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Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Author summary: Infection with the human liver flukes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a public health concern in regions where these parasites are endemic. O. viverrini is endemic in the Mekong River drainage countries including Thailand and the Lao People's Democratic Republic. Infection follows the consumption of undercooked freshwater fish harboring the parasite. Liver fluke infection, opisthorchiasis, is associated with diseases of the liver and bile ducts including cancer of the biliary tract, cholangiocarcinoma, a cancer with a poor prognosis. This report characterizes, for the first time, experimental infection with gene-edited O. viverrini liver flukes during concurrent exposure to a dietary nitrosamine in a rodent model of liver fluke infection-associated cancer. Cancer development was slowed in hamsters infected with the parasite following CRISPR-based knock-out mutation and loss of a parasite gene known to stimulate growth of cells lining the bile ducts. These findings definitely link a parasite factor to cancer etiology, and present a new laboratory model to investigate risk factors for infection-associated cholangiocarcinoma and to assess efficacy of anti-infection/anti-cancer vaccines. [ABSTRACT FROM AUTHOR]

Opisthorchiasis and Clonorchiasis: Major Regional Public Health Problems Liver fluke infection caused by Opisthorchis viverrini, O. felineus, and Clonorchis sinensis is a major public health problem in East Asia and [...]

Schistosoma haematobium is the leading cause of urogenital schistosomiasis and it is recognised as a class 1 carcinogen due to the robust association of infection with bladder cancer. In schistosomes, tetraspanins (TSPs) are abundantly present in different parasite proteomes and could be potential diagnostic candidates due to their accessibility to the host immune system. The large extracellular loops of six TSPs from the secretome (including the soluble excretory/secretory products, tegument and extracellular vesicles) of S. haematobium (Sh-TSP-2, Sh-TSP-4, Sh-TSP-5, Sh-TSP-6, Sh-TSP-18 and Sh-TSP-23) were expressed in a bacterial expression system and polyclonal antibodies were raised to the recombinant proteins to confirm the anatomical sites of expression within the parasite. Sh-TSP-2, and Sh-TSP-18 were identified on the tegument, whereas Sh-TSP-4, Sh-TSP-5, Sh-TSP-6and Sh-TSP-23 were identified both on the tegument and internal tissues of adult parasites. The mRNAs encoding these TSPs were differentially expressed throughout all schistosome developmental stages tested. The potential diagnostic value of three of these Sh-TSPs was assessed using the urine of individuals (stratified by infection intensity) from an endemic area of Zimbabwe. The three Sh-TSPs were the targets of urine IgG responses in all cohorts, including individuals with very low levels of infection (those positive for circulating anodic antigen but negative for eggs by microscopy). This study provides new antigen candidates to immunologically diagnose S. haematobium infection, and the work presented here provides compelling evidence for the use of a biomarker signature to enhance the diagnostic capability of these tetraspanins. Author summary: Schistosoma haematobium, the leading cause of urogenital schistosomiasis, affects millions of people worldwide. Infection with this parasite is associated with different clinical complications such as squamous cell carcinoma and genital malignancy in women. Despite its importance, there is a lack of sensitive and specific diagnostics that support control and elimination initiatives against this devastating disease. Herein, we have characterised six molecules belonging to the tetraspanin family of membrane proteins, providing details about their relative expression during parasite's development and their localization in adult forms of S. haematobium. Furthermore, we have characterised the antibody responses against three of these molecules in urine from infected human subjects from an endemic area, providing compelling evidence for the use of these molecules to diagnose urogenital schistosomiasis. [ABSTRACT FROM AUTHOR]

In 1896, a serendipitous laboratory accident led to the understanding that hookworms propagate infection by penetrating skin, a theory that was then confirmed with the first experimental human infection, reported in 1901. Experimental human infections undertaken in the 20th century enabled understanding of the natural history of infection and the immune response. More recently, experimental hookworm infection has been performed to investigate the immunomodulatory potential of hookworm infection and for the evaluation of hookworm vaccines and chemotherapeutic interventions. Experimental human hookworm infection has been proven to be safe, with no deaths observed in over 500 participants (although early reports predate systematic adverse event reporting) and no serious adverse events described in over 200 participants enrolled in contemporary clinical trials. While experimental human hookworm infection holds significant promise, as both a challenge model for testing anti-hookworm therapies and for treating various diseases of modernity, there are many challenges that present. These challenges include preparation and storage of larvae, which has not significantly changed since Harada and Mori first described their coproculture method in 1955. In vitro methods of hookworm larval culture, storage, and the development of meaningful potency or release assays are required. Surrogate markers of intestinal infection intensity are required because faecal egg counts or hookworm faecal DNA intensity lack the fidelity required for exploration of hookworm infection as a vaccine/drug testing platform or as a regulated therapy. [ABSTRACT FROM AUTHOR]

