ABSTRACT
Occupational fatigue in anaesthetists is recognised as a patient safety risk. Better understanding of the issues surrounding their fatigue is needed. This study aimed to ascertain the sources and effects of occupational fatigue amongst anaesthetists in Australia and New Zealand. An anonymous online survey was sent to 979 anaesthetists. The response rate was 38.0%. Most participants reported regularly working over 40 hours per week; men reported five more hours per week than women. Stated contributors to fatigue included long work hours, mental strain at work, and personal and family demands. Fatigue-related behaviour was reported more by men (OR [odds ratio]=2.6) and less by respondents reporting eight or more hours of sleep before work (OR=0.6). Reporting at least one instance of less than five hours off between shifts was predictive of falling asleep while administering an anaesthetic (OR=1.6). More data are required to support practices and policies that promote more time off between work periods and increased time for sleep to reduce risk of fatigue.
Subject(s)
Anesthetists , Fatigue/epidemiology , Occupational Diseases/epidemiology , Adult , Aged , Aged, 80 and over , Australia/epidemiology , Female , Humans , Male , Middle Aged , New Zealand/epidemiology , Personnel Staffing and Scheduling , Sleep , Surveys and QuestionnairesABSTRACT
Following a large outbreak of community-acquired psittacosis in 2002 in residents of the Blue Mountains, New South Wales, Australia, we reviewed new cases in this area over a 7-year period from 2003 to 2009. Using the 2010 criteria from the Centers for Disease Control National Notifiable Diseases Surveillance System, 85 patients with possible psittacosis were identified, of which 48 were identified as definite or probable infection. Clinical features of these cases are summarized. In addition to Chlamydia-specific serology, specimens, where available, underwent nucleic acid testing for chlamydial DNA using real-time PCR. Chlamydophila psittaci DNA was detected in samples from 23 patients. Four of 18 specimens were culture positive. This is the first description of endemic psittacosis, and is characterized in this location by community-acquired psittacosis resulting from inadvertent exposure to birds. The disease is likely to be under-diagnosed, and may often be mistaken for gastroenteritis or meningitis given the frequency of non-respiratory symptoms, particularly without a history of contact with birds. Clinical characteristics of endemic and outbreak-associated cases were similar. The nature of exposure, risk factors and reasons for the occurrence of outbreaks of psittacosis require further investigation.
ABSTRACT
Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.
Subject(s)
B-Lymphocytes/metabolism , Immunoglobulin G/metabolism , Immunoglobulin M/immunology , Immunoglobulin mu-Chains/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/metabolism , B-Lymphocytes/immunology , CD5 Antigens/immunology , Cloning, Molecular , Epitopes , Escherichia coli , Flow Cytometry , Fluorescein-5-isothiocyanate/metabolism , Humans , Immunoenzyme Techniques , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin M/metabolism , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin kappa-Chains/immunology , Immunoglobulin lambda-Chains/chemistry , Immunoglobulin lambda-Chains/immunology , Immunoglobulin mu-Chains/chemistry , Immunoglobulin mu-Chains/immunology , Ligands , Mice , Protein Conformation , Tumor Cells, CulturedABSTRACT
B-cell chronic lymphocytic leukemia (B-CLL) is characterised by the proliferation and accumulation of sIgM+/CD5+ B-cells that fail to progress to the final stages of B-cell development. Despite their developmental arrest, leukemic CD5+ B-cells can undergo proliferation in vitro in the presence of different activators including phorbol esters, antibodies to cell surface antigens and human cytokines. Interleukin-10 (IL-10) has recently been found to inhibit CLL B-cell function in vitro by inducing apoptosis and down-regulating expression of bcl-2. Here, we examined the effect of IL-10 on proliferation, RNA synthesis, immunoglobulin (IgM) secretion and viability of leukemic CD5+ B-cells induced by activation with the phorbol ester PMA, alone or in combination with anti-Ig. IL-10 reduced PMA and PMA/anti-Ig induced proliferation and RNA synthesis by 50-80% and 15-40% respectively. Although proliferation and RNA synthesis induced by PMA/anti-Ig could be enhanced by the addition of IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha, the presence of these cytokines failed to abrogate the IL-10-mediated inhibition of leukemic CD5+ B-cell activation. In contrast to the effects on proliferation and RNA synthesis, IL-10 did not inhibit IgM secretion, and had only a minimal effect on the viability of activated cells. Our results indicate that IL-10 inhibits proliferation of leukemic CD5+ B-cells by a mechanism distinct from induction of apoptosis and support the proposal for the utilisation of IL-10 in the therapy of B-CLL.
