Schistosome Infection and Schistosome-Derived Products as Modulators for the Prevention and Alleviation of Immunological Disorders.

Parasitic helminths, comprising the flatworms (tapeworms and flukes) and nematodes (roundworms), have plagued humans persistently over a considerable period of time. It is now known that the degree of exposure to these and other pathogens inversely correlates with the incidence of both T helper 1 (Th1)-mediated autoimmunity and Th2-mediated allergy. Accordingly, there has been recent increased interest in utilizing active helminth worm infections and helminth-derived products for the treatment of human autoimmune and inflammatory diseases and to alleviate disease severity. Indeed, there is an accumulating list of novel helminth derived molecules, including proteins, peptides, and microRNAs, that have been shown to exhibit therapeutic potential in a variety of disease models. Here we consider the blood-dwelling schistosome flukes, which have evolved subtle immune regulatory mechanisms that promote parasite survival but at the same time minimize host tissue immunopathology. We review and discuss the recent advances in using schistosome infection and schistosome-derived products as therapeutics to treat or mitigate human immune-related disorders, including allergic asthma, arthritis, colitis, diabetes, sepsis, cystitis, and cancer.

Schistosoma egg antigens suppress LPS-induced inflammation in human IMR-90 cells by modulation of JAK/STAT1 signaling.

BACKGROUND: The regulation of the balance between inflammatory and anti-inflammatory events during the treatment of pulmonary infection is very important. Soluble Schistosoma egg antigens (SEA) can effectively inhibit the expression of cytokines during hepatic acute inflammation. However, the mechanisms by which these proteins suppress the inflammatory responses in lung cells remain unclear. The purpose of this study was to investigate the ability of SEA to inhibit pulmonary inflammation. METHODS: The effects of SEA were investigated in LPS-treated lung IMR-90 cells. The involvement of the JAK/STAT-1 signaling pathway in these effects was evaluated by employing CBA assays, quantitative polymerase chain reaction, and western blotting experiments. RESULTS: Pretreatment of IMR-90 cells with appropriate concentrations of SEA protected cells against the cytotoxic effects of LPS-induced inflammation in a time-dependent manner. SEA pretreatment significantly attenuated the LPS-induced activation of the JAK/STAT1 signaling pathway, including the upregulation of JAK1/2 and STAT1, as well as the production of inflammatory cytokines. The level of phosphorylated STAT1 gradually declined in response to increasing concentrations of SEA. Based on these findings, we hypothesize that SEA-induced anti-inflammatory effects initiate with the downregulation of the IFN-γ-JAK-STAT1 signaling pathway, resulting in the attenuation of LPS-induced inflammation in IMR-90 cells. CONCLUSION: Our study is the first to demonstrate the anti-inflammatory activity of SEA in an in vitro model of pulmonary inflammation, involving the modulation of JAK/STAT1 signaling. We propose SEA as potential therapeutic or preventive agents for the selective suppression of STAT1 and the control of inflammatory response in lung IMR-90 cells.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection.

BACKGROUND: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. OBJECTIVE: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. METHODS: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it's missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. RESULTS: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. CONCLUSION: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

Proteomic and immunomic analysis of Schistosoma mekongi egg proteins.

Schistosomiasis remains a global health problem. In the Mekong river basin, approximately 80,000 people are at risk of infection by Schistosoma mekongi. The parasite's eggs become entrapped in the host's organs and induce massive inflammation, contributing to the pathogenesis of schistosomiasis. In addition, egg antigens are important in circumoval precipitin tests (COPTs) and other diagnostic techniques. Little is known regarding the egg proteins of S. mekongi, and so we applied immunoblotting and mass spectrometry-based proteomic approaches to study these proteins and their antigenicity. A total of 360 unique proteins were identified in S. mekongi eggs using proteomic analyses. The major protein components of S. mekongi eggs were classified into several groups by functions, including proteins of unknown function, structural proteins, and regulators of transcription and translation. The most abundant proteins in S. mekongi eggs were antioxidant proteins, potentially reflecting the need to neutralize reactive oxidative species released from host immune cells. Immunomic analyses revealed that only DNA replication factor Cdt1 and heat shock protein 70 overlap between the proteins recognized by sera of infected mice and humans, illustrating the challenges of knowledge transfer from animal models to human patients. Forty-one immunoreactive protein bands were recognized by either mouse or patient sera. Phosphoglycerate kinase, fructose-1,6-bisphosphate aldolase and elongation factor 1 appeared to be interesting immunogens of S. mekongi eggs as these proteins were recognized by polyclonal IgMs and IgGs in patient sera. Our findings provide new information on the protein composition of S. mekongi eggs as well as the beginnings of a S. mekongi immunogen dataset. These data may help us better understand the pathology of schistosomiasis as well as natural antibody responses against S. mekongi egg proteins, both of which may be useful in including S. mekongi to other schistosoma diagnostic, vaccine and immunotherapy development.