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds. [ABSTRACT FROM AUTHOR]

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 –smp_154600 and Smache2 –smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 –smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate—dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP. Author summary: Cholinesterases—aceytlcholinesterases (AChE)s and butyrylcholinesterases (BChE)s—are multi-functional enzymes that play a pivotal role in the nervous system of parasites by regulating neurotransmission through acetylcholine hydrolysis. Herein, we provide a detailed characterization of schistosome cholinesterases using molecular, enzymatic and gene-silencing approaches and show evidence for these molecules having roles in addition to their neuronal function. Further, we demonstrate the importance of these proteins to parasite development and survival through gene knockdown experiments in laboratory animals, providing evidence for the use of these proteins in the development of novel intervention strategies against schistosomiasis. [ABSTRACT FROM AUTHOR]

Background: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. Methodology/Principal findings: EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. Conclusion/Significance: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogential clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease. Methods and findings: By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection. Conclusion: We provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease. [ABSTRACT FROM AUTHOR]

As part of on-going efforts to control hookworm infection, the “human hookworm vaccine initiative” has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)—the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs. Methodology/Principal findings: We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri—68%, Rubb7-tnl—56%) and were significantly morphologically altered (Rubb12-tri—62% abnormal, Rubb7-tnl—35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Conclusions/Significance: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes. [ABSTRACT FROM AUTHOR]

The article discusses a research on Helminth pathogens infection-induced malignancy. It mentions how infection with helminths leads to cholangiocarcinoma and squamous cell carcinoma of the urinary bladder. It also mentions impact of ingestion of undercooked freshwater fish infected with the metacercaria stage of species of liver flukes. It mentions Cancer occur in the DNA of the genome of the target cell, while the mutations lead to uncontrolled cellular proliferation and metastasis.

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas. [ABSTRACT FROM AUTHOR]

Background: Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties. Methodology/Principal Findings: Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds—luteolin (3) and (3R,6R)-linalool oxide acetate (1)—showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8–36.9 μg/mL) and T. muris (IC50 range = 9.7–20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice. Conclusions/Significance: Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths—T. muris and S. mansoni—and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals. [ABSTRACT FROM AUTHOR]

Background: Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method. Methodology: We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression. Results: When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively. Conclusion: The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites. [ABSTRACT FROM AUTHOR]

Abstract: Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world. [ABSTRACT FROM AUTHOR]

Background: Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. Methodology/Principal Findings: Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. Conclusions/Significance: The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes. Author Summary: Despite recent significant advances in knowledge of the fundamental biology of the carcinogenic liver fluke Opisthorchis viverrini, little is known of the complement of molecular interactions occurring between this parasite and its prosobranch snail intermediate host, Bithynia siamensis goniomphalos. The determination of such interactions is a key, necessary component of the development of future integrated control strategies for liver fluke infection and associated bile duct cancer. Here, we use cutting-edge high-throughput sequencing technologies and advanced bioinformatic analyses to characterize, for the first time, qualitative and quantitative differences in gene expression between uninfected and O. viverrini-infected B. siamensis goniomphalos collected from an endemic region of Northeast Thailand. The analyses led to the identification of a number of molecules putatively involved in immune defense pathways against invading O. viverrini, and of key biological mechanisms potentially implicated in the ability of the parasite to successfully colonize its snail intermediate host. We believe that this ready-to-use molecular resource will provide the scientific community with new tools for the development of strategies to control the spread of liver fluke infection and the resulting bile duct cancer. [ABSTRACT FROM AUTHOR]

