Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Cellular characterization of actin gene concerned with contact-dependent mechanisms in Naegleria fowleri.

Free-living amoeba, Naegleria fowleri, destroys target cells through contact-dependent mechanisms, such as phagocytosis and/or trogocytosis. A previous experiment showed that the nf-actin gene consisted of 1.2 kbp, produced a 50.1 kDa recombinant protein (Nf-actin), and was localized on the cytoskeleton, pseudopodia and amoebastome. In this study, cellular characterization of the nf-actin gene concerned with contact-dependent mechanisms in N fowleri was performed. The nf-actin gene was amplified from a gene-cloned vector, pEXQP5-T7/NT TOPO. The nf-actin gene was introduced into the Ubi-pEGFP-C2 vector, and Ubi-pEGFP-C2/nf-actin was transfected into N fowleri trophozoites. Strong GFP fluorescence was detected in N fowleri trophozoites transfected with Ubi-pEGFP-C2/nf-actin. Expression of EGFP-Nf-actin protein was detected by Western blot analysis. The nf-actin-overexpressing N fowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild-type N fowleri. Moreover, nf-actin-overexpressing N fowleri showed increased phagocytic activity and cytotoxicity in comparison with wild-type N fowleri. In summary, the overexpressed nf-actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by N fowleri.

Fluorescent Immunosorbent Assay for Chikungunya Virus Detection.

BACKGROUND: When infected with the chikungunya virus (CHIKV), 3% to 28% of CHIKV-infected individuals remain asymptomatic, necessitating the development of improved high-throughput screening methods to overcome the limitations of molecular diagnostics or enzyme-linked immunosorbent assays (ELISAs). OBJECTIVE: In this study, two novel monoclonal antibodies (mAbs) targeting envelope 1 (E1) of CHIKV were developed and applied in a fluorescence-linked immunosorbent assay (FLISA) using coumarin-derived dendrimer as the fluorophore. METHODS: The performance of the FLISA was compared with that of ELISA. RESULTS: Using the two novel mAbs (2B5 and 2C8), FLISA could detect 1 × 105 PFU/mL of CHIKV, exhibiting a 2-fold lower limit of detection (LOD) compared to ELISA. The LOD of FICT corresponded to a comparative threshold value of 23.95 and 4 × 106 of RNA copy number/µL. In the presence of human sera and blood, virus detection by FLISA was 3-fold better than ELISA, with an LOD of 2 × 105 PFU/mL. Sera and blood interfered with the ELISA, resulting in 6 × 105 PFU/mL as the LOD. CONCLUSIONS: FLISA using two novel mAbs and coumarin-derived dendrimer is a superior diagnostic assay for detecting CHIKV in human sera and blood, compared to conventional ELISA.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM.

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.

Development of Monoclonal Antibodies for Diagnosis of Plasmodium vivax.

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri.

Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were cocultured with N. fowleri trophozoites in a noncontact system, and consequently, interleukin-1ß (IL-1ß) production was estimated. Caspase-1 activation and IL-1ß production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1ß production via the NLRP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), and N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1ß production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1ß, thus resulting in inflammation.

Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing.

Naegleria fowleri is a pathogenic free-living amoeba, commonly found around the world in warm, fresh water and soil. N. fowleri trophozoites can infect humans by entering the brain through the nose and causing usually fatal primary amebic meningoencephalitis (PAM). Trophozoites can encyst to survive under unfavorable conditions such as cold temperature, starvation, and desiccation. Recent technological advances in genomics and bioinformatics have provided unique opportunities for the identification and pre-validation of pathogen-related and environmental resistance through improved understanding of the biology of pathogenic N. fowleri trophozoites and cysts at a molecular level. However, genomic and transcriptomic data on differential expression genes (DEGs) between trophozoites and cysts of N. fowleri are very limited. Here, we report transcriptome Illumina RNA sequencing (RNA-seq) for N. fowleri trophozoites and cysts and de novo transcriptome assembly. RNA-seq libraries were generated from RNA extracted from N. fowleri sampled from cysts, and a reference transcriptome was generated through the assembly of trophozoite data. In the database, the assembly procedure resulted in 42,220 contigs with a mean length of 11,254 nucleotides and a C+G content of 37.21%. RNA sequencing showed that 146 genes in cysts of N. fowleri indicated 2-fold upregulation in comparison with trophozoites of N. fowleri, and 163 genes were downregulated; these genes were found to participate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The KEGG pathway included metabolic (131 sequences) and genetic information processing (66 sequences), cellular processing (43 sequences), environmental information processing (22 sequences), and organismal system (20 sequences) pathways. On the other hand, an analysis of 11,254 sequences via the Gene Ontology database showed that their annotations contained 1069 biological processes including the cellular process (228 sequences) and metabolic process (214 sequences); 923 cellular components including cells (240 sequences) and cell parts (225 sequences); and 415 molecular functions including catalytic activities (195 sequences) and binding processes (186 sequences). Differential expression levels increased in cysts of N. fowleri compared to trophozoites of N. fowleri, which were mainly categorized as serine/threonine protease, kinase, and lipid metabolism-related proteins. These results may provide new insights into pathogen-related genes or environment-resistant genes in the pathogenesis of N. fowleri.

Evaluating the Diagnostic Potential of Chorismate Mutase Poly-Clonal Peptide Antibody for the Acanthamoeba Keratitis in an Animal Model.

Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis (AK), a vision-threatening parasitic disease whose primary risk factor has been attributed to poor contact lens hygiene. Unfortunately, differential diagnosis of AK is challenging as the clinical manifestations for AK are similar to those of bacterial, fungal, or even viral keratitis. Since delayed AK diagnosis can incur permanent vision impairment, a rapid and sensitive diagnostic method is urgently needed. Here, the diagnostic potential of polyclonal antibodies targeting the chorismate mutase (CM) of Acanthamoeba spp. was evaluated in AK animal models. CM antibody specificity against Acanthamoeba trophozoites and cysts was confirmed by immunocytochemistry after co-culturing Acanthamoeba with Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus, and human corneal epithelial (HCE) cells. Enzyme-linked immunosorbent assay (ELISA) was performed using CM-specific immune sera raised in rabbits, which demonstrated that the antibodies specifically interacted with the Acanthamoeba trophozoites and cysts in a dose-dependent manner. To evaluate the diagnostic potential of the CM antibody, AK animal models were established by incubating contact lenses with an inoculum containing A. castellanii trophozoites and subsequently overlaying these lenses onto the corneas of BALB/c mice for 7 and 21 days. The CM antibody specifically detected Acanthamoeba antigens in the murine lacrimal and eyeball tissue lysates at both time points. Our findings underscore the importance of antibody-based AK diagnosis, which could enable early and differential AK diagnosis in clinical settings.

A Study on the Monitoring of Toxocara spp. in Various Children's Play Facilities in the Republic of Korea (2016-2021).

Toxocara spp. is a zoonotic soil-transmitted parasite that infects canids and felids, which causes toxocariasis in humans, migrating to organ systems, including the lungs, the ocular system, and the central nervous system. Since Toxocara spp. is usually transmitted through soil, children tend to be more susceptible to infection. In order to monitor contamination with Toxocara spp. in children's play facilities in the Republic of Korea, we investigated 11,429 samples of soil from daycare centers, kindergartens, elementary schools, and parks across the country from January 2016 to December 2021. Since the Environmental Health Act in the Republic of Korea was enacted in March 2008, there have been sporadic reports of contamination by Toxocara spp. in children's activity zones. In this study, soil from children's play facilities in regions across the Republic of Korea was monitored according to the Korean standardized procedure to use it as basic data for preventive management and public health promotion. The national average positive rate was 0.16% (18/11,429), and Seoul showed a higher rate of 0.63% (2/318) than any other regions while Incheon, Daegu, Ulsan, Kangwon-do, Jeollabuk-do, and Jeollanam-do were negative (p < 0.05). The positive rates were as follows: 0.37% (4/1089) in daycare centers, 0.13% (3/2365) in kindergartens, 0.2% (7/4193) in elementary schools, 0.09% (1/1143) in apartments, and 0.14% (3/2198) in parks. In addition, it was confirmed that 0.2% (1/498) of elementary schools and 1.17% (2/171) of parks were re-contaminated among play facilities managed with the establishment of a regular inspection cycle. Consequently, there is an essential need for continuous monitoring of Toxocara spp. contamination and regular education for preschool and school children in order to prevent soil-borne parasite infections.

Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

Production of monoclonal antibodies for Plasmodium vivax lactate dehydrogenase and patient sera screening using sandwich ELISA.

Malaria is a worldwide infectious disease. There are many diagnostic kits to detect malaria infection. However, the sensitivity of these diagnostic kits remains a problem. To develop a diagnostic kit for malaria that has high sensitivity, it is necessary to produce monoclonal antibodies (McAbs) with high affinity. The present study was undertaken to produce hybridoma cells that can be used to generate McAbs with high affinity and specificity against Plasmodium vivax lactate dehydrogenase (pvLDH). In this study, BALB/c mice were immunized with purified recombinant polypeptides that encode pvLDH. McAbs against pvLDH were produced according to the protocol of hybridoma technique using myeloma cells (SP2/0 cell lines). The McAbs were characterized by isotyping and by Western blot analysis. Two McAbs (D2H and D7E) against pvLDH antigen were obtained. The isotypes of D2H and D7E were IgG2b. They recognize 33 kDa proteins that were defined as pvLDH by Western blot analysis. In the affinity test, D2H and D7E showed positively optical density value until each McAbs were serially diluted at concentrations of 0.156 and 0.078 µg/ml, respectively. To evaluate sensitivity and specificity against clinical specimens of P. vivax, purified McAbs were tested with alkaline phosphatase-conjugated monoclonal antibodies and blood samples (n = 180) of P. vivax patients using the sandwich enzyme-linked immunosorbent assay, showing the 98% sensitivity. We suggest that McAbs produced in this study may be used for developing efficient and rapid diagnostic kits.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Detection of Acanthamoeba from Acanthamoeba Keratitis Mouse Model Using Acanthamoeba-Specific Antibodies.

Although the prevalence of Acanthamoeba keratitis (AK) is rare, its incidence in contact lens wearers has increased. Acanthamoeba infections can lead to the loss of vision if the diagnosis and treatment are delayed. In this study, we investigated the diagnostic potential of two antibodies raised against the adenylyl cyclase-associated protein (ACAP) and periplasmic binding protein (PBP) of A. castellanii in the AK mouse model. The specificity of ACAP and PBP antibodies to Acanthamoeba was confirmed by immunocytochemistry. AK mouse models were produced by corneal infections with A. castellanii trophozoites for 7 days and 21 days. Enzyme-linked immunosorbent assay results revealed that both ACAP and PBP antibodies successfully detected Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. The detection levels of Acanthamoeba antigens were similar at both infection time points. Anti-Acanthamoeba IgG, IgA, and IgM antibodies were evaluated from the sera of the AK mouse model. Notably, IgM and IgA antibody responses were highest and lowest at both time points, respectively. Our findings revealed that both ACAP and PBP antibodies could detect Acanthamoeba antigens in the tears and eyeball lysates of the AK mouse model. These results provide important information for understanding Acanthamoeba infections and developing a new diagnostic tool for AK.

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.

A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts.

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.