A 27 kDa cysteine protease secreted by newly excysted Paragonimus westermani metacercariae induces superoxide anion production and degranulation of human eosinophils

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic infections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.     

Excretory-secretory product of Paragonimus westermani newly excysted metacercariae inhibits superoxide production of granulocytes stimulated with IgG

It is well known that the cysteine proteases in excretory-secretory product (ESP) of Paragonimus westermani newly excysted metacercariae (PwNEM) are capable of degrading IgG in vitro. Recent evidence suggests that the IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for granulocyte activation. Therefore, this study was designed to investigate the effect of excretory-secretory product (ESP) of PwNEM on superoxide production of granulocytes stimulated with IgG. The 96-well plates were coated with human IgG (0, 10, 30, 100 micrograms/ml) in the absence or presence of ESP. When granulocytes were incubated in the wells coated with human IgG in the presence of ESP, the level of superoxide production of granulocytes was reduced to about 90% when compared to the cells incubated in the wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production of granulocytes was concentration-dependent. These results suggest that ESP secreted by PwNEM may be important in the control of effector functions of granulocytes stimulated with IgG in human paragonimiasis.     

Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.     

Degranulation of human eosinophils induced by Paragonimus westermani-secreted protease

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.     

Early cysteine protease activity in excretory bladder triggers metacercaria excystment of Paragonimus westermani

The cysteine proteases of Paragonimus westermani metacercaria are known to initiate metacercaria excystment. However, their secretory sites, such as the intestine, tegument, and excretory bladder are not well defined. In this study, the metacercariae were labeled with bromodeoxyuridine (BrdU) to distinguish the initial activation sites. BrdU was labeled mainly at the excretory bladder and the excretory granules of the metacercariae and the newly excysted juvenile worms. This result shows that early 'defecation' of the proteases from the excretory bladder may accelerate the excystment of P. westermani metacercariae.     

In Vitro Cultivation of Cymatocarpus solearis (Brachycoeliidae) Metacercariae to Obtain the Adult Stage without the Marine Turtle Definitive Host

In vitro cultivation of trematodes would assist studies on the basic biology of the parasites and their hosts. This is the first study to use the yolk of unfertilized chicken eggs as a simple and successful method of ovocultivation and the first time to obtain the adult-stage of the trematode Cymatocarpus solearis Braun, 1899 (Digenea: Brachycoeliidae). Chicken eggs were inoculated with metacercariae from the muscle of the spiny lobster, Panulirus argus (Latreille, 1804). The metacercariae were excysted and incubated for 576 hr (24 days) at 38℃ to obtain the adult stage. Eggs in utero were normal in shape and light brown color. The metacercariae developed into mature parasites that have been identified as the adult-stage found in marine turtles. The adult lobsters collected in Quintana Roo State, Mexico, showed the prevalence of 49.4% and the mean intensity of 26.0 per host (n = 87). A statistical study was performed to determine that no parasitic preference was detected for male versus female parasitized lobsters. Morphometric measurements of the adult-stage of C. solearis obtained in our study have been deposited in the National Helminths Collection of the Institute of Biology of the National Autonomous University of Mexico. This study is significant because it is the first time that a digenean of the family Brachycoeliidae has been demonstrated to develop in vitro from metacercariae into adults capable of producing eggs using the yolk of unfertilized chicken eggs. Secondly, this technique allows to obtain the adult stage of C. solearis without the presence of its marine turtle host, allows us to describe the mature parasites, and thus contribute to our understanding of the biology of C. solearis.     

Infection status of Paragonimus westermani metacercariae in crayfish (Cambaroides similis) collected from Bogildo (Islet), Wando-gun, Chollanam-do, Korea

During the period from October 1996 to November 1998, the infection status of Paragonimus westermani metacercariae in freshwater crayfish (Cambaroides similis) collected from Bogildo (islet). Wando-gun, Chollanam-do, which is known for an endemic area of P. westermani in Korea, were examined. The average infection rate of Paragonimus metacercariae in crayfish was 88.6%, and mean number of metacercariae per infected crayfish was 30.2. This metacercarial density was the highest in the group of weight in 7.1-9.0 g. These results suggest that the natural life cycle of P. westermani is still well-preserved in Bogildo.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

Eosinophil-mediated destruction of Schistosoma mansoni eggs III. lymphokine involvement in the induction of eosinophil functional abilities

Granulomas isolated from the livers of CBA/J mice infected for 8 weeks with Schistosoma mansoni produced a chemotactic activity for eosinophils, in a manner which correlated with the production of the lymphokine eosinophil stimulation promoter (ESP). ESP and chemotactic activities were also produced when eosinophilrich peritoneal exudative cells from S. mansoni-infected mice were cultured with S. mansoni eggs. These S. mansoni-related eosinophils destroyed approximately 20% of the eggs whereas eosinophils from normal (uninfected) mice did not have this ability. However, normal cells exposed to ESP-containing fluids in the co-cultivation system actively participated in egg destruction. Eosinophil-rich peritoneal exudative cells obtained from Trichinella spiralis-infected mice were incapable of destroying S. mansoni eggs during the normal 24 hr co-cultivation period, but did achieve destruction if the incubation period was extended to 48 hr. Marginal levels of chemotactic activity for eosinophils were detected in the co-cultivation fluids from T. spiralis-related cells and S. mansoni eggs, although these fluids did not contain demonstrable levels of ESP. Together, these data indicate that ESP/chemotactic factor-containing culture fluids can induce in normal, unreactive eosinophils the functional ability to destroy S. mansoni eggs in vitro. This may account for the ability of T. spiralis-related eosinophils to do so upon extended incubation.     

The liver stage of Plasmodium berghei inhibits host cell apoptosis

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite-host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei-infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei-infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-alpha/D-galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-alpha-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.     

Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.     

Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Parasites as indicator of fish behaviour

In the present study two species of gobies, Pomatoschistus microps and P. minutus from southwest Wales were studied and appearance of some unusual parasite of gobies, were used as an indicator for fish behaviour. The occurrence of pleurocercoids of Ligula intestinalis in the body cavity of these fish and Proteocephalus sp., excysted metacercariae and adult of Timoniella spp., excysted metacercariae of Bucephalus baeri , the excysted metacercariae of Podocotyle atomon and progenetic adult of Bucephaloides gracilescens in their gut indicate host anadromous, cannibalistic and scavenging behaviour.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.     

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.