Background: Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. Methodology/Principal Findings: Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. Conclusions/Significance: The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a neglected tropical disease that is responsible for almost 300,000 deaths annually. Mass drug administration (MDA) is used worldwide for the control of schistosomiasis, but chemotherapy fails to prevent reinfection with schistosomes, so MDA alone is not sufficient to eliminate the disease, and a prophylactic vaccine is required. Herein, we take advantage of recent advances in systems biology and longitudinal studies in schistosomiasis endemic areas in Brazil to pilot an immunomics approach to the discovery of schistosomiasis vaccine antigens. We selected mostly surface-derived proteins, produced them using an in vitro rapid translation system and then printed them to generate the first protein microarray for a multi-cellular pathogen. Using well-established Brazilian cohorts of putatively resistant (PR) and chronically infected (CI) individuals stratified by the intensity of their S. mansoni infection, we probed arrays for IgG subclass and IgE responses to these antigens to detect antibody signatures that were reflective of protective vs. non-protective immune responses. Moreover, probing for IgE responses allowed us to identify antigens that might induce potentially deleterious hypersensitivity responses if used as subunit vaccines in endemic populations. Using multi-dimensional cluster analysis we showed that PR individuals mounted a distinct and robust IgG1 response to a small set of newly discovered and well-characterized surface (tegument) antigens in contrast to CI individuals who mounted strong IgE and IgG4 responses to many antigens. Herein, we show the utility of a vaccinomics approach that profiles antibody responses of resistant individuals in a high-throughput multiplex approach for the identification of several potentially protective and safe schistosomiasis vaccine antigens. [ABSTRACT FROM AUTHOR]

The article discusses the need for more research on the molecular biology of snails that harbor parasites, specifically focusing on the liver fluke Opisthorchis viverrini. The liver fluke is a leading cause of bile-duct cancer in Southeast Asia. The authors generated the first reference transcriptome of the Bithynia siamensis goniomphalos snail, which is an intermediate host for O. viverrini. The transcriptome provides valuable information for future studies on the biology of the liver fluke and the interactions between the parasite and the snail. The authors hope that this research will contribute to the development of new strategies to control snail-borne diseases. [Extracted from the article]

Among Oceania's population of 35 million people, the greatest number living in poverty currently live in Papua New Guinea (PNG), Fiji, Vanuatu, and the Solomon Islands. These impoverished populations are at high risk for selected NTDs, including Necator americanus hookworm infection, strongyloidiasis, lymphatic filariasis (LF), balantidiasis, yaws, trachoma, leprosy, and scabies, in addition to outbreaks of dengue and other arboviral infections including Japanese encephalitis virus infection. PNG stands out for having the largest number of cases and highest prevalence for most of these NTDs. However, Australia's Aboriginal population also suffers from a range of significant NTDs. Through the Pacific Programme to Eliminate Lymphatic Filariasis, enormous strides have been made in eliminating LF in Oceania through programs of mass drug administration (MDA), although LF remains widespread in PNG. There are opportunities to scale up MDA for PNG's major NTDs, which could be accomplished through an integrated package that combines albendazole, ivermectin, diethylcarbamazine, and azithromycin, in a program of national control. Australia's Aboriginal population may benefit from appropriately integrated MDA into primary health care systems. Several emerging viral NTDs remain important threats to the region. [ABSTRACT FROM AUTHOR]

Background: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. Methodology/Principal Findings: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Conclusions/Significance: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed. Author Summary: Liver fluke infection is a fish borne disease that afflicts millions of residents in Thailand and Laos. Infection results from eating undercooked freshwater fish contaminated with larvae of the worm Opisthorchis viverrini. Infection can lead to cancer of the bile ducts (cholangiocarcinoma). Indeed, O. viverrini is designated as a Group 1 carcinogen by the World Health Organization, i.e. a definitive cause for cancer. Proteins produced at the surface and/or released from this parasite play pivotal roles in maintaining the infection and disease. These proteins are valid targets for development of vaccines and new drugs. Tetraspanins are prominent in the tegument (the surface covering) of parasites closely related to O. viverrini where they are exposed to immune responses. Similar molecules on the surface of O. viverrini may be vital for the parasite's survival and may make effective vaccines. Here the gene coding for O. viverrini tetraspanin-1 (Ov-TSP-1) was investigated. We used electron microscopy to show that Ov-TSP-1 is expressed in the tegument. We then silenced expression of the gene encoding Ov-TSP-1 and showed that this resulted in malformation of the tegument, highlighting the importance of this molecule for parasite development and its potential as a vaccine target. [ABSTRACT FROM AUTHOR]

