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Biomedical subjects

P Waring

Publications and source records attributed to P Waring.

At least 19 recordsLinked to original sources
PubMed1992

Fragmentation of DNA in the retina of chicken embryos coincides with retinal ganglion cell death.

Neuronal cell death was studied in the developing retina of the chicken embryo. One of the most characteristic indices of the form of cell death termed apoptosis is regular, apparently internucleosomal fragmentation of DNA. When retinae of eight to seventeen day old chicken embryos were dissected out and the DNA from this tissue size fractionated on agarose gels, fragmentation typical of apoptosis was observed on day ten. The maximal amount of fragmentation was reached around day eleven and twelve and declined from day 15 to 17. These findings correlate in time with previous histological data on retinal cell death and demonstrate for the first time the occurrence of DNA fragmentation typical of apoptosis in the developing nervous system.

Aging↗
PubMed1992

Actions of leukaemia inhibitory factor on megakaryocyte and platelet formation.

Leukaemia inhibitory factor (LIF) is able to potentiate megakaryocyte colony formation in cultures of mouse bone marrow cells in the presence of multi-CSF (interleukin 3). Membrane receptors for LIF are present on mouse megakaryocytes and receptor numbers increase with increasing maturation of the cells. When injected into normal mice at doses of 0.2-2 micrograms two to three times daily, LIF induced a rise in platelet numbers, which reached up to twice normal values during the second week of injections. This rise was preceded by a rise first in megakaryocyte progenitor numbers, then in mature megakaryocytes in the bone marrow and spleen. Injections of LIF also marginally accelerated platelet regeneration in mice pre-injected with 5-fluorouracil or subjected to whole-body irradiation and transplantation of marrow cells. In view of similar responses to LIF in parallel studies in primates, clinical trial of LIF in patients with thrombocytopenia is warranted.

Animals↗
PubMed1992

The possible role of gliotoxin in health and disease.

Demonstration of the unique therapeutic properties of penicillin initiated an extensive investigation of other mould metabolites in the hope of finding other useful chemotherapeutic agents. While many hundreds of active substances were studied very few indeed satisfied the criteria necessary for clinical use. Among those whose toxicity ruled them out was gliotoxin. Recently, however a chance observation, in quite a different context, suggests that gliotoxin may prove valuable in human tissue transplantation.

Animals↗
PubMed1992

Leukemia inhibitory factor levels are elevated in septic shock and various inflammatory body fluids.

Leukemia inhibitory factor (LIF) has many biological actions which parallel those of IL-1, IL-6 and tumor necrosis factor-alpha, but its role in the pathogenesis of human disease is unknown. A specific radioreceptor competition assay capable of detecting LIF at concentrations above 1 ng/ml (45 pM) was developed. To identify disease states in which LIF might be involved, a cross-sectional survey of serum and body fluids from approximately 1,500 subjects with a variety of diseases was performed using the LIF radioreceptor competition assay. Serum LIF concentrations were transiently elevated (2-200 ng/ml) in six subjects with meningococcal or Gram-negative septic shock, and in a subject with idiopathic fulminant hepatic failure. Moderately elevated LIF concentrations (> 10 ng/ml) were detected in cerebrospinal fluid from subjects with bacterial meningitis, in effusions associated with pneumonia and peritonitis, and in amniotic fluid from a woman with chorioamnionitis. Low LIF concentrations (1-10 ng/ml) were present in synovial fluid from subjects with inflammatory arthritis, amniotic fluid from women in labor, and some reactive, chronic inflammatory and malignant effusions and cyst fluids, but rarely in transudates. These initial findings suggest that LIF might be involved in the pathogenesis of inflammation and septic shock.

Amniotic Fluid↗
PubMed1991

Gliotoxin treatment selectively spares M-CSF- plus IL-3-responsive multipotent haemopoietic progenitor cells in bone marrow.

