Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 47
Filter
Add more filters










Publication year range
1.
Int J Parasitol ; 43(2): 173-80, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23178997

ABSTRACT

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.


Subject(s)
Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Schizonts/metabolism , Theileria annulata/growth & development , Theileria annulata/metabolism , Theileriasis/parasitology , Animals , Cattle , Mass Spectrometry , Molecular Sequence Data , Proteomics , Protozoan Proteins/genetics , Schizonts/chemistry , Schizonts/growth & development , Theileria annulata/chemistry , Theileria annulata/genetics
2.
Cell Microbiol ; 3(8): 537-50, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11488815

ABSTRACT

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.


Subject(s)
Leukocytes/parasitology , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Theileria/pathogenicity , Animals , Apoptosis , Cattle , Cell Compartmentation , Lymphocyte Activation , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction , Theileria annulata/pathogenicity , Theileria parva/pathogenicity
3.
Microbes Infect ; 2(11): 1311-20, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11018447

ABSTRACT

Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.


Subject(s)
Lymphocyte Activation , NF-kappa B/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/parasitology , Theileria parva/physiology , Activating Transcription Factor 2 , Active Transport, Cell Nucleus , Animals , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , NF-kappa B/genetics , NF-kappa B p50 Subunit , Oligodeoxyribonucleotides/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Signal Transduction , T-Lymphocytes/cytology , Transcription Factor RelA , Transcription Factor RelB , Transcription Factors/metabolism , Ultraviolet Rays
4.
Cell Microbiol ; 2(2): 91-9, 2000 Apr.
Article in English | MEDLINE | ID: mdl-11207566

ABSTRACT

The intracellular parasite Theileria parva infects and transforms bovine T-cells, inducing their uncontrolled proliferation and spread in non-lymphoid as well as lymphoid tissues. This parasite-induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva-transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva-infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T-cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen-activated protein kinase (MAPK), NF-kappaB and phosphoinositide-3-kinase (PI3-K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP-1 and ATF-2. A crucial step in the transformation process is the persistent activation of the transcription factor NF-kappaB, which protects T. parva-transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI-3K, in the continuous proliferation of T. parva-transformed lymphocytes.


Subject(s)
Lymphocyte Activation , T-Lymphocytes/parasitology , Theileria parva/physiology , Theileria parva/pathogenicity , Animals , Cattle , Host-Parasite Interactions , T-Lymphocytes/physiology , Theileriasis/parasitology
5.
Biochem Biophys Res Commun ; 263(1): 63-7, 1999 Sep 16.
Article in English | MEDLINE | ID: mdl-10486254

ABSTRACT

Ceramide is a lipid second messenger which is generated in response to stimulation of a number of surface receptors, treatment with chemotherapeutic agents, or ionising radiation. Depending on the target cell, ceramide induces diverse biological responses including apoptosis, cell-cycle arrest, differentiation, and also proliferation. We studied the effect of ceramide on the degradation of IkappaB, the cytoplasmic inhibitor of the transcription factor NF-kappaB. We show that ceramide treatment results in reduced levels of phosphorylated IkappaBalpha and degradation of both IkappaBalpha and IkappaBbeta. Ceramide synergised with okadaic acid (OA), a compound which interferes with the protein phosphatase 2A-controlled component of the NF-kappaB activation pathway, enhancing OA-induced IkappaB degradation. Ceramide also synergised with phorbol 12-myristate 13-acetate, which mimics protein kinase C activation. Finally, we show that the synergistic effect of ceramide with OA or phorbol ester can be observed in primary lymph node T-cells as well as in transformed T-cells.


Subject(s)
Ceramides/administration & dosage , DNA-Binding Proteins/metabolism , I-kappa B Proteins , Okadaic Acid/administration & dosage , Tetradecanoylphorbol Acetate/administration & dosage , Animals , Cell Line , Ceramides/metabolism , Drug Synergism , Enzyme Activation/drug effects , Humans , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Protein Kinase C/metabolism , Second Messenger Systems , Sphingomyelin Phosphodiesterase/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/parasitology , Theileria parva/pathogenicity
6.
Proc Natl Acad Sci U S A ; 96(13): 7312-7, 1999 Jun 22.
Article in English | MEDLINE | ID: mdl-10377411

ABSTRACT

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.