The nematocyst is a complex intracellular structure unique to Cnidaria. When triggered to discharge, the nematocyst explosively releases a long spiny, tubule that delivers an often highly venomous mixture of components. The box jellyfish, Chironex fleckeri, produces exceptionally potent and rapid-acting venom and its stings to humans cause severe localized and systemic effects that are potentially life-threatening. In an effort to identify toxins that could be responsible for the serious health effects caused by C. fleckeri and related species, we used a proteomic approach to profile the protein components of C. fleckeri venom. Collectively, 61 proteins were identified, including toxins and proteins important for nematocyte development and nematocyst formation (nematogenesis). The most abundant toxins identified were isoforms of a taxonomically restricted family of potent cnidarian proteins. These toxins are associated with cytolytic, nociceptive, inflammatory, dermonecrotic and lethal properties and expansion of this important protein family goes some way to explaining the destructive and potentially fatal effects of C. fleckeri venom. Venom proteins and their post-translational modifications (PTMs) were further characterized using toxin-specific antibodies and phosphoprotein/glycoprotein-specific stains. Results indicated that glycosylation is a common PTM of the toxin family while a lack of cross-reactivity by toxinspecific antibodies infers there is significant divergence in structure and possibly function among family members. This study provides insight into the depth and diversity of protein toxins produced by harmful box jellyfish and represents the first description of a cubozoan jellyfish venom proteome. [ABSTRACT FROM AUTHOR]

Hookworms infect more people than HIV and malaria combined, predominantly in third world countries. Treatment of infection with chemotherapy can have limited efficacy and re-infections after treatment are common. Heavy infection often leads to debilitating diseases. All these factors suggest an urgent need for development of vaccine. In an attempt to develop a vaccine targeting the major human hookworm, Necator americanus, a B-cell peptide epitope was chosen from the apical enzyme in the hemoglobin digestion cascade, the aspartic protease Na-APR-1. The A291 Y alpha helical epitope is known to induce neutralizing antibodies that inhibit the enzymatic activity of Na-APR-1, thus reducing the capacity for hookworms to digest hemoglobin and obtain nutrients. A291Y was engineered such that it was flanked on both termini by a coil-promoting sequence to maintain native conformation, and subsequently incorporated into a Lipid Core Peptide (LCP) self-adjuvanting system. While A291Y alone or the chimeric epitope with or without Freund's adjuvants induced negligible IgG responses, the LCP construct incorporating the chimeric peptide induced a strong IgG response in mice. Antibodies produced were able to bind to and completely inhibit the enzymatic activity of Na-APR-1. The results presented show that the new chimeric LCP construct can induce effective enzyme-neutralising antibodies in mice, without the help of any additional toxic adjuvants. This approach offers promise for the development of vaccines against helminth parasites of humans and their livestock and companion animals. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini is considered among the most important of the food-borne trematodes due to its strong association with advanced periductal fibrosis and bile duct cancer (cholangiocarcinoma). We investigated the relationship between plasma levels of Interleukin (IL)-6 and the risk of developing advanced fibrosis and bile duct cancer from chronic Opisthorchis infection. We show that IL-6 circulates in plasma at concentrations 58 times higher in individuals with advanced fibrosis than age, sex, and nearest-neighbor matched controls and 221 times higher in individuals with bile duct cancer than controls. We also observed a dose-response relationship between increasing levels of plasma IL-6 and increasing risk of advanced fibrosis and bile duct cancer; for example, in age and sex adjusted analyses, individuals with the highest quartiles of plasma IL-6 had a 19 times greater risk of developing advanced periductal fibrosis and a 150 times greater risk of developing of bile duct cancer than individuals with no detectable level of plasma IL-6. Finally, we show that a single plasma IL-6 measurement has excellent positive predictive value for the detection of both advanced bile duct fibrosis and bile duct cancer in regions with high O. viverrini transmission. These data support our hypothesis that common mechanisms drive bile duct fibrosis and bile duct tumorogenesis from chronic O. viverrini infection. Our study also adds a unique aspect to the literature on circulating levels of IL-6 as an immune marker of hepatobiliary pathology by showing that high levels of circulating IL-6 in plasma are not related to infection with O. viverrini, but to the development of the advanced and often lethal pathologies resulting from chronic O. viverrini infection. Author Summary: O. viverrini is among the few parasites considered a Class 1 carcinogen because of its strong association with bile duct cancer (cholangiocarcinoma). Currently, more than 40 million people are infected with O. viverrini worldwide. Thailand has the highest prevalence of O. viverrini at 10 million people infected and also the highest incidence of Opisthorchis-associated bile duct cancer (cholangiocarcinoma) in the world. In the current study, we also show that levels of IL-6 in plasma are associated in a dose-dependent fashion with Opisthorchis-induced bile duct fibrosis (periductal fibrosis) and cholangiocarcinoma. More importantly, we show that O. viverrini infection alone does not elevate IL-6 levels in the plasma. It is only in the presence of these advanced pathologies (advanced fibrosis or cholangiocarcinoma) from chronic O. viverrini infection that significantly elevates plasma levels of IL-6 are observed. Moreover, we show that plasma IL-6 is an easily accessible biomarker for the detection of advanced periductal fibrosis and cholangiocarcinoma, which would be a critical advance for this region of Thailand and other countries in Southeast Asia, where the prevalence of O. viverrini infection can reach as high as 80% and the incidence of bile duct cancer is the highest in the world. [ABSTRACT FROM AUTHOR]

