Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro.

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

Recent Advances in Type-2-Cell-Mediated Immunity: Insights from Helminth Infection.

Type-2-cell-mediated immune responses play a critical role in mediating both host-resistance and disease-tolerance mechanisms during helminth infections. Recently, type 2 cell responses have emerged as major regulators of tissue repair and metabolic homeostasis even under steady-state conditions. In this review, we consider how studies of helminth infection have contributed toward our expanding cellular and molecular understanding of type-2-cell-mediated immunity, as well as new areas such as the microbiome. By studying how these successful parasites form chronic infections without overt pathology, we are gaining additional insights into allergic and inflammatory diseases, as well as normal physiology.

Protease Inhibitors of Parasitic Flukes: Emerging Roles in Parasite Survival and Immune Defence.

Protease inhibitors play crucial roles in parasite development and survival, counteracting the potentially damaging immune responses of their vertebrate hosts. However, limited information is currently available on protease inhibitors from schistosomes and food-borne trematodes. Future characterization of these molecules is important not only to expand knowledge on parasitic fluke biology but also to determine whether they represent novel vaccine and/or drug targets. Moreover, protease inhibitors from flukes may represent lead compounds for the development of a new range of therapeutic agents against inflammatory disorders and cancer. This review discusses already identified protease inhibitors of fluke origin, emphasizing their biological function and their possible future development as new intervention targets.

Plasma autoantibodies against heat shock protein 70, enolase 1 and ribonuclease/angiogenin inhibitor 1 as potential biomarkers for cholangiocarcinoma.

The diagnosis of cholangiocarcinoma (CCA) is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1) and CCA cell lines (M055, M214 and M139) were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70), enolase 1 (ENO1) and ribonuclease/angiogenin inhibitor 1 (RNH1) obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity). Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001). In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05). Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.

Pro-inflammatory cytokines in the serum of silver catfish (Rhamdia quelen) naturally infected by Clinostomum complanatum: a preliminary study.

Clinostomum complanatum is a zoonotic parasite of birds, and its larval form (metacercariae) may cause disease in fish. The immune response of fish infected by C. complanatum is unknown, and therefore the objective of this study was to evaluate serum levels of pro-inflammatory cytokines in silver catfish (Rhamdia quelen) infected by this parasite. The analysis showed that fish infected with metacercariae presented significantly (P < 0.05) higher levels of tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), and interleukin 6 (IL-6) when compared to those not infected. These data suggest that C. complanatum larvae activate the host immune response.

Influence of ß-glucan on innate immunity and resistance of Lutjanus guttatus to an experimental infection of dactylogyrid monogeneans.

The present study investigated the effect of ß-1,3/1,6-glucan on growth, haematology, innate immunity and resistance against dactylogyrids on the spotted rose snapper (Lutjanus guttatus). Fish were fed during 5 weeks with commercial diet (control group) and same diet supplemented with three levels of ß-glucans (0·05%, 0·1% and 0·5%/kg feed). The results showed that at concentrations of 0·05% and 0·1%, fish growth was enhanced, and in weeks 2 and 4, an increase in cellular responses such as percentage of monocytes, neutrophils, respiratory burst activity and nitric oxide activity was observed. In diet with 0·5%ß-glucans, changes were registered at the end of the experiment. At week 2, 0·05%ß-glucans showed the best response to most of the analysed parameters. In a second trial, diet with 0·05%ß-glucans was chosen to prove its effect on the resistance of infected fish with monogeneans. The results showed that fish reduce significantly the number of dactylogyrids, and parameters such as WBC, percentage of lymphocytes, neutrophils and eosinophils were increased. In addition, WBC and percentage of thrombocytes increased in the control (+). The ß-glucans have the potential to be use in diet formulations of the spotted rose snapper and to limit the adverse effects of dactylogyrids.

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer.

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.

Cathepsin B proteases of flukes: the key to facilitating parasite control?

Cysteine proteases are important virulence factors for parasites. This review will focus on the cathepsin B proteases of trematodes (also known as flukes) which are abundant in juvenile and immature flukes. Recent research, primarily in Fasciola, using inhibitors, RNA interference (RNAi) and vaccination studies indicates that cathepsin Bs play a key role in the biology of trematodes. As these proteases are largely expressed by infective parasite stages, their inactivation by chemotherapy or vaccination will greatly reduce the damage wrought by flukes as they invade host tissues. This validates cathepsin Bs as key strategic targets for fluke control.

Similarity and diversity in macrophage activation by nematodes, trematodes, and cestodes.

