Cytopathic changes in rat microglial cells induced by pathogenic Acanthamoeba culbertsoni: morphology and cytokine release.

To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-alpha secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1beta secreted from microglial cells cocultured with A. culbertsoni trophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-alpha and IL-1beta from microglial cells.

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea.

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri.

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies

In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay.

In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml lysate treatments, respectively. Acanthamoeba culbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. castellanii and A. hatchetti which showed 83.6% and 75.5% of cytotoxicity. Acanthamoeba royreba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml lysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.

Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp.

To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.

Pathogenicity of Korean isolates of Acanthamoeba by observing the experimental infection and zymodemes of five isoenzymes.

To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity against target cells and isoenzyme band patterns were observed. Five isolates of Acanthamoeba spp. (YM-2, YM-3, YM-4, YM-5, and YM-7) were used in this study as well as three reference Acanthamoeba spp. (A. culbertsoni, A. hatchetti, and A. royreba). According to the mortality rate of infected mice, Korean isolates could be categorized into three groups high virulent (YM-4), low virulent (YM-2, YM-5, YM-7) and the nonpathogenic group (YM-3). In addition, the virulence of Acanthamoeba spp. was enhanced by brain passage in mice. In the cytotoxicity assay against chinese hamster ovary cells, especially, the cytotoxicity of brain-passaged amoebae was relatively higher than the long-term cultivated ones. The zymodeme patterns of glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), hexokinase (HK), glutamate oxaloacetate transaminase (GOT) and malic enzyme (ME) of Acanthamoeba spp. were different among each isolate, and also between long-term cultured amoebae and brain passaged ones. In spite of the polymorphic zymodemes, a slow band of G6PD and HK, and an intermediate band of MDH were only observed in pathogenic Acanthamoeba spp., which should be used as isoenzymatic makers.

Isoenzyme patterns and phylogenetic relationships in Acanthamoeba spp. isolated from contact lens containers in Korea.

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A, hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. polyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.

Upregulated expression of the cDNA fragment possibly related to the virulence of Acanthamoeba culbertsoni.

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display-polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC #289 and the oligo dT11-C primer, revealed the highest homology (49.8%) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.

Synergy between Entamoeba histolytica and Escherichia coli in the induction of cytokine gene expression in human colon epithelial cells.

Entamoeba histolytica induces the expression and secretion of interleukin 8 (IL-8) by cultured colon epithelial cells. We assessed the array of cytokine genes expressed by human colon epithelial cells in response to co-culture with E. histolytica trophozoites and tested the hypothesis that enteric bacteria may alter the E. histolytica-induced expression of such genes. HT-29 colon epithelial cells were co-cultured with E. histolytica trophozoites in the presence or absence of Escherichia coli. Cytokine gene expression was assessed by quantitative reverse-transcription polymerase chain reaction (RT-PCR). IL-8 mRNA in colon epithelial cells was up-regulated following exposure to E. histolytica and this was paralleled by increased IL-8 secretion. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-1alpha/beta mRNAs were also up-regulated in these cells. When HT-29 cells were co-cultured with E. coli DH5alpha and E. histolytica there was a synergistic increase in the expression of IL-8, IL-1alpha, and GM-CSF. These results suggest that enteric bacteria may significantly affect early proinflammatory signals produced in host tissues in response to E. histolytica infection.

A case of loiasis.

The prevalence of Loa loa infections in non-endemic areas such as Korea is very low, even though it is quite common in the endemic regions of West and Central Africa. We describe a patient who presented with temporary localized edema (classical Calabar swellings) after travelling to Cameroon and in whom the diagnosis of loiasis was made by ELISA. This is the second reported case of loiasis in Korea. As international travel is becoming more frequent, Loa loa infection should be considered in the differential diagnosis for patients with eosinophilia and Calabar swellings in Korea.

[cDNAs encoding the antigenic proteins in pathogenic strain of Entamoeba histolytica].

The differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis was performed to identify the pathogenic strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenic strain YS-27 and the non-pathogenic strain S 16, respectively. Three kinds of first stranded cDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT11M (M: A, C, or G) primers. Each cDNA template was used for DDRT-PCR analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oligo-dT11M primers. Of these, 31 amplicons were verified as the amplicons amplified only from the mRNAs of the pathogenic strain by DNA slot blot hybridization. Further characterization of the 31 pathogenic strain specific amplicons by DNA slot blot hybridization analysis using biotin labeled probes of the PCR amplified DNA of cysteine proteinase genes revealed that 21 of them were amplified from the mRNAs of the cysteine proteinase genes. Four randomly selected amplicons out of the rest 10 amplicons were used for screening of cDNA library followed by immunoscreening and all of them were turned out to be amplified from the mRNA.

Cytokine genes are down-regulated when attachment of Entamoeba histolytica to HT-29 colon epithelial cells is prevented.

