Interaction between Naegleria fowleri and pathogenic Escherichia coli by mannose and changes in N. fowleri protease.

Naegleria fowleri can cause acute primary amoebic encephalitis. It is known that contact-dependent pathogenicity in free-living amoeba may be mediated through a carbohydrate-dependent pathway. In this study, the effect of mannose on the interaction between N. fowleri and pathogenic Escherichia coli O157:H7 and non-pathogenic E. coli DH5α was analyzed. In particular, the changes in proteases expressed by N. fowleri in response to mannose were analyzed. Unlike the conventional method, mannose was treated with N. fowleri for 1 h. The association between N. fowleri and E. coli O157:H7 treated with 50-mM and 100-mM mannose was significantly reduced by approximately 70.9% and 128.5%, respectively. E. coli O157:H7 invasion was reduced by about 10.8% by 100-mM mannose. Moreover, as a result of culturing N. fowleri invaded by E. coli O157:H7 for 24 h, E. coli O157:H7 also grew about 1.2 times in the group not treated with mannose. E. coli DH5α association was reduced by 25.7% by 100-mM mannose. On the other hand, there was almost no inhibitory effect by 100-mM glucose. In the analysis in which mannose bound to either N. fowleri or bacteria and affected the interaction, there was little effect on the interaction between N. fowleri and bacteria. In zymographic analysis, about 135-kDa and 75-kDa bands were observed by 50-mM and 100-mM mannose, and two bands were significantly increased by 100-mM mannose. This study suggests that mannose can be mediated in the contact-dependent pathway of N. fowleri and will serve as a basis for inducing changes in the protease of N. fowleri by other monosaccharides.

Production of a monoclonal antibody against a galactose-binding protein of Acanthamoeba castellanii and its cytotoxicity.

In this study, it was confirmed whether the galactose-binding protein (GBP) was present in Acanthamoeba castellanii, and its function on a target cell was confirmed by production of an antibody against the GBP. Since the genes for GBP have not yet been identified at all, the purification of GBP was done using galactose-beads from amoebial lysates, and monoclonal antibodies were produced using cell fusion. GBP was confirmed to have a size of about 35 kDa. After the third immunization with purified GBP in BALB/c mice, monoclonal antibody production was analyzed. The clone cultured before limiting dilution was named 2AB2 and showed the highest antibody titer in the culture supernatant of a 24-well plate. AF6 clone cultured after limiting dilution showed an antibody titer of 0.259 in a 75-T flask. Antibodies generated by collecting ascites by injecting monoclonal colonies into the abdominal cavity of mice were confirmed through gel analysis and were observed to belong to the isotype of the IgM having kappa chains. Since the cytotoxicity of A. castellanii was inhibited by about 26% by the monoclonal antibody against GBP, it was confirmed that the antibody against GBP had an inhibitory effect on cytotoxicity. This study was the first report on GBP isolated and purified from A. castellanii, and similarly to a mannose-binding protein (MBP), its involvement in contact-dependent cytotoxicity was demonstrated with monoclonal antibody production.

Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification.

Acanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105 A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea.

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

Effect of chlorine dioxide in reduction of harmful microbes on fermented hot pepper paste.

In this study, the in vitro effects of chlorine dioxide (ClO2) in growth reduction against Candia glaebosa, Zygosaccharomyces bisporus, Saccharomycopsis capsularis and Pichia pastoris involving in deterioration of fermented hot pepper paste were studied to assess the applicability of chlorine dioxide to preparation of fermented hot pepper paste, and the concentration of ClO2 required for destruction of harmful microorganisms through the fumigation of fermented hot pepper paste was evaluated. ClO2 was treated by using ClO2 generator for 15 min. C. glaebosa, Z. bisporus and S. capsularis were reduced by ClO2 concentration dependent and not detected by ClO2 over 10 ppmV, whereas the P. pastoris was significantly perished by the treatment of ClO2 over 30 ppmV. We suggest that the ClO2 fumigation in stages of the preparation, disintegration, and fermentation of the paste made of fermented hot pepper might be useful for control of harmful microbes therein.

Report: Effect of macrophage alone or primed with cytokines on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells in vitro.

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated <90% binding and <70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization.

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens.

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® fresh™, FreshKon® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection.

Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae.

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

Infection status of endoparasites in foreigner workers living in Cheonan City, Chungnam Province, Korea.

At present, more than 500,000 foreigner workers, most of them from Asian countries with high parasitic infection rates, are working in Korea. Since investigation into the prevalence of parasitic infections in foreigner workers has not yet been conducted in Korea, the present study was performed to determine the parasitic infection status of foreigner workers living in Cheonan City, Chungcheongnam-do (Chungnam Province) and to plan, on that basis, effective control measures. From October to December 2013, the parasitic infection status of 231 foreigner workers employed at selected Cheonan-si small businesses was investigated by both stool examination and ELISA. A total of 60 individuals (26.0%) were found to be infected with parasites. The stool examination detected 14 positive cases (6.1%), and ELISA revealed 50 positive people (21.6%), for at least a kind of parasitic disease. The most common infection was cysticercosis (8.7%), followed by toxocariasis (7.8%) and clonorchiasis (7.4%). Since it was proved that parasitic infections were prevalent among foreigner workers living in Cheonan City, more comprehensive study is urgently needed in order to understand the nationwide status of parasitic infections in foreigner workers.

