RESUMO
The gene nfa1 was isolated from the free-living pathogenic amoeba Naegleria fowleri. The protein Nfa1 is located in pseudopodia and specifically in food-cups. It is also involved in cytotoxicity. In this study, we used synthetic small interfering RNAs (siRNA) to examine the effects of nfa1 down-regulation. We observed the expression of nfa1 mRNA and Nfa1 protein using Northern and Western blots. We also examined the effects of nfa1 down-regulation on the in vitro cytotoxicity of N. fowleri. Four synthetic siRNAs were constructed, and of those, sinfa1-1 showed the highest down-regulation of an nfa1 mRNA and Nfa1 protein by 70 and 43%, respectively. In order to achieve long-lasting silencing of the transfected genes, we constructed two vectors which were pAct/SAGAH and pAct/asnfa1AGAH cloned with the sinfa1-1 and an antisense RNA to the nfa1 gene. In N. fowleri transfected with pAct/SAGAH, FACS revealed a 60 and 57% reduction in nfa1 mRNA and Nfa1 protein levels, respectively. To determine whether the Nfa1 proteins were related with in vitro cytotoxicity, LDH assays were used and showed that the cytotoxicity of these transfectants to macrophages was reduced by 26.4 and 36.2% at 17 and 24h, respectively. Moreover, after transfection with pAct/asnfa1AGAH, amoebic cytotoxicity decreased by 8.2 and 10% at 17 and at 24h, respectively. This is the first report to show the RNA interference in N. folweri trophozoites and also demonstrate the Nfa1 function in vitro for its cytotoxicity.
Assuntos
Inativação Gênica , Naegleria fowleri/genética , Naegleria fowleri/patogenicidade , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Sequência de Bases , Regulação para Baixo , Regulação da Expressão Gênica , Macrófagos/parasitologia , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transfecção , Trofozoítos/metabolismoRESUMO
Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.
Assuntos
Amebíase/tratamento farmacológico , Amebicidas/uso terapêutico , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Miocamicina/análogos & derivados , Naegleria fowleri , Amebíase/microbiologia , Amebicidas/farmacologia , Animais , Antibacterianos/uso terapêutico , Nitrogênio da Ureia Sanguínea , Infecções Protozoárias do Sistema Nervoso Central/microbiologia , Feminino , Rim/microbiologia , Rim/patologia , L-Lactato Desidrogenase/metabolismo , Fígado/microbiologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Miocamicina/farmacologia , Miocamicina/uso terapêutico , Naegleria fowleri/efeitos dos fármacos , Análise de SobrevidaRESUMO
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 microg/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.
Assuntos
Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Clorpromazina/farmacologia , Naegleria fowleri/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Amebíase/parasitologia , Amebíase/patologia , Anfotericina B/farmacologia , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/patologia , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Naegleria fowleri/patogenicidade , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologiaRESUMO
Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4+/-3.1% and 52+/-2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.
Assuntos
Antígenos de Protozoários/genética , Naegleria fowleri/genética , Proteínas de Protozoários/genética , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/genética , Animais , Antígenos de Protozoários/fisiologia , Northern Blotting , Southern Blotting , Western Blotting , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica , Proteínas de Protozoários/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Cellular adhesion through beta 2-integrin (CD18) is an important step in signal transduction leading to apoptosis of human neutrophils, and NADPH oxidase-derived reactive oxygen species (ROS) are essential for neutrophil apoptosis induced by Entamoeba histolytica. Therefore, we investigated the role of beta 2-integrin-mediated signals in ROS-dependent neutrophil apoptosis induced by E. histolytica. Entamoeba-induced apoptosis was inhibited by pre-incubation of cells with mAb to CD18, but not CD29, suggesting that beta )-integrin plays an important role in this response. Moreover, Entamoeba-induced ROS generation in neutrophils was inhibited by mAbs against CD18 or CD11b, but not by mAbs against CD11a, CD11c, or CD29. A combination of d-galactose plus anti-CD18 mAb had a larger inhibitory effect than d-galactose alone on Entamoeba-induced apoptosis and ROS generation. Furthermore, Entamoeba-induced apoptosis and ROS generation were inhibited by pre-treatment of cells with an inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase). These results indicate that beta 2-integrin and PI-3-kinase are crucial signaling molecules in ROS-dependent apoptosis of neutrophils induced by E. histolytica.
