodium falciparum DNA is the instability of this very A + T-rich DNA in Escherichia coli. We have therefore investigated whether P. falciparum DNA segments over 100 kb in size cloned in the yeast Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) are stable. An analysis of EcoRI fragments adding up to 230 kb, or nearly 1% of the P. falciparum genome, showed that sequences cloned as YACs were indistinguishable from the DNA in the P. falciparum genome, showing neither deletions nor rearrangements. Over 400 YACs have been obtained so far with an average insert size between 110 and 130 kb. Screening the YACs with single-copy probes suggested that most sequences should be represented in a library of less than 10(3) YACs. Hence YACs provide an approach to cloning large segments of P. falciparum DNA.

Animals↗
PubMed1991

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Animals↗
PubMed1987

Fluctuations in the expression of a glycolipid antigen associated with differentiation of melanoma cells monitored by a monoclonal antibody, Leo Mel 3.

A monoclonal antibody, Leo Mel 3, raised against a melanoma cell line (LiBr), binds to a carbohydrate determinant of cell surface gangliosides, the simplest of which is GD3. This monoclonal antibody was screened for by its capacity to block the recognition and lysis of the melanoma cells by cytotoxic T-lymphocytes with anomalous killer cell function, illustrating a novel approach for identifying monoclonal antibody to biologically relevant tumor-associated antigens. Leo Mel 3 reacted selectively with melanoma cells by indirect immunofluorescent and immunoperoxidase staining; it reacted with tissue from all primary and metastatic melanoma tested, and it bound to cells from all but one of six cultured melanoma cell lines. Leo Mel 3 did not react with a variety of carcinomas, lymphomas, leukemias, and other neuroectodermal tumors, nor with adult or fetal tissues, except fetal liver. Very weak staining of cutaneous basal melanocytes was noted in a minority of skin sections, and 50 to 80% of melanocytes in four of seven benign nevi showed weak to moderate reactivity. The antibody was relatively specific for human adherent melanoma cells, since it did not bind to the adherent murine B16 melanoma line nor to a nonadherent human melanoma cell line (PMC-22). Expression of the Leo Mel 3-defined antigen was unrelated to changes in cell cycle. When cells from an adherent melanoma cell line were detached and maintained briefly in suspension culture, the cells became markedly less reactive with Leo Mel 3 and, after readherence to plastic, they rapidly reexpressed higher levels of the ganglioside antigen; since Leo Mel 3 prevented attachment and growth of melanoma cells in vitro, a functional role for the ganglioside is suggested in cell adhesion and metastasis. Differentiation of melanoma cells with dimethyl sulfoxide, retinoic acid, and theophylline resulted in a marked and selective increase in the amount of Leo Mel 3-defined antigen, together with an increase in the target cell binding ability of these cells, assessed by cold target competition assays using anomalous killer cells.

Antibodies, Monoclonal↗
PubMed1987

The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum.

'Knobs' at the surface of erythrocytes infected with mature stages of Plasmodium falciparum are believed to be important in adherence of these cells to capillary walls. They contain at least one parasite protein, designated the knob-associated histidine-rich protein (KAHRP). We present here the sequences of a cDNA and chromosomal clone that predict the complete sequence of KAHRP. The gene contains a single intervening sequence, located at the 3' boundary of the hydrophobic core of a putative signal sequence. Exon two encodes a short region that is rich in histidine as well as two separate regions of repetitive sequence, the 5' repeats (five copies related to SKKHKDNEDAESVK) and the 3' repeats (seven copies related to SKGATKEAST). These repeat blocks were both shown to bear epitopes recognized by the human immune system during natural infection by expressing them separately in Escherichia coli, and reacting human antibodies affinity-purified on lysates of the resulting clones with the corresponding synthetic oligopeptides. The 3' end of the molecule, presumably the repetitive region, is a site of size variation in KAHRP from different isolates.

Amino Acid Sequence↗
PubMed1986

The IIb-IIIa glycoprotein complex that mediates platelet aggregation is directly implicated in leukocyte adhesion.

Evidence is presented that the IIb-IIIa glycoprotein complex, which functions as the receptor for fibrinogen on platelets and is central to platelet aggregation, is expressed on the surface of leukocytes where it may function as a receptor for fibronectin. F(ab')2 fragments of a monoclonal antibody, 25E11, raised against activated large granular lymphocytes, inhibited killing by natural killer cells, blocked the binding of fibronectin-coated particles by monocytes, and stimulated neutrophils to exhibit increased antibody-dependent killing. Immunoprecipitation studies of leukocytes and platelets, and the ability of 25E11 to inhibit platelet aggregation, identified the antigen as an epitope on the IIb-IIIa complex. This glycoprotein thus constitutes the first example of a receptor mediating both platelet aggregation and leukocyte adhesion.

