The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host‐exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti‐apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno‐modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin‐2. Novel roles in pathogenesis were suggested for the tegument–host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.     

Taenia solium: cell reactions to the larva (Cysticercus cellulosae) in naturally parasitized, immunized hogs

In hogs naturally infected with Taenia solium larvae (i.e., Cysticercus cellulosae), we studied the host response induced by antigens obtained from the larvae. Histopathological studies of cysticerci removed after 4 and 8 weeks of immunization showed an intense inflammatory reaction surrounding the larvae. The response was greater in the 8-week specimens. A dense layer of eosinophils was in close contact with the external membrane of the bladder wall and, in several cases, the eosinophils had infiltrated this tegument. Many eosinophils were seen in the spiral canal of larvae. This infiltration by eosinophils increased with time. Preparations from the 8-week samples showed many degenerated and disrupted eosinophils whose granules were found in close contact with the outer membrane of the larval tegument and, in some cases, had entered through the broken surface of this structure. More than 90% of the larvae were found in various stages of degeneration; the rest were completely destroyed and surrounded by a mass of eosinophils. After immunization, peripheral blood eosinophilia increased to 17%, whereas the eosinophilia of the control hog was 4% throughout the study. The larval worms removed from control hogs showed intact structures, with a low degree of infiltration by eosinophils and a discrete inflammatory reaction surrounding the bladder wall of the larvae.     

Developmental surface ultrastructure of Macroorchis spinulosus in albino rats

Developmental surface ultrastructure of Macroorchis spinulosus was studied by scanning electron microscopy. One-day-old juvenile fluke was leaf-shaped and bent ventrally. Body surface was covered densely with peg-like spines and with cobblestone-like cytoplasmic processes. Ciliated sensory papillae were concentrated around oral sucker. Several unciliated sensory papillae occurred equidistantly on oral sucker and acetabulum. The ciliated papillae appeared in two longitudinal lines symmetric bilaterally on dorsal surface. On adult flukes, tegumantal spines became wider in middle of the body surface. The cytoplasmic processes differentiated into more fine velvety form. It is likely that the differentiated fine cytoplasmic processes are an increased absorptive surface to adult M. spinulosus. It is suggested that single pointed tegumental spines on anterior half of the body may be supportive for this fluke to migration.     

Studies on leucocyte migration inhibiton: in vitro enucleation of human eosinophils in reactive cultures.

Peripheral blood eosinophils present in leucocyte migration cultures underwent enucleation when cell migration was inhibited in the presence of specific antigen. Enucleation was not induced by antigen alone or at 22 degrees C or 4 degrees C, and only occurred in the presence of an in vitro cellular immune response.     

Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Arachidonic acid metabolism in the murine eosinophil. III. Effect of nonsteroid anti-inflammatory drugs on lymphokine-directed eosinophil migration in vivo

In vitro studies of murine eosinophils indicated that lymphokine-stimulated metabolism of arachidonic acid by a lipoxygenase pathway was required for a migration response. In this study we tested the effects of drugs that affect arachidonic acid metabolism on lymphokine-dependent eosinophil accumulation in vivo by the use of 111In-labeled eosinophils. Indomethacin at different dosages either stimulated (1 mg/kg) or partially inhibited (5 to 25 mg/kg) eosinophil accumulation. Aspirin had no significant effects in doses up to 50 mg/kg. BW755C, a drug that inhibits both cyclooxygenase and lipoxygenase, dramatically inhibited eosinophil accumulation at 25 to 125 mg/kg. Pretreatment in vitro of 111In-labeled eosinophils with some drugs known to inhibit lipoxygenase reduced their subsequent accumulation at a lymphokine-injected site in vivo, but the high concentrations required for inhibition suggested that the effects of the drugs were at least partially reversible. Pretreatment with indomethacin did not inhibit the ability of 111In-labeled eosinophils to accumulate at the site of lymphokine injection in vivo. It may be anticipated from these results that drugs inhibiting lipoxygenase will have critical effects on manifestations of immunologic reactions that recruit eosinophils.     

Impaired resistance in early secondary Nippostrongylus brasiliensis infections in mice with defective eosinophilopoeisis

