F. Aerts

The activity of ketoconazole in mixed cultures of leukocytes and Candida albicans

A system is described which allows the semi-quantitative investigation of the interaction between Candida albicans and leukocytes in culture with and without the addition of chemotherapeutic agents. Both polymorphonuclear leukocytes and macrophages avidly engulfed added yeast cells. However, they did not succeed in eradicating the fungus even when only 450 yeast cells were added to 3 X 10(6) leukocytes. This is probably due to several factors, including the decline in the functiontional capacity of the leukocytes with time in culture. The major way for the fungus to escape intracellular killing, however, seems to be the switch to the mycellial form in the presence of leukocytes. Engulfed yeasts produce germ tubes, grow out of the leukocytes and form hyphae which are much more resistant to the lytic action of the leukocytes. The leukocytes become necrotic through their interaction with the mycelia. Ketoconazole, a potent, orally active systemic antifungal agent inhibited the growth of C. albicans and completely suppressed the formation of mycelia in culture at very low concentrations (0.01 microgram ml-1). It was toxic to the leukocytes themselves only at 100 microgram ml-1. Addition of ketoconazole (10 (10-1.01 microgram ml-1) to mixed cultures of leukocytes and C. albicans allowed complete elimination of the fungus, probably because the leukocytes could easily remove the remaining yeast cells. The data show the usefulness of the system in the search for systemic antifungals and provide a possible explanation for the efficacy of ketoconazole in vivo.

Cytokine production by phytohemagglutinin-stimulated human blood cells: Effects of corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors

The ability of dexamethasone and prednisolone (corticosteroids), FK506 and cyclosporin A (T cell immunosuppressants), and of nitraquazone and rolipram (phosphodiesterase IV inhibitors) to inhibit cytokine production by stimulated human blood was investigated. Heparinized human blood obtained from normal healthy volunteers was stimulated with phytohemagglutinin (PHA) in the presence or absence of drug. After different incubation times, supernatant levels of interleukin (IL)-2, IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferonγ (IFN-γ) were quantified by ELISA. Dexamethasone strongly inhibited the production of IL-5 (IC50 = 0.004 µM), was less potent against IL-2 and IFN-γ (IC50 = 0.02–0.05 µM) and showed a relatively weak effect against GM-CSF (IC50 = 0.6 µM). Similarly prednisolone potently suppressed IL-5 generation (IC50=0.05 µM), displayed a more modest activity on IL-2 and IFN-γ (IC50 = 0.2–0.3 µM) and exerted only partial effects (43% inhibition at 1 µM) on GM-CSF. FK506 strongly suppressed the production of IL-2 (IC50 = 0.01 µM) and GM-CSF (IC50 = 0.03 µM), but was inactive (<30% inhibition at 1 µM) against IL-5 and IFN-γ. Similarly, cyclosporin A reduced the generation of IL-2 (IC50 = 0.4 µM) and GM-CSF (IC50 = 0.6 µM) while barely affecting the other two cytokines. Nitraquazone and rolipram were most active in reducing the production of IL-5 (IC50 = 0.8 and 1.3 µM, respectively), while their potency against IL-2, GM-CSF and IFN-γ was 3–6 times lower, with IC50s between 2.4 and 8.0 µM. These data indicate that corticosteroids, T cell immunosuppressants and phosphodiesterase IV inhibitors affect cytokine production by PHA-stimulated human blood cells in a differential and “pharmacotypical” manner.

The inhibitory effect of pentamidine on the production of chemotactic cytokines by in vitro stimulated human blood cells.

Pentamidine is an antiprotozoal drug with additional antiinflammatory activities that are not well understood. We now report that pentamidine inhibited the human whole blood production of the chemotactic cytokines (chemokines) interleukin (IL)-8, growth related gene α (GROα) and monocyte chemotactic protein-1 (MCP-1). The title compound dose-dependently suppressed the lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated whole blood generation of these chemokines with IC50-values of 2.1 and 2.2 μM (IL-8), 2.4 and 1.8 μM (GROα) and 2.8 and 2.4 μM (MCP-1). The inhibition was specific: when tested at 10 μM, pentamidine had no significant inhibitory effect on the PHA-induced generation of the non-chemotactic cytokines tumor necrosis factor-α (TNF-α), IL-1β, IL-2, IL-4, IL-5, IL-10 and interferon-ψ (IFN-ψ), except for a partial inhibition on IL-6. Time course experiments indicated that pentamidine (10 μM) retained its ability to inhibit PHA-stimulated IL-8 production even when its addition was delayed for up to 24 h after mitogen stimulation. Furthermore, reverse transcription PCR studies showed that pentamidine had no effect on IL-8 mRNA expression. These finding indicate that pentamidine is a posttranscription acting inhibitor of human chemokine production. This activity may contribute to the antiinflammatory action ascribed to the title compound.