Entamoeba histolytica can cause invasive disease by disruption of the intestinal barriers and subsequent lysis of the intestinal cells. Adherence to and contact dependent killing of host cells requires the galactose inhibitable lectin. To elucidate the mechanism whereby E. histolytica influences host defence, the authors assessed the change of proinflammatory cytokine genes expressed by colon epithelial cells in response to co-culture with E. histolytica trophozoites and carbohydrates, including galactose, N-acetyl-galactosamine or N-acetyl-lactosamine, which prevented E. histolytica from attaching to epithelial cells. After HT-29 human colon epithelial cells were co-cultured with E. histolytica trophozoites in the presence or absence of carbohydrates (0.1-100 mM), RNA was extracted from the epithelial cells by an acid guanidinium thiocyanate-phenol-chloroform method. Cytokine gene expression was assessed by quantitative RT-PCR using a synthetic internal standard, and proteins were determined by ELISA. IL-8 mRNA expressed by HT-29 cells in response to E. histolytica trophozoites was downregulated in the presence of galactose, N-acetylgalactosamine or N-acetyl-lactosamine (0.1-100 mM), and this was paralleled by decreased IL-8 protein secretion. GM-CSF and IL-1 alpha/beta mRNAs were also downregulated in those cells in the presence of these agents. These results suggest that the expression of proinflammatory cytokine genes could be inhibited by preventing E. histolytica from attaching to the host's colon epithelial cells.

[Gastric anisakiasis cases in Cheju-do, Korea].

Human anisakiasis may occur after ingestion of raw marine fish infected with nematode larvae of Anisakidae. Anisakiasis caused by the migration of the larva into the wall of stomach, small intestine and other portion has been reported in Korea. This prospective study was made of all cases referred to parasitological laboratory in Cheju-do between June 1989 and June 1992. Gastric anisakiasis was confirmed if larvae invading the gastric wall were observed by gastrofiberscopy. One hundred and seven cases were diagnosed, most of which were in 30-49 years old. Most of the patients complained acute epigastric pain with history of eating raw marine fish. This symptom usually occurred about 12 hours to 1 day after ingestion of infected marine fish. Edema, erosion or ulcer of the mucosa and hemorrhage from the gastric wall were observed in the involved areas. Ninety larvae removed from the stomach were identified; the larva of Anisakis simplex was the most prevalent species, and the larva of Pseudoterranova decipiens was also detected. The important species of marine fish from which the patients became infected was demonstrated as yellow corvina, sea eel, ling, cuttle fish, yellowtail and others. Five species of marine fish as a possible source of infection were examined, and Anisakis simplex larvae and Pseudoterranova decipiens larvae were collected from the mackerel and rock cod. This study demonstrates that anisakiasis is recognized as a public health problem in Korea.

Cytotoxicity of a cysteine proteinase of adult Clonorchis sinensis.

To clarify the correlation of the proteinase activity with pathogenicity of Clonorchis sinensis, the proteinase activity either in excretory-secretory products (ESP) or in crude extracts of adult C. sinensis was examined. Substrate gel electrophoresis of the ESP and crude extracts revealed four distinct enzyme bands, which were differently inhibited by the specific proteinase inhibitors. The proteinase of the ESP with molecular mass of 24 kDa, was purified 23-fold with 14.5% yield by spectra gel ACA 44 gel filtration. It exhibited optimal pH at 7.5 in sodium phosphate (0.1 M). Its activity was inhibited specifically by N-ethylmaleimide (NEM) and antipain whereas potentiated 1.9 folds in the presence of 5 mM dithiothreitol (DTT). Cytotoxicity of the proteinase increased in a dose-dependent manner up to 120 micrograms/ml while reduced by NEM and antipain, indicating that cysteine proteinase was responsible for the cytotoxicity. This result shows that the 24 kDa cysteine proteinase is deeply correlated with the pathogenicity of C. sinensis infection.

Detection of specific serum IgE in clonorchiasis cases and analysis of Clonorchis sinensis allergens.

Specific serum IgE levels of Clonorchis sinensis in infected humans were measured by avidin-biotin ELISA, and allergens from C. sinensis were identified by immunoblot and autoradiography. Then, allergens fractionated by Sephadex G-200 gel filtration were analyzed, and cross-reactive allergenic components of C. sinensis reacted with paragonimiasis sera were revealed. Fourteen out of 15 C. sinensis egg-positives were found to be serum IgE positive (absorbance > 0.27). Of 14 IgE-reacting allergen bands visualized, major allergens of 66, 61.5, 45, 37, 28.5, 23.5 and 15.5 KD were recognized by more than 50% of the sera of infected humans. The considerable individual variations of IgE immune responses to C. sinensis allergenic components were also noticed. C. sinensis extract was separated into 5 fractions by Sephadex G-200 gel filtration. Seventy-four KD allergen was recognized in the first fraction, 50, 45, 37, 29.5 and 28.5 KD in the third, and 15.5 KD in the fourth. Cross-reactive allergens with sera of paragonimiasis cases were identified as 66, 45, 28.5, 13 and 7.5 KD.