Eggs and Magnetism: New Approaches for Schistosomiasis Diagnosis.

To date, reliable techniques that can provide accurate information on the local and global prevalence of schistosomiasis are still associated with high costs or labour-intensive processes. Here we discuss old and new concepts for diagnostic approaches, and we highlight structural properties of schistosome eggshells that result in their affinity for magnetic materials as a new diagnostic approach.

Identification and functional characterisation of a Schistosoma japonicum insulin-like peptide.

BACKGROUND: Previous studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes. RESULTS: We isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility. CONCLUSIONS: These findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin.

Schistosoma bovis-host interplay: Proteomics for knowing and acting.

Schistosoma bovis is a parasite of ruminants that causes significant economic losses to farmers throughout Africa, Southwestern Asia and the Mediterranean. Additionally, recent studies have reported its zoonotic potential through the formation of S. bovis×Schistosoma haematobium hybrids. As observed in the Schistosoma species infecting humans, it is assumed that S. bovis has also evolved host regulatory molecules that ensure its long-term survival in the bloodstream of its host. Since these molecules could be potential targets for the development of new drugs and anti-schistosome vaccines, their identification and functional characterization were undertaken. With this aim in mind, the molecular interface between S. bovis and its vertebrate host was subjected to a series of proteomic studies, which started with the analysis of the proteomes of the S. bovis moieties exposed to the host, namely, the excretory/secretory products and the tegument surface. Thus, a wealth of novel molecular information of S. bovis was obtained, which in turn allowed the identification of several parasite proteins with fibrinolytic and anticoagulant activities that could be used by S. bovis to regulate the host defensive systems. Following on, the host interface was investigated by studying the proteome of the host vascular endothelium surface at two points along the infection: in the lung vessels during the schistosomula migration and in the portal vein after the parasites have reached adulthood and sexual maturity. These studies have provided original data regarding the proteomes of the endothelial cell surface of pulmonary vasculature and portal vein in S. bovis-infected animals, and have shown significant changes in these proteomes associated with infection. This review compiles current information and the analyses of all the proteomic data from S. bovis and the S. bovis-host interface, including the molecular and functional characterization of S. bovis proteins that were found to participate in the regulation of the host coagulation and fibrinolysis systems.

Schistosome-Derived Molecules as Modulating Actors of the Immune System and Promising Candidates to Treat Autoimmune and Inflammatory Diseases.

It is long known that some parasite infections are able to modulate specific pathways of host's metabolism and immune responses. This modulation is not only important in order to understand the host-pathogen interactions and to develop treatments against the parasites themselves but also important in the development of treatments against autoimmune and inflammatory diseases. Throughout the life cycle of schistosomes the mammalian hosts are exposed to several biomolecules that are excreted/secreted from the parasite infective stage, named cercariae, from their tegument, present in adult and larval stages, and finally from their eggs. These molecules can induce the activation and modulation of innate and adaptive responses as well as enabling the evasion of the parasite from host defense mechanisms. Immunomodulatory effects of helminth infections and egg molecules are clear, as well as their ability to downregulate proinflammatory cytokines, upregulate anti-inflammatory cytokines, and drive a Th2 type of immune response. We believe that schistosomes can be used as a model to understand the potential applications of helminths and helminth-derived molecules against autoimmune and inflammatory diseases.

Highly Sensitive Laser Scanning of Photon-Upconverting Nanoparticles on a Macroscopic Scale.

An upconversion laser scanner has been optimized to exploit the advantages of photon-upconverting nanoparticles (UCNPs) for background-free imaging on a macroscopic scale. A collimated 980 nm laser beam afforded high local excitation densities to account for the nonlinear luminescence response of UCNPs. As few as 2000 nanoparticles were detectable, and the linear dynamic range covered more than 5 orders of magnitude, which is essentially impossible by using conventional fluorescent dyes. UCNPs covered by a dye-doped silica shell were separated by agarose gel electrophoresis and scanned by a conventional fluorescence scanner as well as the upconversion scanner. Both optical labels could be detected independently. Finally, upconversion images of lateral flow test strips were recorded to facilitate the sensitive and quantitative detection of disease markers. A marker for the parasitic worm Schistosoma was used in this study.

