Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of ...Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi.
models for studying interactions between
and host cells are crucial for understanding the pathomechanism of
and assessing differences between strains and cell types. The virulence of
strains is ...usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess
cytopathogenicity.
The viability of human corneal epithelial cells (HCECs) after co-culture with
was evaluated in phase contrast microscopy.
It was shown that
is unable to considerably reduce the tetrazolium salt and the NanoLuc
Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify
cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of
on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.
Our findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc
Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of
with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.
Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards ...these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs).
We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography.
We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba.
This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.
The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces ...keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL‐1β, IL‐10 and/or caspase 3. Intercellular adhesion molecule‐1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post‐inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.
Herein, we report the preparation of a panel of Schiff bases analogues as antiprotozoal agents by modification of the stereoelectronic effects of the substituents on N-1 and N-4 and the nature of the ...chalcogen atom (S, Se). These compounds were evaluated towards
and
. Thiosemicarbazide
showed the best trypanocidal profile (epimastigotes), similar to benznidazole (BZ): IC
(
)=28.72 μM (CL-B5 strain) and 33.65 μM (Y strain), IC
(BZ)=25.31 μM (CL-B5) and 22.73 μM (Y); it lacked toxicity over mammalian cells (CC
> 256 µM). Thiosemicarbazones
,
and
showed remarkable trichomonacidal effects (IC
=16.39, 14.84 and 14.89 µM) and no unspecific cytotoxicity towards Vero cells (CC
≥ 275 µM). Selenoisosters
and
presented a slightly enhanced activity (IC
=11.10 and 11.02 µM, respectively). Hydrogenosome membrane potential and structural changes were analysed to get more insight into the trichomonacidal mechanism.
Ameobae belonging to the genus
are responsible for the human diseases Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the ...existence of a resistance stage (cysts). In an attempt to add new agents that are effective against trophozoites and cysts, tea tree oil (TTO) and dimethyl sulfoxide (DMSO), separately and in combination, were tested In Vitro against two
isolates, T3 and T4 genotypes. The oxygen consumption rate (OCR) assay was used as a drug screening method, which is to some extent useful in amoebicide drug screening; however, evaluation of lethal effects may be misleading when testing products that promote encystment. Trophozoite viability analysis showed that the effectiveness of the combination of both compounds is higher than when either compound is used alone. Therefore, the TTO alone or TTO + DMSO in combination were an amoebicide, but most of the amoebicidal activity in the combination's treatments seemed to be caused mainly by the TTO effect. In fact, DMSO alone seems to be a non-amoebicide, triggering encystment. Regarding cytotoxicity, these compounds showed toxicity in human corneal epithelial cells (HCEpiC), even at low concentrations when tested in combination. In conclusion, the use of TTO and DMSO, in combination or alone, cannot be recommended as an alternative for AK treatment until more cytotoxicity and cyst adhesion tests are performed.
Aerosolized anthrax (
) spores are of extreme health concern and can remain airborne for hours and contaminate all kinds of surfaces, constituting reservoirs from which resuspension is easily ...produced. The assessment of decontamination techniques must therefore consider both air and surfaces. In the present study, several kinds of disinfecting fogs were experimentally tested against
spores, which served as a surrogate for
, both as aerosols released into the air and spread on porous and non-porous surfaces with different positions and orientations. This technology removed
spores from the air in 20 min with just a 1 min application of fog. The dynamics and characteristics of the fog, related to aerosol and surface interactions, proved to be critical for optimal performance and decontamination. An optimal configuration could provide effective disinfection even on indirectly reached surfaces. In all cases, 8% hydrogen peroxide (H
O
) provided a higher disinfection rate than 2% glutaraldehyde.
Cationic carbosilane dendrons and Ag NPs and AuNPs decorated with these dendrons have been tested as antibacterial and antifungal agents.
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Water soluble silver nanoparticles (AgNPs) ...capped with cationic carbosilane dendrons have been synthesized by direct reaction in water of dendrons, silver precursor and a reducing agent. These nanoparticles have been characterized by nuclear magnetic resonance (NMR), transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), ultraviolet spectroscopy (UV), elemental analysis, and zeta potential (ZP). The antibacterial and antifungal properties of the cationic dendrons and dendronized AgNPs and AuNPs with these dendrons have been evaluated against Gram-negative and Gram-positive bacterial –including resistant strains- and yeast strains, respectively. The results stand out for the activity of AgNPs covered with first generation dendron compared with this free dendron and corresponding dendronized AuNPs.
Cationic carbosilane dendrimers are branched molecules with antimicrobial properties. Their activity has been tested against Acanthamoeba polyphaga, a causative agent of Acanthamoeba keratitis, a ...severe ocular disease in humans. A. polyphaga trophozoites and cysts were exposed to different noncytotoxic cationic carbosilane dendrimers with proven antiamoebic activity. The effects of treatment on cell surface and cell ultrastructure were examined by scanning and transmission electron microscopy, respectively. Two of the dendrimers tested induced dramatic alterations of cellular ultrastructure in both trophozoites and cysts, including vacuolization, depletion of cytoplasmic contents, and reduced cell size. Additionally, we observed severe alterations of the plasma membrane with membrane blebbing in trophozoites and disruption in cysts. These alterations were also observed with chlorhexidine, a drug used for treatment of Acanthamoeba keratitis. Our results support that these compounds may target membranes, and their action is critical for parasite integrity.
Aerosolized anthrax (Bacillus anthracis) spores are of extreme health concern and can remain airborne for hours and contaminate all kinds of surfaces, constituting reservoirs from which resuspension ...is easily produced. The assessment of decontamination techniques must therefore consider both air and surfaces. In the present study, several kinds of disinfecting fogs were experimentally tested against Bacillus thuringiensis spores, which served as a surrogate for Bacillus anthracis, both as aerosols released into the air and spread on porous and non-porous surfaces with different positions and orientations. This technology removed Bacillus thuringiensis spores from the air in 20 min with just a 1 min application of fog. The dynamics and characteristics of the fog, related to aerosol and surface interactions, proved to be critical for optimal performance and decontamination. An optimal configuration could provide effective disinfection even on indirectly reached surfaces. In all cases, 8% hydrogen peroxide (Hsub.2Osub.2) provided a higher disinfection rate than 2% glutaraldehyde.
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