Subject(s)
CD5 Antigens/blood , Interleukin-10/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Cell Division/drug effects , Cell Survival/drug effects , Cytokines/pharmacology , Humans , Immunoglobulins/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphocyte Activation , RNA, Neoplasm/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, CulturedABSTRACT
Surface IgM expressed by malignant CD5+ B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) has previously been shown to bind mouse Ig in what appears to be an example of polyreactive antigen-binding activity. This report demonstrates the in vitro and in vivo binding of mouse Ig to the surface of malignant B-cells from a patient with B-cell prolymphocytic leukemia (B-PLL). In vitro studies showed that K121, a mouse monoclonal antibody, bound to the B-PLL cells via the same low-affinity binding interaction demonstrated to occur between mouse Ig and surface IgM expressed by B-CLL cells rather than in the conventional sense against a specific antigen via its antigen-binding site. With the view to using this phenomenon to target malignant B-cells, it was important to determine whether the low-affinity interaction also occurred in vivo. Infusions of K121 totalling 286 mg were administered to a B-PLL patient over 7 days. Binding of K121 to circulating B-PLL cells was demonstrable after the administration of 36 mg of antibody and was preceded by the appearance of free antibody in the serum. Throughout the period of the infusion, the rapid rise in the peripheral blood white cell count normally observed after leukopheresis was abrogated. However, the count rose markedly after cessation of the antibody infusion in parallel with a decrease in both free and cell-bound K121. There were no observable side effects and no host immune response to either species specific or idiotypic determinants on the mouse Ig was detected. The in vivo binding of mouse Ig together with the previous in vitro data suggest the potential for a novel targeting mechanism using a region of the mouse Ig molecule to target polyreactive Ig expressed by malignant cells in B-CLL and B-PLL.
Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin G/immunology , Leukemia, Prolymphocytic/metabolism , Lymphocytosis/metabolism , Animals , Antibodies, Monoclonal/therapeutic use , Humans , Leukemia, B-Cell/immunology , Leukemia, B-Cell/metabolism , Leukemia, B-Cell/therapy , Leukemia, Prolymphocytic/immunology , Leukemia, Prolymphocytic/therapy , Lymphocytosis/immunology , Lymphocytosis/therapy , Male , Mice , Middle Aged , PhenotypeABSTRACT
We have examined the cytolytic effects of the membrane-active peptide, melittin, on a human lymphoblastoid cell line (HMy2) in the context of the use of melittin as the toxic component of an immunotoxin. The toxicity of melittin for HMy2 cells was linear over the concentration range 0.875-3.5 microM. Increased incubation times failed to result in significant cell death at concentrations of melittin below 0.875 microM. Kinetic analysis revealed that the cytolytic activity of melittin was independent of time of exposure beyond 90 min. Flow cytometric analysis of HMy2 cells incubated with FITC-labeled melittin demonstrated that the cells could incorporate up to 2.5 x 10(5) FITC-melittin molecules per cell with no reduction in viability. Extrapolation of this data indicates that 10(6) melittin molecules per cell are required for maximum cytotoxicity to HMy2 cells. Further analysis of HMy2 cells that incorporated melittin, but that remained viable, revealed that these cells were able to reduce the number of melittin molecules per cell over time. The data indicate a potential threshold value for the number of melittin molecules that may be required to be delivered to the cell surface in the form of an immunotoxin if effective selective cell death is to be achieved.