Assuntos
Apoptose , Antígenos CD18/fisiologia , Entamoeba histolytica/fisiologia , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Galactose/farmacologia , Humanos , Neutrófilos/citologia , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
A gene encoding a cytosolic heat shock protein 70 from pathogenic Naegleria fowleri (Nf-cHSP70) was identified. The Nf-cHSP70 was 2,062 bp in length with an open reading frame of 1,980 bp encoding 659 amino acid residues. The deduced amino acid sequence of the gene shared high sequence identities with HSP70s from other parasitic organisms and mammals. The characteristic domains, including N-terminal ATPase domain, calmodulin-binding domain, and EE(D)VD motif, found in HSP70s were also well conserved in this gene. The recombinant Nf-cHSP70 protein showed strong antigenicity against the sera from mice experimentally infected with N. fowleri. Immunofluorescence assay showed that Nf-cHSP70 localized in cytosol of the parasite. The results from semi-quantitative RT-PCR and Western blot analyses demonstrated the expression levels of gene transcripts, and its products were significantly increased at high temperature (42 degrees C). The definitive biological roles of Nf-cHSP70 are not clear, but it may protect the parasite under environmental changes especially high temperature.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Naegleria fowleri/genética , Proteínas de Protozoários/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Citosol/química , DNA de Protozoário/química , DNA de Protozoário/genética , Regulação da Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Fases de Leitura Aberta , Estrutura Terciária de Proteína/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.
Assuntos
Acanthamoeba/genética , Acanthamoeba/patogenicidade , Amebíase/parasitologia , DNA de Protozoário/fisiologia , Regulação da Expressão Gênica , Amebíase/mortalidade , Animais , Northern Blotting/métodos , Encéfalo/parasitologia , Clonagem Molecular/métodos , DNA Complementar/biossíntese , DNA de Protozoário/biossíntese , Perfilação da Expressão Gênica/métodos , Genes de Protozoários/genética , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Inoculações Seriadas , Regulação para Cima , Virulência/genéticaRESUMO
To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.
Assuntos
Antígenos de Protozoários/biossíntese , Regulação da Expressão Gênica/fisiologia , Naegleria/genética , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/biossíntese , Actinas/genética , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Sequência de Bases , Western Blotting , Células CHO , Cricetinae , Cricetulus , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Dados de Sequência Molecular , Naegleria/metabolismo , Plasmídeos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5' upstream regions, the nfa1 open reading frame, and 3' downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.
Assuntos
Antígenos de Protozoários/genética , Naegleria fowleri/genética , Naegleria fowleri/patogenicidade , Proteínas de Protozoários/genética , Regiões 3' não Traduzidas/genética , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/fisiologia , Células CHO , Sobrevivência Celular , Cricetinae , Reação em Cadeia da Polimerase , Proteínas de Protozoários/análise , Proteínas de Protozoários/fisiologia , TransfecçãoRESUMO
The extracellular tissue penetrating protozoan parasite Entamoeba histolytica has been known to induce host cell apoptosis. However, the intracellular signaling mechanism used by the parasite to trigger apoptosis is poorly understood. In this study, we investigated the roles of reactive oxygen species (ROS), and of MAPKs in the Entamoeba-induced apoptosis of human neutrophils. The neutrophils incubated with live trophozoites of E. histolytica revealed a marked increase of receptor shedding of CD16 as well as phosphatidylserine (PS) externalization on the cell surface. The Entamoeba-induced apoptosis was effectively blocked by pretreatment of cells with diphenyleneiodonium chloride (DPI), a flavoprotein inhibitor of NADPH oxidase. A large amount of intracellular ROS was detected after exposure to viable trophozoites, and the treatment with DPI strongly inhibited the Entamoeba-induced ROS generation. However, a mitochondrial inhibitor rotenone did not attenuate the Entamoeba-induced ROS generation and apoptosis. Although E. histolytica strongly induced activation of ERK1/2 and p38 MAPK in neutrophils, the activation of ERK1/2 was closely associated with ROS-mediated apoptosis. Pretreatment of neutrophils with MEK1 inhibitor PD98059, but not p38 MAPK inhibitor SB202190, prevented Entamoeba-induced apoptosis. Moreover, DPI almost completely inhibited Entamoeba-induced phosphorylation of ERK1/2, but not phosphorylation of p38 MAPK. These results strongly suggest that NADPH oxidase-derived ROS-mediated activation of ERK1/2 is required for the Entamoeba-induced neutrophil apoptosis.
Assuntos
Apoptose , Entamoeba histolytica/patogenicidade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Entamebíase , Ativação Enzimática , Humanos , Sistema de Sinalização das MAP Quinases , Neutrófilos/patologia , FosforilaçãoRESUMO
Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.
Assuntos
Apoptose/fisiologia , Entamoeba histolytica/fisiologia , Neutrófilos/ultraestrutura , Animais , Interações Hospedeiro-Parasita/fisiologia , Humanos , Técnicas In Vitro , Neutrófilos/fisiologiaRESUMO
Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.
Assuntos
Acanthamoeba , Amebíase/parasitologia , Naegleria , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/imunologia , Acanthamoeba/patogenicidade , Amebíase/diagnóstico , Amebíase/epidemiologia , Amebíase/terapia , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , DNA Mitocondrial/análise , DNA de Protozoário/análise , Coreia (Geográfico)/epidemiologia , Estágios do Ciclo de Vida , Naegleria/classificação , Naegleria/genética , Naegleria/imunologia , Naegleria/patogenicidade , Filogenia , Polimorfismo de Fragmento de Restrição , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Virulência/genéticaRESUMO
Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin alpha(5)/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin.