Protective effect of levamisole and its sulfhydryl metabolite OMPI against cell death induced by glutathione depletion

Abstract Levamisole protects cultured cells against auto-oxidative necrosis induced by glutathione depletion. The sulfhydryl metabolite, OMPI, is approximately 100 times more active in this respect. As is the case for vitamin E and synthetic antioxidants, the cell rescue is not due to the inhibition of glutathione depletion but probably to a direct radical scavenging effect. The data suggest the following hypothesis: the immunomodulatingg effect of levamisole is at least partially due to its metabolite OMPI which enhances leukocyte functions by scavenging deleterious oxidative radicals the formation of which is enhanced in activated cells.

Study of the parameters of binding of R 61837 to human rhinovirus 9 and immunobiochemical evidence of capsid-stabilizing activity of the compound.

The binding of the antiviral compound R 61837 to human rhinovirus 9 (HRV 9) was studied quantitatively and compared with binding of R 61837 to HRV 9H, a semiresistant variant. For both strains, radiolabelled R 61387 bound to native particles only. The Kd values obtained by Scatchard analysis of saturation binding data were 37 nM for HRV 9 and 172 nM for HRV 9H, whereas the concentrations resulting in a 50% reduction of cytopathic effect were 42 nM and 840 nM, respectively. Reversibility experiments showed that 65% of the compound could be extracted with chloroform from HRV 9H but less than 5% could be extracted from HRV 9. Dissociation studies demonstrated that in the presence of excess unlabelled compound, the half-lives of the virus compound complex HRV 9 and HRV 9H were 385 and 15 min, respectively. The effect of this antirhinoviral compound on the formation of subviral particles induced by low pH or heat was also investigated. Rate zonal centrifugation experiments using [35S]methionine-labelled HRV 9 showed that binding of R 61837 protected the virus against heat (56 degrees C) and acid (pH 5.0) and that at the same concentration of R 61837 the semiresistant strain was stabilized to a lesser extent. This observation was confirmed immunochemically with nonneutralizing and neutralizing monoclonal antibodies. Both 80S and 130S subviral particles have C antigenic determinants, whereas native particles (150S) have been designated D. R 61837 prevented the switch from D to C antigenicity which can be induced by exposure of rhinoviruses to mild denaturing conditions. These findings indicate that the compound is able to prevent a conformational change of the capsid which may be a prerequisite for infection. Images

Lack of immunosuppression by ketoconazole and itraconazole

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.

Ketanserin in Wound Healing and Fibrosis: Investigations into its Mechanism of Action

Ketanserin, an 5-HT2 receptor blocker, has a paradoxical effect in wound healing. It stimulates wound closure but inhibits hypertrophic scar formation. The drug stimulates proliferation and collagen synthesis of dermal fibroblasts but inhibits the 5-HT-induced DNA synthesis and retraction in myofibroblasts. Ketanserin is also bifunctional regulator of collagen synthesis in a sponge granuloma model. It increases the collagen content at day 4 but it lowers the collagen concentration in 14 day old granulomas. It is speculated that this differential response is due to a switch in serotonin receptor-type.

R 17934 : A New Synthetic Anti-cancer Drug Interfering with Microtubules

As has been shown by Atassi (1) R 17934 is active against many experimental tumors and leukemias. Preliminary clinical observations have already established its activity against human neoplastic cells in vivo.

Microtubule Dynamics and the Mitotic Cycle: A Model

In what follows we will give a personal view of how organized microtubule (MT) systems may evolve during the cell cycle. It is based on some of our own experiments and ideas and on those of many other investigators that have devoted attention to these matters. We will not reiterate the argumentation which has led to the various facets of the following hypothesis, which is based on our personal conjecture. This chapter is similar to the concluding remarks of a review in press (9). For a detailed account of the data we refer to this review and Ref. 2–8. Figures 1–4 provide the basic illustrations necessary to follow this short review.