Subject(s)
Melitten/pharmacology , Cell Line , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Humans , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/metabolism , Melitten/metabolism , Melitten/toxicity , Stem Cells/drug effects , Stem Cells/metabolism , Time FactorsSubject(s)
DNA-Binding Proteins/genetics , Mesoderm/physiology , T-Box Domain Proteins , Transcription Factors/genetics , Vertebrates/embryology , Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Body Patterning , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Embryo, Nonmammalian/physiology , Embryonic Induction , Fetal Proteins/biosynthesis , Gene Expression Regulation, Developmental , Transcription Factors/biosynthesis , Transcription Factors/physiology , Transcription, GeneticABSTRACT
Purified leukaemic CD5+ B cells obtained from patients with B cell chronic lymphocytic leukaemia (B-CLL) undergo activation and differentiation following in vitro stimulation with optimal concentrations of the phorbol ester PMA. This paper examines the ability of exogenous cytokines, anti-Ig antibodies, or combinations of these, to enhance or replace the activation signal provided by PMA to different populations of leukaemic B cells. Proliferation induced by PMA was enhanced 2-20-fold when the cells were co-cultured with either anti-Ig, IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha. Moreover, the combination of anti-Ig, PMA and any one of the above cytokines further enhanced proliferation. Anti-Ig and exogenous cytokines were also capable of inducing proliferation in leukaemic B cells cultured with a non-mitogenic concentration of PMA. When taken together with the finding that IL-2, IL-4, IL-13, IFN-gamma and TNF-alpha prevent in vitro apoptosis of leukaemic CD5+ B cells, the results presented here suggest that these cytokines, in conjunction with signals delivered via sIg, may play a role in the proliferation of leukaemic B cells in vivo and, consequently, the pathogenesis of B-CLL.
Subject(s)
B-Lymphocytes/immunology , CD5 Antigens/immunology , Cytokines/immunology , Immunoglobulin M/immunology , Lymphocyte Activation/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Animals , Antibodies/immunology , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cross-Linking Reagents , Cytokines/pharmacology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphocyte Activation/immunology , Rabbits , Tumor Cells, CulturedABSTRACT
BACKGROUND: The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES: We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN: The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS: Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION: The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.
Subject(s)
Antigens/metabolism , Immunotoxins/metabolism , Immunotoxins/pharmacology , Melitten/metabolism , Melitten/pharmacology , Amino Acid Sequence , Animals , Artificial Gene Fusion , Base Sequence , Cytotoxicity, Immunologic , Genes, Immunoglobulin , Humans , Hybridomas , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/metabolism , Immunoglobulin Fragments/pharmacology , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Immunoglobulin Variable Region/pharmacology , Immunotoxins/genetics , Melitten/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacologyABSTRACT
The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.
Subject(s)
DNA-Binding Proteins/genetics , Fetal Proteins/genetics , Gene Expression Regulation, Developmental , T-Box Domain Proteins , Trans-Activators/genetics , Animals , Base Sequence , Chromosome Mapping , DNA , DNA-Binding Proteins/metabolism , Drosophila Proteins , Fetal Proteins/metabolism , Homeodomain Proteins/genetics , Insect Hormones/genetics , Mesoderm/metabolism , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/genetics , Transcriptional Activation , Xenopus , ZebrafishABSTRACT
Thiophosphate induced apoptosis in human HL-60 cells. HL-60 cell proliferation was inhibited with an IC50 of about 60 microM. Typical morphological changes of apoptosis were observed by phase contrast microscopy and DNA laddering was observed after agarose gel electrophoresis. Thiophosphate-induced DNA fragmentation was time and concentration-dependent. After exposure to thiophosphate (100 microM) apoptosis occurred as early as 4 h after treatment and 90% of cells were apoptotic by 24 h. dbcAMP-differentiated HL-60 cells as well as undifferentiated HL-60 cells were susceptible. Thiophosphate was also effective in inducing apoptosis in other leukemia cell lines including CEM and K562 and a lymphoma cell line, Raji.
Subject(s)
Apoptosis/drug effects , Phosphates/pharmacology , Cell Differentiation , Cell Division/drug effects , DNA Damage , DNA, Neoplasm/drug effects , DNA, Neoplasm/isolation & purification , DNA, Neoplasm/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Agar Gel , HL-60 Cells , Humans , KineticsABSTRACT
B cell chronic lymphocytic leukaemia (B-CLL) is characterized by the proliferation and accumulation of sIgM+ CD5+ lymphocytes that fail to progress to the final stages of B cell development. Stimulation of unfractionated PBL from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and IgM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of IgM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis was also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on CD5+ CLL B cells was independent of T cells. In contrast, activation and differentiation of the malignant CD5+ B cells into IgM-secreting cells following culture with mitogens did not occur in the absence of T cells.
Subject(s)
B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Immunoglobulin M/biosynthesis , Lymphocyte Cooperation/immunology , Tetradecanoylphorbol Acetate/pharmacology , Antigens, CD/immunology , CD5 Antigens , Flow Cytometry , Humans , Immunoglobulin M/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , RNA, Messenger/biosynthesis , T-Lymphocytes/immunology , Tumor Cells, CulturedABSTRACT
A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.