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic. Author Summary: There are currently no vaccines available to combat helminth (worm) infections in humans. The most devastating of the diseases caused by human helminths are schistosomiasis (or bilharzia) and hookworm disease. By fusing one of the lead schistosomiasis vaccine antigens, Sm-TSP-2, with a protective fragment from one of the lead hookworm vaccine antigens, Na-APR-1, we have produced a chimeric vaccine, termed Sm-TSP-2/5B that might provide protection against two debilitating and co-endemic neglected tropical diseases. Sm-TSP-2/5B provided increased protection compared to Sm-TSP-2 alone when formulated with human approved adjuvants and tested in a mouse model of schistosomiasis. Moreover, IgE against Sm-TSP-2 or Na-APR-1 has not been detected in the blood of residents from an area in Brazil that is endemic for schistosomes and hookworms, indicating that vaccines based on these molecules would be unlikely to generate allergic reactions in recipients from developing countries. [ABSTRACT FROM AUTHOR]

Background: SCP/TAPS proteins of parasitic helminths have been proposed to play key roles in fundamental biological processes linked to the invasion of and establishment in their mammalian host animals, such as the transition from freeliving to parasitic stages and the modulation of host immune responses. Despite the evidence that SCP/TAPS proteins of parasitic nematodes are involved in host-parasite interactions, there is a paucity of information on this protein family for parasitic trematodes of socio-economic importance. Methodology/Principal Findings: We conducted the first large-scale study of SCP/TAPS proteins of a range of parasitic trematodes of both human and veterinary importance (including the liver flukes Clonorchis sinensis, Opisthorchis viverrini, Fasciola hepatica and F. gigantica as well as the blood flukes Schistosoma mansoni, S. japonicum and S. haematobium). We mined all current transcriptomic and/or genomic sequence datasets from public databases, predicted secondary structures of full-length protein sequences, undertook systematic phylogenetic analyses and investigated the differential transcription of SCP/TAPS genes in O. viverrini and F. hepatica, with an emphasis on those that are up-regulated in the developmental stages infecting the mammalian host. Conclusions: This work, which sheds new light on SCP/TAPS proteins, guides future structural and functional explorations of key SCP/TAPS molecules associated with diseases caused by flatworms. Future fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new approaches for the control of parasitic diseases. [ABSTRACT FROM AUTHOR]