[Proteinase activity in the isolates of Trichomonas vaginalis according to their pathogenicity].

Ten axenic isolates of Trichomonas vaginalis were subcutaneously injected to the BALB/c mice in order to assess their pathogenicity by means of so-called "mouse assay" method. All the isolates revealed neutral and acid proteinase activities both in their lysates and in culture media, but the specific activities of both proteinases in the severely pathogenic group were significantly higher than the mildly pathogenic group (p < 0.05). In the SDS-PAGE system in which the electrophoretic gels contained 0.4% gelatin as the substrate, five different banding patterns of trichomonal proteinases were detected, and the patterns were closely related with the pathogenicity of the isolates of T. vaginalis. All five bands might be regarded as cysteine proteinases group in the inhibitor assays. The cytotoxicity of the lysates of T. vaginalis to the target Chinese hamster ovarian (CHO) cell line was also significantly different according to the pathogenicity of the isolates, and generally lower in the lysates treated with cysteine proteinase inhibitors than in the control lysates. In summarizing the results, it might be considered that the proteinases of T. vaginalis showing five electrophoretic banding patterns are closely related with the pathogenicity and cytotoxicity of the isolates of T. vaginalis.

[The effect of active immunization with Acanthamoeba culbertsoni in mice born to immune mother].

Acanthamoeba culbertsoni is a pathogenic free-living amoeba causing primary amoebic meningoencephalitis (PAME) in human and mouse. Several reports on the immune responses in mice with this amoebic infection have been published, but the effects of transferred passive immunity on the active immunization in offspring mice have not been demonstrated. This experiment was done to observe the effect of active immunization with Acanthamoeba culbertsoni in mice born to immune mothers. Acanthamoeba culbertsoni was cultured in the CGV medium axenically. Female BALB/c mice weighing about 20g were immunized through the intraperitoneal injection of Acanthamoeba culbertsoni trophozoites 1 x 10(6) each three times at the interval of one week. Offspring mice were immunized two times. The mice were inoculated intranasally with 1 x 10(4) trophozoites under secobarbital anesthesia. There was a statistical difference in mortality between the transferred immunity group and the active immunization group. Statistical differences were not demonstrated in antibody titer between both groups. But L3T4+ T cell/Ly2+ T cell ratio was increased in the transferred immunity group more than active immunization group of the offspring mice at the age of 5 weeks. There was no differences statistically in mortality between both groups. It was recognized that active immunization in offspring mice born to immune mother could modulate the immune status according to the time of immunization.

[Immunological approach for classification of free-living amoeba in Korea].

Acanthamoeba spp., free-living amoebae inhabited in moist soil, pond, freshwater, sewage, atmosphere and swimming pool, may be causative protozoa of the fatal primary amoebic meningoencephalitis in experimental animals and humans. In this study, Acanthamoeba spp., including Acanthamoeba sp. YM-4 (isolated strain from Korea) had been compared by the two-dimensional electrophoresis and hybridoma technique as well as the difference of morphological characteristics. Trophozoite of Acanthamoeba sp. YM-4 is usually uninucleate and show the hyaline filamentous projections (acanthopoda). No flagellate stage observed. Cysts have two walls, the outer wall is nearly circular, but inner wall is oval or some irregular. As results of SDS-PAGE for lysate of Acanthamoeba sp. YM-4, 16 major protein fractions are similar to those of A. culbertsoni, but different to A. royreba and A. polyphaga. Findings of two-dimensional electrophoretic patterns of Acanthamoeba sp. YM-4 are almost same to those of A. culbertsoni, The isotype of monoclonal antibodies produced from McAY 6, McAY 7, McAY 8, McAY 13 and McAY 16 clones were IgG1, and McAY 10 and McAY 11 clones were IgM. As results of the cross-reactivity among various amoebae using ELISA with monoclonal antibodies, McAY 7 monoclonal antibody (molecular weight 43 kDa by EITB) was only reacted with Acanthamoeba sp. YM-4, but McAY 6 and McAY 10 monoclonal antibodies were reacted to A. culbertsoni as well as Acanthamoeba sp. YM-4.

Mass treatment of head louse infestation with Sumithrin powder in primary schools in Korea.

A mass treatment of head louse infestation with Sumithrin powder (0.4% phenothrin) in primary school children was implemented during the period of September 1991-May 1992. The infestation rate of total 2,515 children was 38.6% in average (21.2% in boys and 57.2% in girls). The reduction rate of head louse infestation was 93.4% with a single treatment and 94.8% with double consecutive treatments with about 10 days interval, which indicated that a single treatment would be recommended for the mass treatment in the community. Long term follow-up after Sumithrin powder application for head louse control in a primary school showed that the infestation rate dropped from 33.1% before treatment to 5.4% by seven months after treatment, giving a 83.4% reduction rate.