Subject(s)
Complement System Proteins/genetics , Complement System Proteins/immunology , Hagfishes/immunology , Opsonin Proteins/immunology , Phagocytes/immunology , Phylogeny , Receptors, Immunologic/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Complement System Proteins/metabolism , Fluorescent Antibody Technique , Hagfishes/blood , Hagfishes/genetics , Phagocytes/metabolism , Phagocytosis , Receptors, Immunologic/immunology , Zymosan/pharmacologyABSTRACT
The 90 degrees light scatter parameter of the flow cytometer was used to observe changes in the membrane of human lymphoblastoid cells (HMy2) as a result of the action of the cytolytic peptide, melittin. There was a rapid and concentration-dependent increase in 90 degrees light scatter after incubation of the cells with melittin, with the level of 90 degrees light scatter reaching a maximum after 2 min. Even after all the cells were killed, as determined by ethidium bromide incorporation, the 90 degrees light scatter continued to increase. Further, the 90 degrees light scatter changes were temperature dependent. The data are consistent with the formation of lipid vesicles, either attached to the cell membrane or intracellular, as confirmed by electron microscopy of cells treated with melittin. The results demonstrate the use of the flow cytometer to detect changes in the integrity of the cell membrane.
Subject(s)
Cell Membrane/drug effects , Flow Cytometry , Lasers , Melitten/pharmacology , Cell Death , Cell Line , Cell Membrane/ultrastructure , Humans , Lymphocytes/drug effects , Lymphocytes/ultrastructure , Microscopy, Electron , Scattering, Radiation , TemperatureABSTRACT
Polyreactive immunoglobulin (Ig) secreted by CD5-bearing B cells has the capacity to bind a broad range of self and foreign antigens. Flow cytometric analysis was used to detect low-affinity binding of mouse Ig molecules to the surface of CD5-bearing B cells from patients with chronic lymphocytic leukaemia (CLL). Mouse Ig isotypes G, A, and M, and IgG subclasses G1, G2a, and G3 bound to the B cell surface via a mechanism not involving the antigen-binding site of the mouse Ig molecule. Fab and F(ab')2 fragments of mouse Ig associated with the CLL cells in a similar manner to intact Ig, indicating that Fc receptor interactions were not involved. Dissociation of the mouse Ig from the B cell surface by three washing steps distinguished this lower-affinity binding from high-affinity binding which occurs through the antigen-binding site of a mouse monoclonal antibody (MoAb) when it recognizes a specific cell surface epitope. Blocking studies suggest that the low-affinity binding occurred via surface IgM and surface IgD (sIgM/sIgD) on the CD5-bearing B cells. The results are consistent with the expression of polyreactive Ig on the surface of CD5-bearing B cells in CLL.
Subject(s)
B-Lymphocytes/immunology , Immunoglobulins/metabolism , Animals , Antigens, CD , Binding Sites , Binding, Competitive , CD5 Antigens , Cell Line , Humans , In Vitro Techniques , Kinetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , MiceABSTRACT
Lewis rats sensitized with guinea-pig spinal cord in Freund's complete adjuvant developed an acute-phase protein response. This was characterized by a marked increase in plasma alpha 2-macroglobulin (alpha 2 M) levels which, however, declined towards normal values before the onset of clinical signs of experimental allergic encephalomyelitis (EAE). In contrast, levels of two other acute-phase proteins, fibrinogen and caeruloplasmin, remained variably elevated over the entire study period. Recovery from EAE coincided with an increase in alpha 2 M levels. Infusion of purified alpha 2 M effectively protected the rats against clinical EAE and this was associated with a restimulation of the acute-phase response. The protected rats were shown to be sensitized to myelin basic protein and to have comparable mononuclear infiltration of the central nervous system with the diseased animals. It is postulated that the infusion of alpha 2 M leads to the inhibition of the effector pathways of the delayed type hypersensitivity response.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/prevention & control , alpha-Macroglobulins/therapeutic use , Acute-Phase Proteins/metabolism , Animals , Encephalomyelitis, Autoimmune, Experimental/blood , Encephalomyelitis, Autoimmune, Experimental/etiology , Freund's Adjuvant , Guinea Pigs , Male , Rats , Rats, Inbred Lew , Spinal Cord/immunology , alpha-Macroglobulins/metabolismABSTRACT
The v-myb oncogene and its cellular progenitor c-myb are both DNA binding proteins capable of transcriptional activation, and are implicated in the regulation of the switch between growth and differentiation in hematopoietic cells. Studies attempting to define the oncogenic determinants of v-myb and activated c-myb genes implicate N- and/or C-terminal truncation as important; both these events appear to increase the affinity of the myb protein for DNA. Myb-like genes have been found in organisms ranging from yeast, through plants, to humans; in the more distantly related cases, only the myb DNA binding domain, situated at the N-terminus of the protein, has been conserved.