Gliotoxin, an epipolythiodioxopiperazine, is a fungal metabolite that causes genomic DNA degradation preferentially in certain blood cell types including T lymphocytes and macrophages. Gliotoxin has previously been used to treat murine allogeneic bone marrow prior to transplantation into irradiated recipients, and in this situation the drug prevents development of graft-versus-host disease, and permits the establishment of allogeneic bone marrow chimeras. We have examined the nature of the cells that survive gliotoxin treatment and report here that gliotoxin selectively spares a unique class of haemopoietic stem cell that forms large (HPP) colonies in the presence of mixtures of M-CSF and IL-3. We confirm that the cells which survive gliotoxin treatment are capable of reconstituting the haemopoietic system in allogeneic lethally irradiated mice.

Animals↗
PubMed1991

Langerhans cell depletion in gliotoxin-treated murine epidermis.

Langerhans cells (LC) are dendritic antigen presenting cells of bone marrow origin which reside in the suprabasal layer of the epidermis. They express high concentrations of Class II MHC glycoproteins on their plasma membrane and transport cutaneous antigen to local lymph nodes for presentation to helper T cells. They are thus essential for the induction of cutaneous immunity. Gliotoxin is a member of the epipolythiodioxopiperazine (ETP) group of fungal metabolites, derived from the human pathogen Aspergillus fumigatus. It has been shown to have immunomodulating properties in vivo and in vitro, and has been proposed as a potential immunosuppressant for transplantation therapy. Epicutaneous application of gliotoxin reduced the numbers of epidermal LC by 30-35 per cent with an associated morphological change from highly dendritic to a more rounded form. Electron microscopic studies showed selective damage to LC at very low (nM) concentrations of gliotoxin, with no obvious effect on adjacent keratinocytes. LC numbers remained depleted for 13 weeks after initial treatment, suggesting that systemic suppression or prolonged retention of gliotoxin within the skin may play a role in its mechanism of action.

Animals↗
PubMed1990

DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels.

We have shown that the immunomodulating agent gliotoxin induces DNA fragmentation in macrophages characteristic of programmed cell death or apoptosis (Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) J. Biol. Chem, 263, 18493-18499). In addition, morphological changes and DNA fragmentation characteristic of apoptosis are induced in 48 h concanavalin A-stimulated T blasts by gliotoxin and these changes are inhibited by Zn2+ (Waring, P., Egan, M., Braithwaite, A., Mullbacher, A., and Sjaarda, A. (1990) Int. J. Immunopharmacol., in press). We have studied the effects of actinomycin D and the protein synthesis inhibitor cycloheximide on apoptosis induced by gliotoxin in these cells, and these studies demonstrate no effect on apoptosis induced by gliotoxin. Cycloheximide and actinomycin D alone induce DNA fragmentation in these cells. Gliotoxin itself proved to be a potent inhibitor of protein synthesis. The fragmentation caused by cycloheximide correlated with the extent of protein synthesis inhibition. The toxin ricin also induced DNA fragmentation in T blasts characteristic of apoptosis. These results indicate that protein synthesis is not required for induction of apoptosis in macrophages or T blasts by gliotoxin. Gliotoxin caused elevated levels of inositol triphosphate in treated macrophages which may be related to mobilization of Ca2+ levels during apoptosis.

Animals↗
PubMed1990

Apoptosis induced in macrophages and T blasts by the mycotoxin sporidesmin and protection by Zn2+ salts.

Incubation of 48 h concanavalin A stimulated spleen cells (T blasts) and murine peritoneal macrophages with the mycotoxin sporidesmin results in DNA fragmentation characteristic of apoptosis. Morphological changes, particularly condensed chromatin, observed following incubation of these cells with sporidesmin and the immunotoxin gliotoxin and related epipolythiodioxopiperazines (ETP) also show changes characteristic of apoptosis. The presence of Zn2+ salts in the culture medium at concentrations non toxic to the cells over the time period studied protects against DNA damage and morphological change. Interaction between Zn2+ and the reduced form of a simple ETP compound assessed by spectral changes demonstrated the formation of a weak complex between the two molecules. Complex formation between zinc and thiol however was insufficient to prevent oxidative damage to plasmid DNA in vitro by inhibiting auto-oxidation of the reduced ETP compound because of the looseness of the interaction. Cd2+, which appears to form a tighter complex with the dithiol does inhibit cleavage of plasmid DNA. These results establish that the toxicity of sporidesmin may be due in part to its ability to induce apoptosis or programmed cell death in sensitive cells. In addition the immunotoxin gliotoxin and related compounds have now been shown to induce the same characteristic morphological changes in cells of haemopoietic origin. The inhibition of apoptosis induced by ETP compounds by Zn2+ appears to be due to direct inhibition of apoptosis rather than Zn2+ acting as an antioxidant. These results demonstrate the inhibition of apoptosis induced by ETP compounds by Zn2+ and suggest an alternate explanation for the known prophylactic effect of Zn2+ on sporidesmin induced tissue damage.

