Duk-Young Min

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.

NADPH Oxidase-Derived Reactive Oxygen Species-Mediated Activation of ERK1/2 Is Required for Apoptosis of Human Neutrophils Induced by Entamoeba histolytica

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.

Production of Interleukin-8 by Human Neutrophils Stimulated with Trichomonas vaginalis

ABSTRACT Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with Trichomonas vaginalis. Although chemoattractants, such as leukotriene B4 and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute T. vaginalis infection. We examined IL-8 production in neutrophils activated by T. vaginalis and evaluated the factors involved in T. vaginalis adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites, T. vaginalis lysate, or T. vaginalis excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of T. vaginalis to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-κB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live T. vaginalis, particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-κB and MAP kinase signaling pathways. In other words, T. vaginalis-induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live T. vaginalis.

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.

Toxoplasma gondii Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-κB Activation

Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracellular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/10% FBS at 37℃, in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-κB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respectively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-κB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis-associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-κB activation in mouse spleen cells.

Caspase-3-Mediated Apoptosis of Human Eosinophils by the Tissue-Invading Helminth Paragonimus westermani

Background: Eosinophils are important cells in host immunity to infections by parasitic worms. Tissue-invasive helminthic parasites have been known to induce apoptosis of immune cells for their successful establishment in vivo. We have previously found that high doses of excretory-secreted products (ESP) secreted by lung fluke Paragonimus westermani newly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, accelerate eosinophil cell death. However, little is known about the mechanism of eosinophil apoptosis induced by the Paragonimus-derived products. Objective: We examined involvement of caspase 3 activation in eosinophil cell death induced by ESP produced by PwNEM. Methods: Eosinophils were isolated from the peripheral blood of healthy donors by CD16-negative immunomagnetic selection. We examined the inhibitory effect of pan-caspase inhibitor on ESP-triggered phosphatidylserine (PS) externalization on the outer surface of eosinophils and intracellular activation of caspase 3 in cell lysates treated with the ESP. Results: When ESP secreted by PwNEM were incubated for up to 3 h with eosinophils, they increased surface exposure of PS on eosinophils in a time- and dose-dependent manner. This proapoptotic effect of the ESP on eosinophils was significantly inhibited by pretreatment of cells with pan-caspase inhibitor z-VAD-fmk, and was completely abolished by heat inactivation of ESP at 56°C. The activated forms of caspase 3 were also clearly detected in eosinophils incubated with ESP. Moreover, ESP potently inhibited prolonged survival of eosinophils induced by cytokines such as IL-5, IL-3, and GM-CSF. Conclusion: These results suggest that ESP secreted by PwNEM contain biological active factors causing caspase-3-mediated apoptosis of human eosinophils, thereby enabling the larvae to evade and to subvert the tackle by eosinophils during the early phase of the infection.

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm.

Three new members of the cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The amino acid alignment showed conservation of the cysteine, histidine, and asparagine residue that form the catalytic triad. With 57 cysteine proteinases including PwCP1-5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1-5 were clustered with cruzipain-like or cathepsin L-like cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22 cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported cruzipain-like cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to cathepsin L-like cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six cruzipain signature sequences known previously, whereas PwCP5 do not have any cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175--human cathepsin L numbering) for cathepsin L-like group are conserved in PwCP5, which are conserved within cathepsin L-like group and also different from those of cruzipain and other cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser-Tyr-Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr-Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1-4 (cruzipain) and PwCP5 (cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1-4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the cruzipain and cathepsin L-like cysteine proteinases. In addition, the fact that most of cysteine proteinases from P. westermani are cruzipain-like type implies strongly that a new powerful drug for paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani cruzipain-like cysteine proteinases.

Indochinamon ou (Crustacea: Potamidae) as a New Second Intermediate Host for Paragonimus harinasutai in Luang Prabang Province, Lao PDR

Paragonimus harinasutai metacercariae were found in a species of freshwater crab, Indochinamon ou, collected in a small stream of Namback District, Luang Prabang Province, Lao PDR. Adult flukes were recovered after experimental infection of the metacercariae to dogs. Metacercariae were round or slightly elliptical, 0.666 x 0.626 mm in average size, and had a thin cyst wall of about 20 microm in thickness, a black excretory bladder, convoluted ceca, and some pinkish materials in the body. Adults were somewhat elongated, 95.2 x 36.5 mm in average size, covered with single-tipped tegumental spines, had a smaller oral sucker than the ventral sucker, a moderately branched ovary, and 5-6 lobulated testes. Eggs were ovoid and bilaterally symmetrical in shape, 79 x 45 microm in average size, and had a uniformly thickened shell. By the present study, it has been confirmed that I. ou is a new second intermediate host for P. harinasutai.