Subject(s)
Oncogenes/genetics , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Oncogenes/physiologyABSTRACT
Human cytomegalovirus (HCMV) is a herpesvirus with a genome of 230 kilobases (Kb) encoding about 200 genes. Although infection is generally innocuous, HCMV causes serious congenital and neonatal disease, and is a dangerous opportunistic pathogen in immune-deficient individuals. We have identified a family of three HCMV genes which encode polypeptides containing seven putative membrane-spanning domains, and a series of well-defined motifs characteristic of the rhodopsin-like G protein-coupled receptors (GCRs). By these criteria all three of the HCMV sequences are homologous to cellular GCRs. Members of this receptor family function in visual signal transduction, regulation of homeostasis, and development, and include known and potential oncogenes. These receptors are activated by photons or small molecules such as neurotransmitters, and glycoprotein hormones. The finding of viral-encoded GCR homologues implies a further level of complexity in the interactions between HCMV and its host, and may provide a potential pathway for virally transformed cell proliferation. Their identification could permit the development of a novel class of antiviral drugs analogous to beta-adrenergic receptor antagonists.
Subject(s)
Cytomegalovirus/genetics , GTP-Binding Proteins/physiology , Genes, Viral , Receptors, Cell Surface/genetics , Amino Acid Sequence , Base Sequence , Cell Membrane/metabolism , Glycosylation , Humans , Molecular Sequence Data , Phosphorylation , Sequence Homology, Nucleic Acid , Signal TransductionABSTRACT
Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.
Subject(s)
Antigen-Presenting Cells/immunology , Lymphocyte Activation , Lymphokines/biosynthesis , T-Lymphocytes/immunology , Animals , Antigen-Presenting Cells/radiation effects , Antigens, Surface/analysis , B-Lymphocytes/immunology , B-Lymphocytes/radiation effects , Crosses, Genetic , Interferon-gamma/biosynthesis , Interleukin-1/biosynthesis , Interleukin-2/biosynthesis , Interleukin-3/biosynthesis , Lymph Nodes/cytology , Lymphocyte Activation/radiation effects , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Phenotype , T-Lymphocytes/classification , T-Lymphocytes/metabolismABSTRACT
Abnormal T cells from autoimmune MRL/Mp-lpr/lpr mice express T-cell-receptor complexes on their surfaces. These receptors are nonfunctional, since monoclonal anti-T-cell-receptor antibody-conjugated beads, which normally activate receptor-bearing T cells, were unable to activate these abnormal T cells. In addition, these abnormal T cells are unresponsive to the synergistic effect of phorbolmyristate acetate (PMA) and calcium ionophore A23187, as quantitated by proliferation, interleukin-2(IL-2) production, and the expression of IL-2 receptors. The failure of abnormal T cells to respond to PMA is not due to the absence of PMA receptors since Scatchard plot analysis reveals that there are 1-1.5 X 10(5) PMA-binding sites/cell with a Kd of 6-10 nM on these abnormal T cells. Similar to normal T lymphocytes, protein kinase c activity can be readily detected in the cytosolic fraction of these abnormal T cells. More importantly, in vitro culture of the abnormal T cells with PMA results in translocation of protein kinase c activity from the cytosolic fraction to the membrane fraction. From these experiments we concluded that the defect in the signal-transducing machinery in MRL/Mp-lpr/lpr T cells is not due to the lack of PMA receptors or the absence of protein kinase c activity, but may result from events which occur after the activation and translocation of protein kinase c. However, whether defects in response to a physiological stimulus (i.e., anti-receptor antibody) could occur in a step prior to protein kinase c activation remains to be determined.