Eosinophils are an important feature of immune responses to infections with many of the tissue-invasive helminth parasites. The cytokine IL-5 and a high-affinity double GATA-binding site within the GATA-1 promoter are critical for eosinophilopoiesis. In this study, we believe we demonstrate for the first time that defects in eosinophilopoiesis are associated with impaired resistance to Nippostrongylus brasiliensis. Primary and secondary infections were established in wildtype (WT), IL-5(-/-) and DeltadblGATA mice. Resistance to secondary infections was impaired in IL-5(-/-) and DeltadblGATA mice, with significantly more larvae able to reach the lungs 2 days p.i. Pulmonary inflammation was minimal in all strains in the first 2 days of both primary and secondary infections, suggesting that eosinophil-dependent resistance occurred before larvae reached this site. Intestinal worm burdens and/or parasite egg production in primary infections were greater in animals with defective eosinophilopoiesis. While larvae did reach the gut by day 3 of secondary infections of WT and IL-5(-/-) mice, worms were expelled by day 7, even in the complete absence of eosinophils in tissues of the small intestine. This and our previous studies indicate that N. brasiliensis are likely to be exquisitely sensitive to attack by eosinophils soon after entry into the skin. Eosinophils in the gut may make a modest contribution to resistance on first exposure to the parasite, but are not required for expulsion in either primary or secondary infections. In order to mount an effective immune response it may be vital for the host to identify and attack the parasite before it implements immune evasion strategies and migrates to other anatomical sites. These observations may be of particular significance for the development of successful vaccines against hookworms and other nematodes.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

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.     

Analysis of eosinophil turnover in vivo reveals their active recruitment to and prolonged survival in the peritoneal cavity

Eosinophils are potent effector cells associated with allergic inflammation and parasite infections. However, limited information exists about their turnover, migration, and survival in vivo. To address these important questions, we determined murine eosinophil turnover under steady state and inflammatory conditions by flow cytometric analysis of BrdU incorporation and analyzed their migration pattern and survival in different tissues after adoptive transfer into recipient mice. In naive mice approximately 50% of bone marrow eosinophils were labeled with BrdU during a 15-h pulse, whereas only 10% of splenic eosinophils were labeled within this time frame. Unexpectedly, the rate of eosinophil production did not change during acute infection with the helminth parasite Nippostrongylus brasiliensis despite massive eosinophilia in several tissues. Eosinophils present in lung and peritoneum remained largely BrdU negative, indicating that eosinophilia in end organs was mainly caused by increased survival of already existing eosinophils rather than increased production of new eosinophils in the bone marrow. Adoptive transfer experiments revealed that eosinophils preferentially migrated to the peritoneum in a macrophage-independent and pertussis toxin-sensitive manner, where they survived for several days. Peritoneal eosinophils expressed high levels of the inhibitory receptor Siglec-F, released less eosinophil peroxidase compared with eosinophils from the spleen, and could recirculate to other organs. These results demonstrate that the peritoneum serves as reservoir for eosinophils.     

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.     

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.     

Fasciola hepatica: Migration of newly excysted juveniles in resistant rats

Using transmission and scanning electron microscopy, the early migration of juvenile Fasciola hepatica was examined in naive and resistant rats. In naive rats, the migration of flukes to the peritoneal cavity was uneventful. In resistant rats, flukes were rapidly coated with antibody whilst still in the gut lumen and a proportion of the flukes were unable to penetrate the intestinal wall. Those that did penetrate were unharmed as they crossed the gut wall, but on entering the peritoneal cavity they were coated with antibody and host cells including eosinophils, neutrophils, macrophages, and mast cells. Eosinophils were seen degranulating onto the fluke surface, and this appeared to result in the erosion of the tegumental syncytium.     

Fasciola hepatica: a technique for monitoring in vitro motility

An apparatus utilizing a force and displacement transducer is described for the direct and long-term recording of the motility in vitro of Fasciola hepatica. Normal movement is typically rhythmical, with bursts of more powerful contractions alternating with periods of lesser activity. Such rhythms and the overall level of activity are maintained for more than 30 hr. The fluke remains active for much longer periods of time: recordings of fluke movements have been made for up to 4 days. Potential damage to the fluke caused by the attachment system within the recording apparatus has been determined by the Evans' Blue Technique and scanning electron microscopy. It is restricted to the attachment sites, and does not spread to other parts of the body over the 30-hr normal activity period. Transmission electron microscope studies have shown that the tegument retains its structural and functional integrity over this period of time. There are advantages of the recording apparatus over previous kymographic methods for studying fluke motility.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Complement-mediated leucocyte adherence to newly excysted and 4-day-old Hymenolepis diminuta

Four-day-old and newly excysted H. diminuta were exposed in vitro (37 degrees C; 5% CO2/air atmosphere) to serum and peritoneal cells (1.7-4.4 X 10(5] obtained from rats. Four-day-old worms incubated in serum alone were lysed in titres of less than 16. In assays containing peritoneal cells, leucocytes, predominantly eosinophils and macrophages, adhered to the posterior end of the parasite in serum titre 32, but not in serum titres 64 and 128. In this region of the worms phagocytosis of microtriches by macrophages, microthrix denudation and loss of tegument were noted. Serum-mediated lysis of newly excysted cysticercoids occurred at a serum titre of 64 and leucocyte adherence and phagocytosis of microtriches occurred in serum titres 128 and 256. Attachment of peritoneal cells to worms did not occur in assays containing heat-inactivated serum and it is suggested that regional leucocyte adherence and subsequent parasite damage is complement-mediated.     

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.