Subject(s)
Apoptosis , Calcium-Binding Proteins , T-Lymphocytes/pathology , T-Lymphocytes/parasitology , Theileria parva/parasitology , Theileriasis/immunology , Animals , Apoptosis/immunology , Cattle , Cell Line , DNA-Binding Proteins/immunology , Humans , I-kappa B Proteins , Membrane Glycoproteins/immunology , NF-kappa B/immunology , Nerve Tissue Proteins/immunology , Synaptotagmin I , Synaptotagmins , T-Lymphocytes/immunology , Theileriasis/pathology
7.
Cell Death Differ ; 6(4): 342-50, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10381628

ABSTRACT

The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).


Subject(s)
Acetylcysteine/pharmacology , Apoptosis/drug effects , Free Radical Scavengers/pharmacology , Jurkat Cells/cytology , Serine Proteinase Inhibitors/pharmacology , Tosylphenylalanyl Chloromethyl Ketone/pharmacology , Animals , Annexin A5/metabolism , Cell Division/drug effects , Cell Line, Transformed/cytology , Cell Line, Transformed/drug effects , Cell Line, Transformed/parasitology , Cysteine/pharmacology , DNA-Binding Proteins/metabolism , Flow Cytometry , Humans , I-kappa B Proteins , Jurkat Cells/drug effects , Jurkat Cells/parasitology , NF-kappa B/metabolism , Phosphorylation , Theileria parva , Theileriasis/immunology , Transcriptional Activation/drug effects
8.
Biol Chem ; 380(12): 1383-94, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10661865

ABSTRACT

Nuclear factor-kappaB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-kappaB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-kappaB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-kappaB inhibitor, can actually enhance rather than inhibit IkappaB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-kappaB inhibition. This was observed at the level of IkappaB degradation, NF-kappaB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-kappaB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.


Subject(s)
Acetylcysteine/pharmacology , Apoptosis/drug effects , NF-kappa B/metabolism , Pyrrolidines/pharmacology , T-Lymphocytes/drug effects , Thiocarbamates/pharmacology , Activating Transcription Factor 2 , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Division/drug effects , Cell Line, Transformed , Cyclic AMP Response Element-Binding Protein/metabolism , DNA/metabolism , DNA Primers , Disulfiram/antagonists & inhibitors , Disulfiram/pharmacology , Enzyme Activation , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/antagonists & inhibitors , Pyrrolidines/antagonists & inhibitors , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thiocarbamates/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic/drug effects , p38 Mitogen-Activated Protein Kinases
9.
Proc Natl Acad Sci U S A ; 94(23): 12527-32, 1997 Nov 11.
Article in English | MEDLINE | ID: mdl-9356483

ABSTRACT

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor kappaB (NFkappaB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFkappaB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IkappaB molecules which normally sequester NFkappaB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IkappaBalpha. However, IkappaBalpha reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFkappaB-mediated positive feedback loop which restores cytoplasmic IkappaBalpha. In contrast, T. parva mediated continuous degradation of IkappaBbeta resulting in persistently low cytoplasmic IkappaBbeta levels. Normal IkappaBbeta levels were only restored following T. parva killing, indicating that viable parasites are required for IkappaBbeta degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IkappaB degradation and consequent enhanced expression of NFkappaB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IkappaB levels or NFkappaB activation, indicating that the parasite subverts the normal IkappaB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.


Subject(s)
Lymphoproliferative Disorders/parasitology , NF-kappa B/immunology , T-Lymphocytes/parasitology , Theileria parva , Theileriasis/immunology , Animals , Cattle , Cell Division/immunology , Cell Line , Lymphocyte Activation , Lymphoproliferative Disorders/immunology , Lymphoproliferative Disorders/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology
10.
Clin Exp Immunol ; 109(3): 431-8, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9328118