Technical approaches of a natural dye extracted from Phytolacca americana L.-berries with chemical mordants.

Phytolacca americana L. is a large semi-succulent herbaceous plant which reaches three meters in height. It is native to eastern North America, the Midwest, and the Gulf Coast, with more scattered populations in the far West. It is imported into Korea and has been frequently used as a traditional natural drug for diseases such as systemic edema and nephritis. Its berries, that is, fruits are shiny dark purple held in racemous clusters on pink pedicels with a pink peduncle. They are round with a flat indented top and bottom. Immature berries are green, maturing into white and then blackish purple. It is not well known how the berries are used for a natural staining yet. In this study, using Phytolacca americana L.-berries, a natural staining was analyzed. Moreover, due to the broad use of chemical mordants, five different mordants including copper acetate, aluminum potassium sulfate, sodium tartrate plus citric acid, Iron II sulfate and potassium dichromate were combined. Extracted dye from the berries stained silk fabrics with ivory. The original purple color from the berries disappeared and transformed into ivory. Although the silk fabrics were differentially stained by the berries that were combined with mordants of aluminum potassium sulfate, sodium tartrate plus citric acid and potassium dichromate, only differences in lightness and darkness were observed. Interestingly, the combination of the dye from the berries with a mordant of copper acetate and Iron II sulfate induced the staining of the silk fabrics into khaki and dark khaki, respectively. This study is the first systemic report on staining silk fabrics with Phytolacca americana L.-berries and chemical mordants and suggests application of natural products to the fiber industry.

In vitro assessment of cytokines interactions with Balamuthia mandrillaris using human brain microvascular endothelial cells.

Balamuthia amoebic encephalitis (BAE) is a life threatening human disease which, always lead to death. Amoebae invasion of the bloodstream is considered an important step in BAE followed by their haematogenous spread. It is more likely that Balamuthia mandrillaris enters into the central nervous system through blood-brain barrier (BBB) sites. The objective of the present study was to determine the impact of cytokines on biological properties of Balamuthia in vitro. Human brain microvascular endothelial cells (HBMEC), which constitutes the BBB were used in vitro test model for the present investigation. It was observed that Balamuthia exhibited >90 % binding and >70% cytotoxicity to HBMEC. However, cytokines did not affect amoebic binding and cytotoxicity except lipopolysaccharide (LPS) which reduced Balamuthia-mediated HBMEC cytotoxicity. It is also important to note that amoebic numbers were reduced in the presence of LPS within 24 h. We have shown previously the bacterial uptake by Balamuthia is very limited which is further investigated in the presence of cytokines and observed a slight reduction of bacterial uptake during phagocytosis assay. Zymography assays revealed there is no effect of cytokines on proteolytic activity of Balamuthia. Overall we described for the first time that cytokines has no inhibitory effects on biological properties of Balamuthia in vitro.

Isolation and molecular characterization of potentially pathogenic Acanthamoeba genotypes from diverse water resources including household drinking water from Khyber Pakhtunkhwa, Pakistan.

Acanthamoeba, an opportunistic protozoan pathogen, is ubiquitous in nature, and therefore plays a predatory role and helps control microbial communities in the ecosystem. These Acanthamoeba species are recognized as opportunistic human pathogens that may cause blinding keratitis and rare but fatal granulomatous encephalitis. To date, there is not a single report demonstrating Acanthamoeba isolation and identification from environmental sources in Pakistan, and that is the aim of this study. Acanthamoeba were identified by morphological characteristics of their cysts on non-nutrient agar plates seeded with Escherichia coli. Additionally, the polymerase chain reaction (PCR) was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Furthermore, our PCR and sequencing results confirmed seven different pathogenic and nonpathogenic genotypes, including T2-T10, T4, T5, T7, T15, T16, and T17. To the best of our knowledge, we have identified and isolated Acanthamoeba sp., for the first time, from water resources of Khyber Pakhtunkhwa, Pakistan. There is an urgent need to address (1) the pathogenic potential of the identified genotypes and (2) explore other environmental sources from the country to examine the water quality and the current status of Acanthamoeba species in Pakistan, which may be a potential threat for public health across the country.

Evaluation of inhibitory potential of some selective methanolic plants extracts on biological characteristics of Acanthamoeba castellanii using human corneal epithelial cells in vitro.