Animals↗
PubMed1989

Alloreactive cytotoxic T cells induce DNA fragmentation in peritoneal macrophages: evidence for target cell killing by cytotoxic T cells in vivo.

This report addresses the question whether cytotoxic T cells can cause target cell death in vivo by examining target cell DNA fragmentation. The results show that alloreactive cytotoxic T cells induced significant DNA fragmentation in peritoneal macrophages in vivo and that the DNA fragment was a multiple of 180 +/- 30 bp. Furthermore, the effector cells which caused this characteristic DNA fragmentation were CD8+ T cells. These results demonstrate that cytotoxic T cells can cause target cell death in vivo.

Animals↗
PubMed1989

Natural killer cells and cytotoxic T cells induce DNA fragmentation in both human and murine target cells in vitro.

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC-1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC-1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.

Animals↗
PubMed1988

Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties.

We have previously shown that the fungal metabolite and immunomodulating agent gliotoxin induces apparently random double-stranded fragmentation of genomic DNA in a variety of cell types and double- and single-stranded scission in isolated plasmid DNA. The in vitro damage to plasmid DNA appears to be mediated by reactive oxygen species, but the mechanism of damage to genomic DNA is not yet known. In this paper we show that treatment of macrophages with gliotoxin and some analogues gives rise to discrete DNA fragments with molecular weight 170 +/- 30 base pairs. This pattern of DNA fragmentation has the characteristics of apoptosis, a programmed form of cell death. Three structural analogues of gliotoxin and two S-acetylated precursors capable of intracellular hydrolysis to the thiol form induce identical DNA degradation patterns. Only those compounds with the epipolythiodioxopiperazine (ETP) bridged disulfide structure or those capable of extracellular conversion to ETP compounds are equipotent with gliotoxin in their effects on macrophage phagocytosis, although all are capable of generating reactive oxygen species intracellularly. These results suggest that the effect of gliotoxin on macrophage function as assessed by adherence to plastic surfaces is unrelated to DNA damage and in addition suggests a new mechanism by which the toxin and other ETP compounds may damage cells.

Animals↗
PubMed1988

Gliotoxin causes oxidative damage to plasmid and cellular DNA.

The cytotoxic effects of gliotoxin (Müllbacher, A., and Eichner, R. D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3835-3837), a fungal secondary metabolite, and related epipolythiodioxopiperazines have been investigated using plasmid and eukaryotic DNA. Incubation of the dithiol derivative of these compounds with DNA and Fe3+ is sufficient to cause single- and double-stranded breaks as determined by neutral agarose gel electrophoresis. The disulfide form is inactive except in the presence of a suitable reducing agent, such as reduced glutathione, dithiothreitol, or reduced pyridine coenzymes. The autooxidation of these dithiols produces reducing equivalents as evidenced by (i) the production of H2O2 and (ii) the generation of thiobarbituric acid reactive products when incubated with deoxyribose. The latter process is inhibited by ethanol and desferrioxamine. The DNA damage is abrogated by metal chelators and catalase. We conclude that the antiproliferative action of gliotoxin may be caused by DNA damage effected by reactive oxygen species or other radicals generated through redox cycling.

Animals↗
PubMed1988

Replication of donor lymphocytes in recipients is not essential for the passive transfer of allergic encephalomyelitis.