ABSTRACT

Cytokine-mediated modulation of nitric oxide (NO) production by bacteria-stimulated bovine macrophages was studied. When Salmonella dublin, as a prototypic gram-negative organism, was used, NO generation was barely enhanced by recombinant bovine and ovine IFN-gamma, but was suppressed by IL-4. Salmonella dublin-induced NO generation was not influenced by a panel of nine other cytokines. The panel included IL-1, tumour necrosis factor (TNF) and IFN-alpha, which are active in a similar mouse macrophage model. The tested cytokines were either homologous or known to interact with bovine cytokine receptors. Recombinant bovine and ovine IFN-gamma were the only cytokines which strongly enhanced NO synthesis by macrophages exposed to the gram-positive organism, Listeria monocytogenes. Listeria-induced NO generation was strongly suppressed by recombinant human and bovine IL-4, but not by IL-10 and transforming-growth-factor-beta. Thus, two cytokines characterizing a Th1 and a Th2 response up- and down-regulate, respectively, bacteria-induced NO generation in bovine macrophages, whereas nine other cytokines had little activity in this regard. This modulation was reflected in changes in the steady state levels of mRNA coding for inducible nitric oxide synthase. Combinations of IFN-gamma and IL-4 suggested that the relative proportion of these cytokines determined whether bacteria-induced NO generation was up- or down-regulated. At saturating IL-4 concentrations, stimulation of bacteria-induced NO generation in macrophages by T cell supernatants was solely dependent on IFN-gamma. This was shown by antibody neutralization experiments and by a close correlation between the capacity of supernatants to stimulate NO generation and the IFN-gamma content, as determined by immunoassay.


Subject(s)
Interferon-gamma/immunology , Interleukin-4/immunology , Macrophages/immunology , Macrophages/metabolism , Nitric Oxide Synthase/metabolism , Animals , Cattle , Cells, Cultured , Culture Media, Conditioned , Down-Regulation , Gram-Negative Bacteria/immunology , Interferon-alpha/immunology , Interferon-gamma/genetics , Interleukins/immunology , Listeria/immunology , Macrophages/microbiology , Neutralization Tests , RNA, Messenger/metabolism , Recombinant Proteins/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Tumor Necrosis Factor-alpha/immunology , Up-Regulation
11.
J Virol ; 71(10): 7488-97, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9311828

ABSTRACT

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.


Subject(s)
Arthritis-Encephalitis Virus, Caprine/immunology , Cytokines/biosynthesis , Gene Expression Regulation/immunology , HIV/immunology , Macrophages/immunology , Macrophages/virology , Transcription, Genetic , Animals , Apoptosis , Arthritis-Encephalitis Virus, Caprine/genetics , Cells, Cultured , Chemokine CCL2/biosynthesis , Gene Products, tat/metabolism , Genes, tat , Goats , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Humans , Interferon-gamma/pharmacology , Interleukin-8/biosynthesis , Interleukins/biosynthesis , Lipopolysaccharides/pharmacology , Listeria monocytogenes/immunology , Macrophages/cytology , Staphylococcus aureus/immunology , Transcription, Genetic/drug effects , tat Gene Products, Human Immunodeficiency Virus
12.
J Leukoc Biol ; 61(4): 459-68, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9103233

ABSTRACT

Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage. Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated. Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation). Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mphi transformed by T. annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite.


Subject(s)
Macrophages/physiology , Macrophages/parasitology , Theileria annulata , Animals , Antigens, Surface/analysis , Antiprotozoal Agents/pharmacology , Cattle , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line, Transformed , Down-Regulation , Female , Flow Cytometry , Host-Parasite Interactions , Lipopolysaccharides/pharmacology , Macrophages/cytology , Naphthoquinones/pharmacology , Phenotype , Sensitivity and Specificity , Theileriasis/drug therapy , Theileriasis/pathology
13.
Gene ; 169(2): 165-71, 1996 Mar 09.
Article in English | MEDLINE | ID: mdl-8647441

ABSTRACT

CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.


Subject(s)
CD3 Complex/genetics , Membrane Proteins/genetics , Receptor-CD3 Complex, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , CD3 Complex/chemistry , Cattle , Cell Line , Cloning, Molecular , DNA, Complementary , Escherichia coli , Gene Expression , Humans , Membrane Proteins/chemistry , Membrane Proteins/immunology , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptor-CD3 Complex, Antigen, T-Cell/chemistry , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/genetics , Sequence Analysis , T-Lymphocytes/cytology
14.
Cell Immunol ; 163(2): 268-79, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7606798

ABSTRACT

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.


Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , Interleukin-4/biosynthesis , Interleukin-4/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Animals , Antibody Formation/drug effects , B-Lymphocytes/drug effects , Babesia bovis/immunology , Cattle , Cell Differentiation/drug effects , Cell Line , DNA, Complementary/biosynthesis , Fasciola hepatica/immunology , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/drug effects , Immunoglobulin E/biosynthesis , Immunoglobulin E/drug effects , Immunoglobulin G/biosynthesis , Immunoglobulin G/drug effects , Immunoglobulin M/biosynthesis , Immunoglobulin M/drug effects , Interleukin-4/genetics , Lymphocyte Activation/drug effects , Receptors, IgE/biosynthesis , Receptors, IgE/drug effects , Receptors, Interleukin-2/biosynthesis , Receptors, Interleukin-2/drug effects , Recombinant Proteins/pharmacology , T-Lymphocytes/drug effects , Th1 Cells/drug effects , Th2 Cells/drug effects
16.
Mol Biochem Parasitol ; 64(1): 11-23, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8078514

ABSTRACT

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.


Subject(s)
Endopeptidases/genetics , Fasciola hepatica/genetics , Genes, Helminth , Helminth Proteins/genetics , Multigene Family , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Escherichia coli , Fasciola hepatica/growth & development , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid
17.
Infect Immun ; 62(3): 818-27, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7509319

ABSTRACT

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.


Subject(s)
Antigens, Helminth/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Fascioliasis/immunology , Animals , Cattle , Cell Adhesion Molecules/analysis , Cell Line , Chronic Disease , Clone Cells , Fasciola hepatica/immunology , L-Selectin , Leukocyte Common Antigens/analysis
19.
Cell Immunol ; 153(1): 9-27, 1994 Jan.
Article in English | MEDLINE | ID: mdl-7507005

ABSTRACT

Functional studies with WC1+ gamma/delta T cell lines were performed to clarify the role of this subpopulation of gamma/delta T cells in the in vitro immune response to Babesia bovis. As CD4+ T cells decreased and gamma/delta T cells increased in B. bovis-stimulated T cell lines, antigen-specific proliferation declined to background levels. One irradiated gamma/delta T cell line inhibited proliferation of autologous Th1 cells, although unirradiated gamma/delta T cells either synergized with or had no effect on Th cell proliferation. gamma/delta T cells were not cytolytic for bovine alpha/beta T cells, but expressed natural killer (NK)-like cytotoxicity when assayed on xenogeneic NK-sensitive target cells. The gamma/delta T cells were IL-2 dependent and expressed IFN-gamma and TNF-alpha, but not TNF-beta, IL-2, or IL-4 mRNA. Together, these results raise the possibility that WC1+ gamma/delta T cells respond in vitro to autoantigens present on CD4+ T cells or to cytokines secreted by activated CD4+ T cells, resulting in modulation of the CD4+ T cell response and outgrowth of the gamma/delta T cells in parasite-stimulated lines.


Subject(s)
Babesia bovis/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , Animals , Antigens, Differentiation, T-Lymphocyte/analysis , Antigens, Protozoan/immunology , Cattle , Cell Adhesion Molecules/metabolism , Cell Line , Cytokines/genetics , Cytokines/metabolism , Cytotoxicity, Immunologic , Gene Expression , Interferon-gamma/metabolism , L-Selectin , Leukocyte Common Antigens/analysis , Lymphocyte Activation , RNA, Messenger/genetics , T-Lymphocytes, Helper-Inducer/immunology , Tumor Necrosis Factor-alpha/metabolism
20.
Semin Cell Biol ; 4(5): 363-71, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8257788

ABSTRACT

The protozoan intracellular parasites, Theileria parva and Theileria annulata, infect cattle and cause severe and fatal leukocytic proliferative diseases. The proliferation is dependent on the presence of the parasites in the host cell cytoplasm. T. parva-infected cells proliferate permanently in cell culture and exhibit many features characteristic of tumor cells. The proliferation is reversible by treatment with parasite-specific drugs. Constitutive expression of interleukin-2, its receptor and their transcription factor, NF-kappa B, are dependent on the parasite and suggest autocrine growth. Cell-cell contact possibly via T cell adhesion molecules has been shown to stimulate proliferation.


Subject(s)
Lymphocyte Activation , Lymphocytes/parasitology , Theileria parva/physiology , Animals , Cattle , Cell Communication , Cell Line, Transformed , Cytokines/physiology , Cytoplasm/parasitology , Lymphocytes/physiology
SELECTION OF CITATIONS
SEARCH DETAIL
...