Ronald D. Eichner

Identification of an agent in cultures of Aspergillus fumigatus displaying anti-phagocytic and immunomodulating activity in vitro.

When cultured in vitro, Aspergillus fumigatus generated a metabolite(s) with anti-phagocytic activity as tested by macrophage adherence to plastic and phagocytosis of particulate matter. The metabolite(s) appeared after 3 d culture and reached a peak concentration after 5-6 d. The action of the anti-phagocytic agent(s) was rapid (5-15 min) and appeared not to alter membrane permeability or cause rapid cell death. Treatment of stimulator spleen cells with the agent(s) inhibited their ability to induce alloreactive and major histocompatibility complex restricted cytotoxic T cells. The metabolite(s) was chloroform-soluble and separated into three biologically active compounds on thin-layer chromatography. These compounds were purified greater than 1000-fold and one of them was identified as gliotoxin, a known metabolite of A. fumigatus, based upon NMR and IR spectroscopy, mass spectrometry, biological properties and other data.

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.

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.

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 allogeneic 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.

The isolation and identification of a new metabolite from Aspergillus fumigatus related to gliotoxin

Summary Gliotoxin G, the tetrasulphide analogue of gliotoxin has been characterized from a laboratory isolate of Aspergillus fumigatus .

Activation of cyclic AMP-dependent protein kinase in macrophages.

The activity of cyclic AMP-dependent protein kinase (cyclic AMP-PK) was significantly higher (P less than 0.001) in thioglycollate-elicited than in resident rat peritoneal macrophages. The activity ratio of the enzyme (its activity in the absence of added cyclic AMP divided by that in the presence of 5 microM cyclic AMP) was similar in the two cell types. The divalent ion ionophore A23187 induced a rapid increase in the activity ratio of cyclic AMP-PK in both macrophage types. This effect was blocked by pretreating the cells with indomethacin or aspirin (inhibitors of cyclo-oxygenase) and bromo-phenacyl bromide (an inhibitor of phospholipase A2), implicating the synthesis of a prostanoid as an intermediary step. Prostaglandin (PG) E2, 8-bromo cyclic AMP and cholera toxin, all of which inhibit chemiluminescence and/or PG formation in macrophages, increased the activity ratio of cyclic AMP-PK in these cells. We propose that the activation of cyclic AMP-PK plays a central role in the response of macrophages to both endogenously-generated and exogenously added PGE.

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.

Glycogen synthesis in resident and thioglycollate-elicited rat peritoneal exudate cells

The values of the A0.5 for glucose-6-P, apparent Km for UDPglucose and -/+glucose-6-P activity ratio are similar for glycogen synthase derived from rat resident and thioglycollate-elicited peritoneal macrophages; the specific activity is 7-fold higher for the enzyme from thioglycollate-elicited macrophages. The rate of incorporation of [14C]glucose into macrophage glycogen is 7-fold greater in the elicited population that that in the resident one; the values of the S0.5 for glucose are similar. The in vitro activation of glycogen synthase proceeds at a greater rate and extent for the enzyme from elicited macrophages; thus, phosphatase activity may be reduced in resident macrophages relative to that in thioglycollate-elicited ones.

The Molecular Mechanism of Toxicity of Gliotoxin and Related Epipolythiodioxopiperazines

Gliotoxin (Fig. la) belongs to the epipolythiodioxopiperazine (ETP) class of fungal metabolites having a bridged polysulphide piperazine ring in common.10,11,14 Gliotoxin was the first member of this class of metabolites to be described being isolated from a strain of Trichoderma lignosum in 1932.17 The tri12 and tetra13 sulphide analogues of gliotoxin have also been described. Gliotoxin and a number of related ETP compounds have been shown to be responsible for a number of mycotoxicoses following ingestion of contaminated feed by ruminants.3

Glycogen synthesis in bovine lymphocytes from efferent prefemoral lymph

Abstract 1. 1. The values of the A0.5 for glucose-6-P, −/+glucose-6-P activity ratio, apparent Km for UDP-glucose and Vmax for glycogen synthase from bovine lymphocytes are 0.68 ± 0.11. 0.042 ± 0.009, 0.10 ± 0.02 mM and 783 ± 155nmol/min per 1010 cells, respectively. 2. 2. The reversible interconversion (as measured by kinetic criteria) of bovine lymphocyte glycogen synthase is consistent with a phosphorylation-dephosphorylation mechanism. 3. 3. The levels of glycogen, glucose-6-P and UDP-glucose per 1010 bovine lymphocytes are 9.2 ± 0.1 mmol glucosyl units, 2.3 ± 0.30 mmol glucose-6-P and 0.30 ± 0.12 mol UDP-glucose, respectively. 1.4. The incorporation of [14C]glucose into bovine lymphocyte glycogen is characterized by an S0.5 for glucose of 2.5 ± 0.35 mM, nH of 1.3 ± 0.06, and Vmax of 13.5 ± 1.1 nmol glucose incorporated/min per 1010 cells.