Assuntos
Fibronectinas/metabolismo , Integrinas/metabolismo , Naegleria fowleri/fisiologia , Naegleria fowleri/patogenicidade , Proteína Quinase C/metabolismo , Animais , Células CHO , Adesão Celular , Cricetinae , Regulação da Expressão Gênica , Transdução de SinaisAssuntos
Antígenos de Protozoários/genética , Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , Naegleria fowleri/genética , Naegleria fowleri/imunologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , DNA Complementar , DNA de Protozoário/química , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The nfa1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polyclonal antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1:100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dose-dependent manner.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Naegleria fowleri/patogenicidade , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/genética , Células CHO , Cricetinae , Relação Dose-Resposta Imunológica , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Naegleria fowleri/crescimento & desenvolvimento , Naegleria fowleri/imunologia , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologiaRESUMO
A new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Acanthamoeba sp. YM-4 (Korean isolate YM-4). The trophozoites were 11.0-23.0 micrometer in length and had hyaline filamentous projections. Cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Acanthamoeba YM-4 can survive at 40 degrees C, and its generation time was 19.6 hr, which was longer than that of A. culbertsoni. In terms of the in vitro cytotoxicity of lysates, Acanthamoeba YM-4 was weaker than A. culbertsoni, but stronger than A. polyphaga. On the basis of the mortality of experimentally infected mice, Acanthamoeba YM-4 was found to be highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. An anti-Acanthamoeba YM-4 monoclonal antibody, McAY7, was found to react only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster on the basis of phylogenetic distances. Thus the Acanthamoeba Korean isolate YM-4 was identified as a new species, and assigned as Acanthamoeba sohi.
Assuntos
Acanthamoeba/classificação , Acanthamoeba/patogenicidade , Amebíase/veterinária , Doenças dos Peixes/parasitologia , Carpa Dourada/parasitologia , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Amebíase/parasitologia , Animais , DNA Mitocondrial/análise , DNA de Protozoário/análise , Brânquias/parasitologia , Coreia (Geográfico) , Camundongos , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , VirulênciaRESUMO
We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Citotoxicidade Imunológica , Naegleria fowleri/imunologia , Proteínas de Protozoários/imunologia , Amebíase/imunologia , Animais , Antígenos de Protozoários/genética , Western Blotting , Encéfalo/microbiologia , Células CHO , Clonagem Molecular , Técnicas de Cocultura , Cricetinae , Testes Imunológicos de Citotoxicidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Naegleria fowleri/ultraestrutura , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologiaRESUMO
Since the Gordius worm is a parasite of crickets and several arthropods, cases of humans infected with this worm have been rare and accidental. A Gordius worm was obtained from a three-year-old girl who consulted a local clinic in Gwangju, Kyunggi-do, Korea. She lived in a rural area, and had eaten an insect that looked like a cricket. She expelled the worm in vomitus 15 minutes later; in fact, she expelled two worms, but one was discarded. The worm had a grayish white color and an intact outer surface. It was 16 cm in length and 0.6 cm wide. The posterior end of the worm was spirally enrolled and furcated into two caudal lobes, which were nearly cylindrical but showed a somewhat concave medio- ventral surface. The cloacal aperture was round and situated anterior to the point of bifurcation of the lobes. The cloacal aperture was encircled by a dark ring, which was a little removed from the aperture. The crescent fold was reddish brown, and no hairs were noticed over the entire body surface. The worm had the morphological features of a male Gordius. Accidental human cases involving the Gordius worm are rare and this is the first such case in Korea.
Assuntos
Helmintos , Vômito/parasitologia , Administração Oral , Animais , Pré-Escolar , Feminino , Gryllidae/parasitologia , Humanos , MasculinoRESUMO
We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.
Assuntos
DNA Mitocondrial/genética , DNA Espaçador Ribossômico/genética , Diploide , Complexo IV da Cadeia de Transporte de Elétrons/genética , Paragonimus/genética , Filogenia , Poliploidia , Animais , Evolução Molecular , Genes de Helmintos/genética , Coreia (Geográfico)RESUMO
Total of 7,495 children including 3,908 boys and 3,587 girls from a kindergarten and 15 primary schools were examined for head lice infestation (HLI). The overall prevalence of HLI in this study was found to be 5.8%. Head lice were much more commonly detected in girls than in boys with prevalence of 11.2% and 0.9%, respectively. Sixty-nine children with HLI were treated with 1% lindane shampoo alone (group 1), and 45 children with HLI were treated with 1% lindane shampoo and oral trimethoprim/sulfamethoxazole (group 2), and follow-up visits were conducted 2 and 4 weeks later. The children who still had HLI 2 weeks after the primary treatment were treated again. At the 2-week follow-up visit, the treatment success rates of groups 1 and 2 were 76.8% and 86.7%, respectively, and at the 4-week follow-up visit, the rates were 91.3% and 97.8%, respectively. No statistically significant synergistic effect was observed for the combination of a 1% lindane shampoo and oral trimethoprim/sulfamethoxazole.