Schistosome sirtuins as drug targets.

The sirtuins form a superfamily of evolutionarily conserved NAD(+)-dependent protein N-ϵ-acyl-lysine (AcK) deacylases with roles in a variety of key cellular processes. Sirtuins have a broadly conserved overall structure with a catalytic site formed by a hydrophobic channel between the NAD(+)-binding Rossmann fold domain and a smaller Zn(2+)-binding domain. Schistosomes express five members of the sirtuin family and generic sirtuin inhibitors induce apoptosis and death in schistosome larvae, the disruption of adult worm pairs, inhibition of egg laying and damage to the male and female worm reproductive systems. Sirtuins in schistosomes and other parasitic flatworms present structural differences from their human orthologues that should allow the development of selective inhibitors that can be developed as drug leads.

Drugging the schistosome zinc-dependent HDACs: current progress and future perspectives.

Schistosomes, like many eukaryotic pathogens, typically display morphologically distinct stages during their life cycles. Epigenetic mechanisms underlie the pathogens' morphological transformations, and the targeting of epigenetics-driven cellular programs therefore represents an Achilles' heel of parasites. To speed up the search for new antiparasitic agents, drugs validated for other diseases can be rationally optimized into antiparasitic therapeutics. Specifically, zinc-dependent histone deacetylases (HDACs) are the most explored targets for epigenetic therapies, notably for anticancer treatments. This review focuses on the development of drug-leads inhibiting HDACs from schistosomes. More precisely, current progress on Schistosoma mansoni HDAC8 (smHDAC8) provided a proof of concept that targeting epigenetic enzymes is a valid approach to treat diseases caused by schistosomes, and possibly other eukaryotic pathogens.

The role of glycans in immune evasion: the human fetoembryonic defence system hypothesis revisited.

Emerging data suggest that mechanisms to evade the human immune system may be shared by the conceptus, tumour cells, persistent pathogens and viruses. It is therefore timely to revisit the human fetoembryonic defense system (Hu-FEDS) hypothesis that was proposed in two papers in the 1990s. The initial paper suggested that glycoconjugates expressed in the human reproductive system inhibited immune responses directed against gametes and the developing human by employing their carbohydrate sequences as functional groups. These glycoconjugates were proposed to block specific binding interactions and interact with lectins linked to signal transduction pathways that modulated immune cell functions. The second article suggested that aggressive tumour cells and persistent pathogens (HIV, H. pylori, schistosomes) either mimicked or acquired the same carbohydrate functional groups employed in this system to evade immune responses. This subterfuge enabled these pathogens and tumour cells to couple their survival to the human reproductive imperative. The Hu-FEDS model has been repeatedly tested since its inception. Data relevant to this model have also been obtained in other studies. Herein, the Hu-FEDS hypothesis is revisited in the context of these more recent findings. Far more supportive evidence for this model now exists than when it was first proposed, and many of the original predictions have been validated. This type of subterfuge by pathogens and tumour cells likely applies to all sexually reproducing metazoans that must protect their gametes from immune responses. Intervention in these pathological states will likely remain problematic until this system of immune evasion is fully understood and appreciated.

cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs.

RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.

Segmenting the etiological agent of schistosomiasis for high-content screening.

Schistosomiasis is a parasitic disease with a global health impact second only to malaria. The World Health Organization has classified schistosomiasis as an illness for which new therapies are urgently needed. However, the causative parasite is refractory to current high-throughput drug screening due to the diversity and complexity of shape, appearance and movement-based phenotypes exhibited in response to putative drugs. Currently, there is no automated image-based approach capable of relieving this deficiency. We propose and validate an image segmentation algorithm designed to overcome the distinct challenges posed by schistosomes and macroparasites in general, including irregular shapes and sizes, dense groups of touching parasites and the unpredictable effects of drug exposure. Our approach combines a region-based distributing function with a novel edge detector derived from phase congruency and grayscale thinning by threshold superposition. The method is sufficiently rapid, robust and accurate to be used for quantitative analysis of diverse parasite phenotypes in high-throughput and high-content screening.