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Methods/Findings: Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. Conclusions: These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen. Author Summary: More people are infected with Schistosoma haematobium than other major human schistosomes yet it has been less studied because of difficulty in maintaining the life cycle in the laboratory. S. haematobium might be considered the 'neglected schistosome' since minimal information on the genome and proteome of S. haematobium is available, in marked contrast to the other major schistosomes. In this report we describe tools and protocols to investigate the genome and genetics of this neglected schistosome. We cultured developmental stages of S. haematobium, and investigated the utility of introducing gene probes into the parasites to silence two model genes. One of these, firefly luciferase, was a reporter gene whereas the second was a schistosome gene encoding a surface protein, termed Sh-tsp-2. We observed that both genes could be silenced – a phenomenon known as experimental RNA interference (RNAi). These findings indicated that the genome of S. haematobium will be amenable to genetic manipulation investigations designed to determine the function and importance of genes of this schistosome and to investigate for novel anti-parasite treatments. [ABSTRACT FROM AUTHOR]

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs. [ABSTRACT FROM AUTHOR]

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)&ggr; and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation andmucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-c and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection. [ABSTRACT FROM AUTHOR]

Background and Aims: The association between hygiene and prevalence of autoimmune disease has been attributed in part to enteric helminth infection. A pilot study of experimental infection with the hookworm Necator americanus was undertaken among a group of otherwise healthy people with celiac disease to test the potential of the helminth to suppress the immunopathology induced by gluten. Methods: In a 21-week, double-blinded, placebo-controlled study, we explored the effects of N. americanus infection in 20 healthy, helminth-naïve adults with celiac disease well controlled by diet. Staged cutaneous inoculations with 10 and 5 infective 3rd stage hookworm larvae or placebo were performed at week-0 and -12 respectively. At week-20, a five day oral wheat challenge equivalent to 16 grams of gluten per day was undertaken. Primary outcomes included duodenal Marsh score and quantification of the immunodominant α-gliadin peptide (QE65)-specific systemic interferon-γ-producing cells by ELISpot pre- and post-wheat challenge. Results: Enteric colonisation with hookworm established in all 10 cases, resulting in transiently painful enteritis in 5. Chronic infection was asymptomatic, with no effect on hemoglobin levels. Although some duodenal eosinophilia was apparent, hookworm-infected mucosa retained a healthy appearance. In both groups, wheat challenge caused deterioration in both primary and several secondary outcomes. Conclusions: Experimental N. americanus infection proved to be safe and enabled testing its effect on a range of measures of the human autoimmune response. Infection imposed no obvious benefit on pathology. [ABSTRACT FROM AUTHOR]

Background: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings: Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. Author Summary: Parasitic worms cause untold morbidity and mortality on billions of people and livestock. Drugs are available but resistance is problematic in livestock parasites and is a looming threat for human helminths. Currently, new drug discovery and resistance monitoring is hindered as drug efficacy is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods evaluated by eye using microscopy. Here we describe a novel application for a cell monitoring device (xCELLigence) that can simply and objectively assess real time anti-parasite efficacy of drugs on eggs, larvae and adults in a fully automated, label-free, high-throughput fashion. This technique overcomes the current low-throughput bottleneck in anthelmintic drug development and resistance detection pipelines. The widespread use of this device to screen for new therapeutics or emerging drug resistance will be an invaluable asset in the fight against human, animal and plant parasitic helminths and other pathogens that plague our planet. [ABSTRACT FROM AUTHOR]

Background: Iron has an integral role in numerous cellular reactions and is required by virtually all organisms. In physiological conditions, iron is abundant in a largely insoluble ferric state. Ferric reductases are an essential component of iron uptake by cells, reducing iron to the soluble ferrous form. Cytochromes b561 (cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. The identification of the ferric reductase duodenal cytochrome b (dcytb) and recent observations that other cyts-b561 may be involved in iron metabolism have opened novel perspectives for elucidating their physiological function. Methodology/Principal Findings: Here we have identified a new member of the cytochrome b561 (Sjcytb561) family in the pathogenic blood fluke Schistosoma japonicum that localises to the outer surface of this parasitic trematode. Heterologous expression of recombinant Sjcyt-b561 in a Saccharomyces cerevisiae mutant strain that lacks plasma membrane ferrireductase activity demonstrated that the molecule could rescue ferric reductase activity in the yeast. Significance/Conclusions: This finding of a new member of the cytochrome b561 family further supports the notion that a ferric reductase function is likely for other members of this protein family. Additionally, the localisation of Sjcytb561 in the surface epithelium of these blood-dwelling schistosomes contributes further to our knowledge concerning nutrient acquisition in these parasites and may provide novel targets for therapeutic intervention. Author Summary: Parasites acquire their food from their hosts, either by feeding directly on tissues of the host, or by competing for ingested food. Adult schistosomes live within the vasculature of humans and rely on the blood cells and plasma they ingest and dissolved solutes they derive across their body surface, the tegument, for their nutrition. Schistosomes require host trace elements, notably iron, which is used as a co-factor in many biological reactions. Iron is especially important for schistosomes, for it has a significant role in egg formation and embryogenesis. In human tissues, iron predominates in the trivalent (ferric) form; however, it is the divalent (ferrous) form that is used as an essential co-factor for multiple biomolecules and enzymes. In order to be acquired from the host environment, the valency of iron must be modified to render it suitable for transport across the parasite membrane. This paper describes the molecular characterisation of a schistosome molecule that is crucial for bringing about this change in iron. Schistosoma japonicum Cytb561 is the first ferric reductase characterised in any parasitic helminth and emphasises the importance of iron, and other divalent cations, in these organisms. [ABSTRACT FROM AUTHOR]

