In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

The effect of synthetic iron chelators on bacterial growth in human serum

Abstract The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms ( Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa ); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis , but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa . Desferrioxamine enhanced the growth chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergiong chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

The effect of synthetic iron chelators on bacterial growth in human serum

The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms (Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis, but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa. Desferrioxamine enhanced the growth of all except E. coli. These results suggest that the chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergoing chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

In vitro measurement of pollen tube growth inhibition

A method for estimating inhibition of pollen tube growth was developed. Pollen is placed in straight lines on an agar surface where it responds uniformly and predictably to aqueous solutions of germination-inhibiting substances located in wells at the ends of the lines. A scale of ratings, roughly corresponding to serial, doubled concentrations of inhibiting substances, was devised. Water-soluble organic solvents are relatively noninhibitory, salts are variable, and metabolic inhibitors have strong inhibitory effects. Pollens differ in their susceptibility to inhibition and in their response to particular substances.     

In vitro growth inhibition of human cancer cells by novel honokiol analogs

Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells.     

In vitro inhibition of Candida albicans growth by plant extracts and essential oils

The opportunistic Candida albicans yeast strain ATCC 10261 grows at 37 °C and gives germ tubes after 3 h on corn meal agar and blood plasma. It produces chlamydospores and assimilates sucrose, dextrose, galactose, maltose, trehalose and xylose among the tested carbon sources. Other growth characters were also investigated. The agar diffusion cut plug technique revealed that 200 l of Foeniculum vulgare Miller (fennel), Mentha piperita L. (peppermint) and Citrus limon (lemon) essential oils showed inhibitory actions. Fumigation test of the three essential oils had complete inhibition on the growth. The essential oil of Eucalyptus occidentalis End1 (eucalyptus, eucalypt) had no influence on C. albicans growth by the two tests. Meanwhile, methanol extracts of both leaves and male cones of the conifer Thuja orientalis (eastern thuya) had an inhibitory influence on the growth by two tests (cut plug and filter paper disc assay). In comparative tests the antibiotics mycostatin and dermatin had an inhibitory activity on C. albicans at the concentrations of 50–80 g per hole. Crude methanol extract of leaves of eastern thuya (10–80 g/ml) reduced C. albicans growth and intercellular protein nitrogen on liquid media.     

In vitro effects on bacterial growth of phenoloxidase reaction products

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.     

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.     

In vitro inhibition of Trichomonas vaginalis growth by some ortho-phenacyloxy-benzyl alcohols and their derivatives

Some phenacyl ethers of different ortho-hydroxy-benzyl alcohols and analogues have been synthesized and tested for the in vitro activity towards Trichomonas vaginalis. The most active compounds had a minimum inhibitory concentration of 6.25 micrograms/ml and appeared to be of a certain interest as representative of a new type of anti-Trichomonas substances not containing a nitro group.     

Release of iron from ferritin molecules and their iron-cores by 3-hydroxypyridinone chelators in vitro

Ferritin molecules contain 24 subunits forming a shell around an inorganic iron-core. Release of iron(III) from ferritin and its isolated iron-cores by a series of hydroxypyridinone chelators with high affinities for iron(III) has been compared. The results collectively suggest that the chelators act by penetrating the protein shell and interacting directly with the iron-core in ferritin. Iron(III) is probably removed bound to a single ligand, but once outside the protein shell, the trihydroxypyridinone iron(III) complex predominates. The order of effectiveness of a group of pyridinones found for iron removal from ferritin molecules in solution differs from that obtained with hepatocytes in culture or with whole animals, where membrane solubility and other factors may modulate the response.     

The inhibition of bacterial growth by ochratoxin A.

A series of bacterial species was examined for their sensitivity to ochratoxin A. Only grampositive bacteria could be inhibited, generally at a pH lower than 7.0. Bacillus subtilis did not show any reduction of growth rates in presence of ochratoxin A, but had a prolonged lag phase. With Staphylococcus pyogenes var. aureus and Streptococcus faecalis, a prolonged lag phase and a reduction of the growth rate was observed. Most sensitive was Streptococcus faecalis in the exponential-growth phase. The inhibition could be diminished by changing the pH to neutral, or by addition of yeast extract, tetrahydrofolate, or MgSO4. With MgSO4 a complete abolition of the inhibitory effect was achieved, but not with CaCl2. During growth inhibition, protein and RNA synthesis were reduced simultaneously, but not DNA synthesis. Even with the very high concentration of 1 mg/ml, no lethal effect was observed.     

In vitro inhibition of Helicobacter pylori by micromycetes

The anti-Helicobacter pylori effect of 22 micromycetes were studied against one standard strain and 11 clinical isolates of H. pylori. Penicillium ochlochloron and Penicillium funiculosum have been proven the most active fungi against this microorganism. Further bio-guided chemical analysis of P. funiculosum afforded an active component identified as (-) 2,3,4-trihydroxybutanamide.     

In vitro inhibition of mumps virus by retinoids

Background

Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules α_vβ₁, α_vβ₃, α_vβ₆, or α_vβ₈ by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the α_vβ₆ interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted α_vβ₆ (ssα_vβ₆) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction.

Results

Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssα_vβ₆. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssα_vβ₆ neutralization, relative ability to utilize α_vβ₆ for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for α_vβ₆. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssα_vβ₆. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssα_vβ₆ resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding.

Conclusions

These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.     

Prevention of oxidant-induced cell death by lysosomotropic iron chelators

Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of alpha-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (alpha-lipoic acid-plus [LAP]; pKa = 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1000 and 5000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 microM) whereas LM (200 microM) is much less effective.     

In vitro growth inhibition of Helicobacter pylori by lactobacilli belonging to the Lactobacillus plantarum group

AIMS: The aim of this study was to test and locate the in vitro anti-Helicobacter activity of seven Lactobacillus strains belonging to Lactobacillus plantarum group. METHODS AND RESULTS: Growth inhibition of H. pylori was tested using a well-plate assay. Of the strains displaying the strongest growth inhibition, a L. plantarum isolated from sauerkraut (MLBPL1) was chosen for further studies. The detected anti-Helicobacter activity of MLBPL1 was mainly associated with cell wall, and to a minor extent with the culture supernatant. The active component, which was determined to be between 3 and 10 kDa in size, retained its activity after 10 min treatment at 100 degrees C. The activity was present when MLBPL1 was cultivated in rich laboratory cultivation medium MRS and in different food matrices. CONCLUSIONS: The strains belonging to L. plantarum group showed anti-Helicobacter activity in vitro. The main activity seemed to be associated with cell wall rather than culture supernatant or intracellular fraction. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the rapid spread of resistant H. pylori strains caused by antibiotic therapy, addition of a fermented food containing L. plantarum to the conventional antibiotic treatment of Helicobacter infection could establish a potential complementary means to suppress the infection.