This review summarizes current knowledge of macrophages in helminth infections, with a focus not only on delineating the striking similarities in macrophage phenotype between diverse infections but also on highlighting the differences. Findings from many different labs illustrate that macrophages in helminth infection can act as anti-parasite effectors but can also act as powerful immune suppressors. The specific role for their alternative (Th2-mediated) activation in helminth killing or expulsion versus immune regulation remains to be determined. Meanwhile, the rapid growth in knowledge of alternatively activated macrophages will require an even more expansive view of their potential functions to include repair of host tissue and regulation of host metabolism.

Antigenic properties of cystatin-binding cysteine proteinases from Neodiplostomum seoulense.

The antigenic properties of cysteine proteinases binding to cystatin were analyzed in Neodiplostomum seoulense, an intestinal trematode that infects humans and rodents in the Republic of Korea. Cystatin was found to effectively capture cysteine proteinases present in the crude extract of N. seoulense. The IgG levels against cystatin-binding cysteine proteinases in sera of mice infected with N. seoulense were higher than those in sera of mice immunized with the crude extract of N. seoulense. The production of IgG antibodies against cystatin-binding cysteine proteinases increased according to the length of infection period. In immunoblots of purified cystatin-binding proteinases, 2 molecules, approximately 50 kDa and 60 kDa, reacted with N. seoulense-infected mouse sera. Of the sera from patients infected with various helminths, those of sparganum-infected patients showed the strongest affinities for cystatin-binding cysteine proteinases of N. seoulense. Cystatin-binding cysteine proteinases of N. seoulense are suggested to be putative antigens for serodiagnosis of human N. seoulense infection.

The divergent roles of alternatively activated macrophages in helminthic infections.

Macrophages play crucial roles in the immune response, as they can initiate, modulate and also be final effector cells during immune responses to infections. Macrophages are derived from myeloid precursor cells in bone marrow and are widely distributed in every tissue of the body. Over the past 10 years, the concepts about macrophage activation have clearly changed; macrophages are not called activated or inactivated as they used to be. These changes in the concept of macrophage response is the result of many in vitro and in vivo studies, but the major support for the current concept of alternatively activated macrophages (AAMphi) comes from parasitic helminth infections. Parasitic helminths have developed complex mechanisms to evade and modulate host immunity. Infections with these parasites induce strong polarized Th2-type immune responses frequently associated with impaired T-cell proliferative responses to parasitic or unrelated antigens. Given the recent advances in understanding the immunoregulatory capabilities of helminthic infections, it has been suggested that macrophages can be a target for immunomodulation. Furthermore, they become altered when a host experiences chronic exposure to helminth parasites or their by-products, which favour the induction of AAMphi. How AAMphi participate in modulating host immunity during helminth infections and what their roles are in clearing or favouring parasite survival remains elusive. Here we review the most recent advances in the literature on AAMphi at the host-parasite interface, including three classes of helminths: nematodes (Brugia, Nippostrongylus, Litomosoides, Heligmosomoides), trematodes (Schistosoma, Fasciola) and cestodes (Taenia, Echinococcus, Hymenolepis).

[The effect of trematodes on the cellular immunity of the dragonfly Aeschna grandis (Odonata) larvae].

The high percent of the infestation by the trematodes of the families Plagiorchidae and Prosthogonimidae is recorded in the dragonfly Aeschna grandis larvae of the last stage. About 30% of the trematode cysts were melanized. It is established, that the parasitising of the trematodes do not effects on the process of the incapsulation of foreign bodies, but it suppresses partly the formation of oxygen free radicals and phenoloxidase activity in the haemocytes of dragonfly larvae.

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults.

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.

Epizootiology of spirorchiid infection in green turtles (Chelonia mydas) in Hawaii.

We describe the epizootiology of spirorchiid trematode infections in Hawaiian green turtles (Chelonia mydas) by quantifying tissue egg burdens in turtles submitted for necropsy and by assessing antibody response to crude adult worm and egg antigens among a variety of age groups. Hapalotrema sp. and Laeredius sp. predominated in turtles infected with spirorchiids. Tissue egg burdens decreased with increasing size and increased with deteriorating body condition of turtles. No relationship was found between tissue egg burdens and sex or fibropapillomatosis status. Tissue egg burdens increased in turtles from southeast to northwest in the main Hawaiian Islands (Hawaii to Kauai). Hatchling and captive-reared turtles had significantly lower levels of antibodies against crude worm and egg antigens. Based on tissue egg burdens and antibody status, we hypothesize that immature turtles become infected with spirorchiids shortly after recruiting into coastal foraging pastures from the pelagic environment, that infection levels decrease with age, and that spirorchiids detrimentally affect the body condition of sea turtles independent of tumor burden. The low intensity of infection in turtles with the endemic trematode Carettacola hawaiiensis suggests either that turtles are less susceptible to infection with this parasite or that the parasite is outcompeted by species of Hapalotrema and Laeredius. Given that the 2 latter species are found in the Pacific and other oceans, they are not likely endemic and were probably introduced into Hawaii through an undetermined route.