The two parasitic trematodes, Clonorchis sinensis and Opisthorchis viverrini, have a major impact on the health of tens of millions of humans throughout Asia. The greatest impact is through the malignant cancer (= cholangiocarcinoma) that these parasites induce in chronically infected people. Therefore, both C. sinensis and O. viverrini have been classified by the World Health Organization (WHO) as Group 1 carcinogens. Despite their impact, little is known about these parasites and their interplay with the host at the molecular level. Recent advances in genomics and bioinformatics provide unique opportunities to gain improved insights into the biology of parasites as well as their relationships with their hosts at the molecular level. The present study elucidates the transcriptomes of C. sinensis and O. viverrini using a platform based on next-generation (high throughput) sequencing and advanced in silico analyses. From 500,000 sequences, >50,000 sequences were assembled for each species and categorized as biologically relevant based on homology searches, gene ontology and/or pathway mapping. The results of the present study could assist in defining molecules that are essential for the development, reproduction and survival of liver flukes and/or that are linked to the development of cholangiocarcinoma. This study also lays a foundation for future genomic and proteomic research of C. sinensis and O. viverrini and the cancers that they are known to induce, as well as novel intervention strategies. Author Summary: The parasitic worms, Clonorchis sinensis and Opisthorchis viverrini, have a serious impact on the health of tens of millions of people throughout Asia. The greatest impact, however, is through the malignant, untreatable cancer (cholangiocarcinoma) that these parasites induce in chronically infected people. These liver flukes are officially classified by the World Health Organization (WHO) as Group 1 carcinogens. In spite of their massive impact on human health, little is known about these parasites and their relationship with the host at the molecular level. Here, we provide the first detailed insight into the transcriptomes of these flukes, providing a solid foundation for all of the molecular/-omic work required to understand their biology, but, more importantly, to elucidate key aspects of the induction of cholangiocarcinoma. Although our focus has been on the parasites, the implications will extend far beyond the study of parasitic disease. Importantly, insights into the pathogenesis of the infection are likely to have major implications for the study and understanding of other cancers. [ABSTRACT FROM AUTHOR]

Background: The blood-feeding hookworm Necator americanus infects hundreds of millions of people worldwide. In order to elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of the adult stage of Necator americanus was explored using next-generation sequencing and bioinformatic analyses. Methodology/Principal Findings: A total of 19,997 contigs were assembled from the sequence data; 6,771 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and most of them encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that the C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%); most of these molecules were predicted to be involved in more than one biological pathway. Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. For instance, proteinase inhibitors were inferred to be highly represented in the former species, whereas SCP/Tpx-1/Ag5/PR-1/Sc7 proteins (= SCP/TAPS or Ancylostoma-secreted proteins) were predominant in the latter. In N. americanus, essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have homologues in the human host. These candidate targets were inferred to be linked to mitochondrial (e.g., processing proteins) or amino acid metabolism (e.g., asparagine t-RNA synthetase). Conclusions: This study has provided detailed insights into the transcriptome of the adult stage of N. americanus and examines similarities and differences between this species and A. caninum. Future efforts should focus on comparative transcriptomic and proteomic investigations of the other predominant human hookworm, A. duodenale, for both fundamental and applied purposes, including the prevalidation of anti-hookworm drug targets. Author Summary: The blood-feeding hookworm Necator americanus infects hundreds of millions of people. To elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of adult Necator americanus was studied using next-generation sequencing and in silico analyses. Contigs (n = 19,997) were assembled from the sequence data; 6,771 of them had known orthologues in the free-living nematode Caenorhabditis elegans, and most encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%). Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. Essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have human homologues. These candidate targets were inferred to be linked to mitochondrial metabolism or amino acid synthesis. This investigation provides detailed insights into the transcriptome of the adult stage of N. americanus. [ABSTRACT FROM AUTHOR]