Gliotoxin is a fungal metabolite belonging to the class of epipolythiodioxopiperazines which possesses both immunomodulating and anti-phagocytic activities. We have examined the effect of gliotoxin on passively induced allergic encephalomyelitis and report here that pulse treating activated experimental allergic encephalomyelitis (EAE) effector lymphocytes with gliotoxin inhibits, in a dose-dependent manner, their ability to transfer disease. Cells treated with 300 ng/ml are unable to replicate in vitro in response to concanavalin A stimulation, nor did they produce interleukin-2 (IL-2) following stimulation. Furthermore this concentration of gliotoxin also causes complete fragmentation of genomic DNA in treated cells, yet these cells are still capable of transferring clinical EAE. These data suggest that replication of donor lymphocytes in the recipient is not essential for the development of EAE.

Animals↗
PubMed1988

Prevention of graft-versus-host disease by treatment of bone marrow with gliotoxin in fully allogeneic chimeras and their cytotoxic T cell repertoire.

Gliotoxin, a secondary fungal metabolite, at nanomolar concentrations, irreversibly inhibits murine T cell proliferation to mitogen. Treatment of allogeneic spleen cells with gliotoxin allows their transfer into sublethally irradiated recipients without inducing a GVH reaction. Gliotoxin treatment of bone marrow allows the establishment of fully allogenic bone marrow chimeras free of GVH disease. The cytotoxic T cell repertoire against influenza virus in these animals is restricted to both host- and donor-type MHC. However, their immune competence is severely compromised by their lack of host MHC-type stimulator cells.

Animals↗
PubMed1987

The immunomodulating agent gliotoxin causes genomic DNA fragmentation.

Gliotoxin, a member of the class of secondary fungal metabolites characterized by the presence of an epipolythiodioxopiperazine ring, caused fragmentation of spleen cell DNA as observed by flow cytometry and gel electrophoresis. Gliotoxin was found to cause substantial double-stranded DNA breakage in spleen cells which was dose- and time-dependent. The ability of gliotoxin to cause DNA breakage was also found to be specific to cell type. DNA breakage occurred in all cell types in which gliotoxin inhibited proliferation and so provides a general explanation as to how gliotoxin prevents cell proliferation. Other results showed that gliotoxin bound to a similar extent to both sensitive and resistant cells, indicating that differential uptake is not a likely mechanism to explain cell type selectivity. The results are discussed in terms of a mechanism for gliotoxin action involving genomic DNA as the central target.

Animals↗
PubMed1987

Selective resistance of bone marrow-derived hemopoietic progenitor cells to gliotoxin.

The fungal metabolite gliotoxin at low concentrations prevents mitogen stimulation of mature lymphocytes as a result of gliotoxin-induced genomic DNA degradation. Bone marrow, on the other hand, contains a subpopulation of cells resistant to gliotoxin at similar concentrations. This population includes the hemopoietic progenitor cells that grow in vitro in response to appropriate colony-stimulating factors and cells that form colonies in the spleens of lethally irradiated recipients. Gliotoxin treatment of lymph node cell-enriched bone marrow significantly delayed the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras.

Animals↗
PubMed1986

Structural relationship of epipolythiodioxopiperazines and their immunomodulating activity.

Epipolythiodioxopiperazines were tested for their immunoregulatory activity in vitro. Using the macrophage adherence test as a measure of inhibition of phagocytosis, their effect on stimulator cells in mixed lymphocyte cultures and their ability to inhibit mitogen stimulation of T lymphocytes, a hierarchy of activity was observed, with sporidesmin being the most active, followed by gliotoxin and 1,4-dimethyl-3,6-epidithio-2,5-dioxopiperazine. Derivatives of gliotoxin such as dehydro-, trisulfide and tetrasulfide gliotoxin have activities comparable to gliotoxin. The dimethylthioether derivative of gliotoxin was devoid of activity. The presence of reducing agents abrogated the activity of epipolythiodioxopiperazines. This suggests that the bridged disulfide moiety is the single most important chemical entity for their activity. The differential activities of the active compounds may be attributable to their variations in lipophilic properties.

Animals↗