Immunity in rainbow trout, Oncorhynchus mykiss, against the monogenean Discocotyle sagittata following primary infection.

Rainbow trout (Oncorhynchus mykiss) were experimentally infected by continuous or single exposure with the monogenean Discocotyle sagittata. To determine whether immunity follows primary infection, fish were exposed to a secondary challenge by one of two modes: (1) primary infections were cleared with praziquantel (PZQ) and hosts re-infected with 100 oncomiracidia; (2) parasites were allowed to reach maturity and hosts super-infected with 100 oncomiracidia. Fish challenged after initial continuous exposure developed significant partial resistance to re-infection, carrying burdens 35% lower than controls. PZQ treatment controls demonstrated that the drug did not account for the protection observed. Single exposure did not significantly modify the outcome of secondary challenges. Super-infection experiments suggested that no concomitant immunity develops. No correlation was found between initial burdens and the outcome of secondary challenges in the same individual. Significantly elevated anti-D. sagittata antibodies were detected in infected fish, but there was no correlation between immunoglobulin levels and parasite burdens.

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer.

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electro-phoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from <29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be <14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and <14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.

Serological association between spirorchidiasis, herpesvirus infection, and fibropapillomatosis in green turtles from Florida.

Serodiagnostic tests for detecting green turtle (Chelonia mydas) antibody responses were developed to test the strength of association between exposure to spirorchid trematode antigens or herpesvirus antigens and having green turtle fibropapillomatosis (GTFP). Plasma samples from 46 captive-reared green turtles, including paired pre- and 1-yr post-inoculation samples from 12 turtles with experimentally induced GTFP, were found by enzyme-linked immunosorbent assay (ELISA) to be negative for antibodies to adult spirorchid (Learedius learedi) antigens. In contrast, all 12 turtles that developed experimentally induced GTFP converted within 1 yr from having negative to positive antibody reactivity to GTFP-associated herpesvirus antigens, whereas the three controls and four turtles that failed to develop tumors remained negative. Plasma samples from 104 free-ranging green turtles from two Florida (USA) coastal feeding grounds with different GTFP prevalences were tested by ELISA for antibodies to L. learedi adult antigens; and there was no statistically significiant association between antibody prevalence and sampling site. When a low optical density cutoff value (0.15) was used to interpret ELISA results, 98% of the turtles from each site were spirorchid antibody-positive and there was no association between antibody reactivity to spirorchids and GTFP status. When a higher negative cutoff value was used, however, a statistically significant association between antibody reactivity to spirorchids and GTFP-free status was found. These results suggest that spirorchids do not have a role in GTFP pathogenesis. All 20 of the tumor-bearing lagoon turtles had antibodies to herpesvirus antigens whereas only two (10%) of the tumor-free reef turtles had detectable anti-herpesvirus reactivity. The strong association between antibody reactivity to herpesvirus antigens and GTFP status in both captive-reared and free-ranging turtles is consistent with the hypothesis that the transmissible agent that causes GTFP is a herpesvirus.

Intestinal mastocytosis and goblet cell hyperplasia in BALB/c and C3H mice infected with Neodiplostomum seoulense.

Mucosal mast cell (MMC) and goblet cell (GC) responses were observed in the small intestine of two strains of mice (BALB/c and C3H) infected with Neodiplostamum seoulense, and their roles in the host defense and worm expulsion were studied. From day 3 to 28 post-infection (PI) with 200 metacercariae, the worm recovery rate from BALB/c mice was consistently and remarkably higher than that from C3H mice. In the duodenum of both strains of mice, the main habitat of the flukes, mastocytosis was pronounced on day 7 PI but quickly diminished thereafter. Similar kinetics were observed in the jejunum and ileum, although the extent of mastocytosis was lesser in the ileum than other two areas. These MMC kinetics were not different between the two strains of mice. Moreover, the extent of mastocytosis was stronger in BALB/c mice than in C3H mice. GC hyperplasia was remarkable in the duodenum of BALB/c mice throughout the course of infection except day 14 PI, whereas it was recognizable only in the jejunum and ileum of C3H mice on day 7 PI. Mucin activation was evidently demonstrated in both strains of mice throughout the course of infection, but more marked in BALB/c than in C3H mice. The results strongly suggest that mastocytosis and GC hyperplasia are local immune responses against N. seoulense, however, they play a minor role in the host defense and worm expulsion.

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes.

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H-type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.