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%-75% (p = 0.011) and 69%-89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability. [ABSTRACT FROM AUTHOR]

Background: Schistosomes cause more mortality and morbidity than any other human helminth, but control primarily relies on a single drug that kills adult worms. The newly transformed schistosomulum stage is susceptible to the immune response and is a target for vaccine development and rational drug design. Methodology/Principal Findings: To identify genes which are up-regulated during the maturation of Schistosoma mansoni schistosomula in vitro, we cultured newly transformed parasites for 3 h or 5 days with and without erythrocytes and compared their transcriptional profiles using cDNA microarrays. The most apparent changes were in the up-regulation of genes between 3 h and 5 day schistosomula involved in blood feeding, tegument and cytoskeletal development, cell adhesion, and stress responses. The most highly up-regulated genes included a tegument tetraspanin Sm-tsp-3 (1,600-fold up-regulation), a protein kinase, a novel serine protease and serine protease inhibitor, and intestinal proteases belonging to distinct mechanistic classes. The inclusion of erythrocytes in the culture medium resulted in a general but less pronounced increase in transcriptional activity, with the highest up-regulation of genes involved in iron metabolism, proteolysis, and transport of fatty acids and sugars. Conclusions: We have identified the genes that are up-regulated during the first 5 days of schistosomula development in vitro. Using a combination of gene silencing techniques and murine protection studies, some of these highly up-regulated transcripts can be targeted for future development of new vaccines and drugs. Author Summary: Schistosome blood flukes cause more mortality and morbidity than any other human worm infection, but current control methods primarily rely on a single drug. There is a desperate need for new approaches to control this parasite, including vaccines. People become infected when the free-swimming larva, the cercaria, enters through the skin and becomes the schistosomulum. Schistosomula are susceptible to immune responses during their first few days in the host before they become adult parasites. We characterised the genes that these newly transformed parasites switch on when they enter the host to identify molecules that are critical for survival in the human host. Some of these highly up-regulated genes can be targeted for future development of new vaccines and drugs. [ABSTRACT FROM AUTHOR]

The human liver fluke, Opisthorchis viverrini, infects millions of people throughout south-east Asia and is a major cause of cholangiocarcinoma, or cancer of the bile ducts. The mechanisms by which chronic infection with O. viverrini results in cholangiocarcinogenesis are multi-factorial, but one such mechanism is the secretion of parasite proteins with mitogenic properties into the bile ducts, driving cell proliferation and creating a tumorigenic environment. Using a proteomic approach, we identified a homologue of human granulin, a potent growth factor involved in cell proliferation and wound healing, in the excretory/secretory (ES) products of the parasite. O. viverrini granulin, termed Ov-GRN-1, was expressed in most parasite tissues, particularly the gut and tegument. Furthermore, Ov-GRN-1 was detected in situ on the surface of biliary epithelial cells of hamsters experimentally infected with O. viverrini. Recombinant Ov-GRN-1 was expressed in E. coli and refolded from inclusion bodies. Refolded protein stimulated proliferation of murine fibroblasts at nanomolar concentrations, and proliferation was inhibited by the MAPK kinase inhibitor, U0126. Antibodies raised to recombinant Ov- GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of murine fibroblasts and a human cholangiocarcinoma cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumorigenic environment that may ultimately manifest as cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Background: Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. Methods and Findings: We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056) and fecal egg counts (p = 0.018) in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood. Conclusion: To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine. [ABSTRACT FROM AUTHOR]

Background: Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. Methodology/Principal Findings: Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. Conclusions/Significance: The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes. [ABSTRACT FROM AUTHOR]