Comparative proteomic analysis of Fasciola hepatica juveniles and Schistosoma bovis schistosomula.

Protein interactions between host and parasites can influence the infection success and severity. The aim of this investigation was to identify the proteins from two trematodes potentially localized at the host-parasite interface. We performed the proteomic profiles from in vivo obtained immature lung stage Schistosoma bovis schistosomula and in vitro excysted juveniles from Fasciola hepatica, parasites of ruminants and man usually giving rise to chronic infections. Proteomes from those parasites were obtained after digestion with trypsin and the peptides generated were identified by mass spectrometry, both before and after parasites' treatment with 70% methanol. The comparison of the two proteome sets from each parasite and between them, the analysis of their relative abundance and of their potential exposure to the host from living parasites, together with the specific immunolocalization of two of the identified molecules, show that this approach could assist in the identification of parasite exposed proteins and in the definition of molecules common for the two parasites with potential interaction with the host. Further characterization of these molecules could guide to define new common anti-parasitic targets and potential vaccine candidates.

Modeling and analysis of Schistosoma Argonaute protein molecular spatial conformation.

OBJECTIVE: To analyze the amino acid sequence composition, secondary structure, the spatial conformation of its domain and other characteristics of Argonaute protein. METHODS: Bioinformatics tools and the internet server were used. Firstly, the amino acid sequence composition features of the Argonaute protein were analyzed, and the phylogenetic tree was constructed. Secondly, Argonaute protein's distribution of secondary structure and its physicochemical properties were predicted. Lastly, the protein functional expression form of the domain group was established through the Phyre-based analysis on the spatial conformation of Argonaute protein domains. RESULTS: 593 amino acids were encoded by Argonaute protein, the phylogenetic tree was constructed, and Argonaute protein's distribution of secondary structure and its physicochemical properties were obtained through analysis. In addition, the functional expression form which comprised the N-terminal PAZ domain and C-terminal Piwi domain for the Argonaute protein was obtained with Phyre. CONCLUSIONS: The information relationship between the structure and function of the Argonaute protein can be initially established with bioinformatics tools and the internet server, and this provides the theoretical basis for further clarifying the function of Schistosoma Argonaute protein.

Total synthesis of cyanolide A and confirmation of its absolute configuration.

The tandem allylic oxidation/oxa-Michael reaction promoted by the gem-disubstituent effect and the 2-methyl-6-nitrobenzoic anhydride (MNBA)-mediated dimerization were explored for the efficient and facile synthesis of cyanolide A.

Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor.

A novel inhibitor of Schistosoma PNP was identified using an "in silico" approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC(50) of 1.3 microM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.

Morphometric and molecular characterizations of schistosome populations in Estuaire province Gabon.

The aim of this study was to test the hypothesis of the presence of hybrids between Schistosoma guineensis and S. haematobium in the Estuaire province (Western Gabon). Egg morphometry and single-stranded conformational polymorphism (SSCP) analysis on adult worms were used in order to characterize the schistosome populations of two sites. The morphology of the eggs showed three morphotypes: S. haematobium, S. guineensis and intermediate morphotypes, but the eggs of the morphotype S. guineensis were smaller compared to the values found in the literature. Furthermore, the SSCP analysis of the adult schistosomes showed that all the patterns corresponded to that of S. haematobium and gave evidence that hybrids were absent from our samples.

Schistosoma spp.: Isolation of microtubule associated proteins in the tegument and the definition of dynein light chains components.

Schistosomes are parasitic blood flukes that reside in human mesenteric veins or urinary bladder veins, depending on species of the parasite. The syncytial tegument of these parasites represents a dynamic interface that regulates nutritional and immunological interactions between the parasite and the host. It is known that the components for biogenesis and maintenance of the tegument are supplied via vesicles from the nucleated cell bodies beneath the syncytium and muscle layer. To investigate the common motor components of vesicular transport in the tegument of schistomes, we extracted Schistosoma mansoni tegumental microtubule associated proteins utilizing detergent/high-salt procedure and raised antiserum against these proteins. The antiserum was applied to screen Schistosoma haematobium lambda gt11 expression library and some of the isolated clones were sequenced. Blast search for the sequences against NCBI database identified clones that are dynein light chains and myosin genes. Further analysis of schistosome dynein genes in the databases identified three families of dynein light chains (Dlcs). The Tctex family protein sequences are significantly different from the mammalian homologs and, therefore, offer a potential vaccine/drug target against schistosomes.