Acanthamoeba is an opportunistic protozoan pathogen and known to be one of the most ubiquitous organisms, play a vital role in ecosystem, and recognized to cause blinding keratitis and rare but fatal granulomatous encephalitis involving the central nervous system with a very poor prognosis. This is due to limited availability of effective anti-Acanthamoeba drugs. The objective of the present study was to determine the efficacy of methanolic plants crude extracts on the viability and biological properties of Acanthamoeba castellanii (T4 genotype) and its cytotoxic effects on human corneal epithelial cells (HCEC). Using HCEC, it was observed that Acanthamoeba exhibited binding (>90 %) and cytotoxicity (>80 %) to host cells. However, plant crude extracts remarkably inhibited more than 70 and 60 % of Acanthamoeba binding and cytotoxicity to HCEC, respectively. It was further established that crude extracts (ranging from 0.1 to 1.5 mg/ml) exhibited amoebicidal effects, i.e., >50 % of trophozoites were killed/reduced at maximum dose (1.5 mg/ml) within 1 h incubation. However, the residual subpopulation remained static over longer incubations. Furthermore, growth assay demonstrated crude extracts inhibited >50 % Acanthamoeba numbers up to 7 days. Our results confirmed that plant crude extracts has inhibitory effects on Acanthamoeba growth and viability. Overall, these findings revealed that tested plant extracts is inhibitory to Acanthamoeba properties associated with pathogenesis. To the best of our knowledge, our findings demonstrated for the first time that selected methanol plant crude extracts exhibits inhibitory effects on biological properties of Acanthamoeba without any toxic effects on HCEC cells in vitro.

Association between the SPRY1 gene polymorphism and obesity-related traits and osteoporosis in Korean women.

BACKGROUND: Emerging evidence has revealed a close relationship between obesity and osteoporosis. It was reported recently that conditional knockout of the Spry1 gene in mice adipocytes causes an increase in body fat and a decrease in bone mass, and that these phenotypes are rescued by Spry1 overexpression in adipose tissue. In this study, we investigated whether genetic variation in the human SPRY1 gene is associated with obesity-related phenotypes and/or osteoporosis in humans. METHODS: We performed a candidate gene association analysis between the four single nucleotide polymorphisms (SNPs) and 14 imputed SNPs in the SPRY1 gene and obesity-related traits and osteoporosis in a Korean women cohort (3013 subjects). RESULTS: All four SPRY1 gene SNPs were significantly associated with either obesity-related traits or osteoporosis. The TGCC haplotype in the SRPY1 gene showed simultaneous association with an increased risk for obesity-related traits, percentage body fat (p=0.0087) and percentage abdominal fat (p=0.047), and osteoporosis (odds ratio=1.50; p=0.025) in the recessive genetic model. CONCLUSIONS: Our results support a previous finding in conditional Spry1 gene knockout mice and suggest that the SPRY1 gene is an important genetic factor for determining the risk of both obesity and osteoporosis in humans.

Effects of mannose on pathogenesis of Acanthamoeba castellanii.

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.

Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii.

Acanthamoeba castellanii is a single-celled protozoan that is widely distributed in the environment and is a well-known of causing human keratitis, a vision-threatening infection. In this study, an ethyl methane sulfonate (EMS) and a selection of saccharide were applied to A. castellanii by chemical mutagenesis. To understand the functional roles of a mannose-binding protein (MBP). A. castellanii were treated with methyl-alpha-D-mannopyranoside abbreviated Man, with and without the EMS pre-treatment, and their adhesion and cytotoxicity were analyzed, using a human brain microvascular endothelial cell (HBMEC) as the target cell. Both EMS and Man mutants exhibited significantly decreased levels of MBP expression and cytotoxicity to HBMEC, but showed similar levels of binding to HBMEC, as compared with the wild type. Of interest was that the exogenous mannose inhibited amoebae (i.e., Man mutant) binding to the HBMEC by <20%. Only the mutant Man exhibited a significant decrease in bacterial uptake, as compared to the wild type, 0.020 vs 0.032 (p<0.05) and proteolytic activity. The results showed that MBP should be clearly provided as the pathogenic target candidate, to further target-based therapy, but EMS mutation should not be associated with initial adhesion and phagocytosis of A. castellanii.

Simple and efficient model systems of screening anti-Toxoplasma drugs in vitro.

INTRODUCTION: A lot of in vitro technologies have been developed to screen drugs for toxoplasmosis, which is caused by Toxoplasma gondii and is one of the most serious infectious diseases in the world. However, developed screening methods still have limitation such as inaccuracy, labor-intensive and time-consuming procedure. Therefore, the development of simpler, more efficient and accurate high-throughput screening assay is needed. AREAS COVERED: The present review gives the overview of in vitro screening technologies described in literatures so far including morphological assay, incorporation of [(3)H]uracil assay, enzyme-linked immunosorbent assay (ELISA), colorimetric microtiter assay (ß-galactosidase assay), flow cytometric quantification assay, yellow fluorescent protein assay and cell viability assay. The authors discuss how these methods are efficient and/or limited for screening anti-T. gondii drugs. The authors further suggest brand-new technologies which are faster, simpler, more effective and available for high-throughput screening. EXPERT OPINION: Options for clinical treatment of toxoplasmosis are currently very limited. Thus, more accurate in vitro screening methods must be established to identify the most effective anti-T. gondii drugs from random screening of compounds. At the same time, based on genome information, combination of an appropriate screening technology, combinatorial chemistry and computational biology may increase the efficiency of target-based drug discovery against T. gondii.

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

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 (51)Cr 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-1ß, 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.