Adult↗
PubMed1986

Cloned human cytotoxic T lymphocytes develop anomalous killer cell function.

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein-Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes; CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these, 13 (7%) retained specific function, 29 (16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy.

Antigens↗
PubMed1985

TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

Adult↗
PubMed1985

The isolation of natural killer (NK)-resistant variants of the K562 cell line by mutagenesis and selection with antibodies which inhibit NK cell-mediated lysis.

Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.

Antibodies, Monoclonal↗
PubMed1985

In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture.

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.

Acetylglucosamine↗
PubMed1985

Lymphocytes from patients with hairy cell leukaemia enable the distinction of two separate subpopulations of activated lymphocyte killer cells generated in mixed lymphocyte culture.

Blood from patients with hairy cell leukaemia (HCL) was used to investigate subpopulations of the non-specific activated lymphocyte killer (ALK) cells generated in mixed lymphocyte culture (MLC). Mononuclear cells from four of five patients with HCL had decreased natural killer (NK) cell activity and, of these, all showed a decrease in ALK cell activity against K-562 target cells (NK like cells), but three had normal levels of ALK cells able to kill melanoma cells. One patient who had received splenectomy had normal NK cell activity, and in MLC his cells generated normal levels of both subpopulations of ALK cells. The results support the hypothesis of separate subsets of ALK cells: NK like cells with NK cell precursors and which preferentially lyse the K-562 target cell and a second separate population which arises independent of NK cell precursors and which has strong cytolytic activity against an NK insensitive melanoma cell line.

Cell Line↗
PubMed1985

Antibodies to the T3 antigen complex on human T cells render thymocytes unresponsive to mitogen.

Human thymocytes differed from peripheral blood T cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of T-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the T3 antigen complex.

Adolescent↗
PubMed1985

Identification of a structure on human melanoma cells recognized by CTL exhibiting anomalous killer cell function.

The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.

Animals↗
PubMed1985

Rapid changes in surface antigen expression by blood monocytes cultured in suspension or adherent to plastic.

Cell surface antigens used as markers of in vivo differentiation may not be stable on monocytes maintained under different conditions of in vitro culture. Monocytes were isolated from blood by centrifugation over Percoll or by adherence to plastic dishes, and the cells cultured in suspension or as adherent monolayers. Initially, monocytes obtained by both methods were similar in size, morphology, and surface antigen expression detected with the antimonocyte monoclonal antibodies OKM1, FMC17, PHM2, and PHM3. After culture, cells maintained in suspension were predominantly small, whereas those adherent to plastic rapidly increased in size; however, cytochemical staining for nonspecific esterase and acid phosphatase showed increased enzymic activity by monocytes in both systems, possibly reflecting increased cell maturation. The most striking difference was a substantial loss of FMC17 antigen by most monocytes within four hours in suspension culture, as compared with a qualitative and quantitative increase in expression by plastic adherent cells within two hours. These changes occurred even if the cells were first reacted with lipopolysaccharide. Monocytes taken from suspension culture and allowed to adhere to plastic rapidly synthesized the antigen, a process inhibited by cycloheximide, and conversely, cells removed from plastic progressively displayed decreased FMC17 antigen expression when transferred to suspension culture. No functional role in adherence or phagocytosis has been found for the FMC17 antigen. The results suggest that antigen expression may depend as much on the physical state of the cells as on apparent activation or maturation events.

Acid Phosphatase↗
PubMed1984

High frequency of precursors of anomalous killer cells in human peripheral blood: evidence for T-cell regulation.

The frequency of precursors (P) of the anomalous killer (AK) cells able to kill a melanoma target cell line without prior sensitization was determined by limiting dilution analysis. The frequencies obtained from the peripheral blood mononuclear cells of six healthy individuals ranged from 1/250 to 1/750, which was considerably higher than those of alloreactive cytolytic T-lymphocyte (CTL) precursors induced in the same cultures (range 1/900 to 1/7500). The presence of phytohemagglutinin (PHA) inhibited the appearance of both CTL and AK in bulk cocultures, and in limiting dilution analysis the presence of the lectin resulted in multiphasic cell dose-response curves rather than linear single hit responses for both types of precursor cells. The results suggest that AK-P are under the same type of regulation as are CTL-P.

Cell Differentiation↗