Naegleria fowleri is a ubiquitous, pathogenic free-living amoeba; it is the most virulent Naegleria species and causes primary amoebic meningoencephalitis (PAME) in laboratory animals and humans. Although amphotericin B is currently the only agent available for the treatment of PAME, it is a very toxic antibiotic and may cause many adverse effects on other organs. In order to find other potentially therapeutic agents for N. fowleri infection, the present study was undertaken to evaluate the in vitro and in vivo efficacies of miltefosine and chlorpromazine against pathogenic N. fowleri. The result showed that the growth of the amoeba was effectively inhibited by treatment with amphotericin B, miltefosine, and chlorpromazine. When N. fowleri trophozoites were treated with amphotericin B, miltefosine, and chlorpromazine, the MICs of the drug were 0.78, 25, and 12.5 g/ml, respectively, on day 2. In experimental meningoencephalitis of mice that is caused by N. fowleri, the survival rates of mice treated with amphotericin B, miltefosine, and chlorpromazine were 40, 55, and 75%, respectively, during 1 month. The average mean time to death for the amphotericin B, miltefosine, and chlorpromazine treatments was 17.9 days. In this study, the effect of drugs was found to be optimal when 20 mg/kg was administered three times on days 3, 7, and 11. Finally, chlorpromazine had the best therapeutic activity against N. fowleri in vitro and in vivo. Therefore, it may be a more useful therapeutic agent for the treatment of PAME than amphotericin B.
PurposeThe characteristics of oxidized titanium (Ti) surfaces varied according to treatment conditions such as duration time and temperature. Thermal oxidation can change Ti surface characteristics, which affect many cellular responses such as cell adhesion, proliferation, and differentiation. Thus, this study was conducted to evaluate the surface characteristics and cell response of thermally treated Ti surfaces.MethodsThe samples were divided into 4 groups. Control: machined smooth titanium (Ti-S) was untreated. Group I: Ti-S was treated in a furnace at 300℃ for 30 minutes. Group II: Ti-S was treated at 500℃ for 30 minutes. Group III: Ti-S was treated at 750℃ for 30 minutes. A scanning electron microscope, atomic force microscope, and X-ray diffraction were used to assess surface characteristics and chemical composition. The water contact angle and surface energy were measured to assess physical properties.ResultsThe titanium dioxide (TiO2) thickness increased as the treatment temperature increased. Additional peaks belonging to rutile TiO2 were only found in group III. The contact angle in group III was significantly lower than any of the other groups. The surface energy significantly increased as the treatment temperature increased, especially in group III. In the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, after 24 hours of incubation, the assessment of cell viability showed that the optical density of the control had a higher tendency than any other group, but there was no significant difference. However, the alkaline phosphatase activity increased as the temperature increased, especially in group III.ConclusionsConsequently, the surface characteristics and biocompatibility increased as the temperature increased. This indicates that surface modification by thermal treatment could be another useful method for medical and dental implants.
To evaluate the role of heat shock 70 protein (HSP70) in free-living amoeba, a constitutive and inducible heat shock 70 gene of pathogenic Naegleria fowleri has previously been cloned, characterized, and named as NfcHSP70. The Nf-cHSP70 is localized in the cytoplasm, pseudopodia, and phagocytic food-cups. To investigate the role of Nf-cHSP70 in the pathogenicity of N. fowleri, the synthesis of N. fowleri HSP70 was first inhibited with benzylidene lactam compound (KNK437), and Nf-cHSP70 gene was knock-downed with antisense oligomers, which were designed with a start region-specific antisense oligonucleotides (24 oligomers) and modified with phosphorothioate. KNK437 inhibited the induction of N. fowleri HSP70 in a dose-dependent manner. In addition, 300 μM KNK437 reduced the proliferation of N. fowleri to 79.4% of untreated control (100%). Nf-cHSP70 knock-downed N. fowleri with antisense oligomers showed 68.5% reduction of proliferation in comparison with untreated control (100%). The cytotoxicity of N. fowleri against CHO target cells was reduced to 42.1% by KNK437 and 68.6% by antisense oligomers. These results suggest that the cloned Nf-cHSP70 plays an important role in the proliferation and cytotoxicity of pathogenic N. fowleri.
Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51 Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after coincubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.
Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.
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