Extraction and Fractionation of Human Gastric Mucins from Gastric Juice.

Mucin, a major component of the mucus, is considered to be one of the principal factors in the physiological defense mechanism of the gastrointestinal mucosa. Measuring the mucin content of human gastric mucus is a useful tool for the assessment of Helicobacter pylori (H. pylori) eradication or the involvement of mucus secretion in various gastroduodenal diseases. Here, we describe a methodology for the isolation of the mucin fraction from human gastric juice and the quantification of mucin.

Structural Elucidation of Sialylated O-Glycan Alditols Obtained from Mucins by Mass Spectrometry.

Acidic O-glycans having sialic acid and/or sulfate residue are abundantly expressed in intestinal mucins. However, structural elucidation of acidic O-glycans is a laborious and time-consuming task due to their large structural variations. Here, we describe a methodology of structural elucidation for sialylated O-glycan alditols from intestinal mucins using tandem mass spectroscopy. Methylesterification and mild periodate oxidation of sialylated O-glycan alditols assist mass analysis. This description includes the purification process of O-glycan alditols for structural analysis.

Biochemical characterization of mitochondria from adult worms and plerocercoid larvae of Spirometra mansoni shows mixed populations of anaerobic and aerobic mitochondria.

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg-1 for adult, and 0.677 and 0.137 nmol mg-1 for plerocercoid, respectively. Inhibition studies on the succinate-oxidase system of adult mitochondria showed that they possessed both cyanide-sensitive and -insensitive succinate oxidases, the latter of which produces hydrogen peroxide. Adult mitochondria, when NADH was used as a substrate, were shown to produce hydrogen peroxide, and the production of hydrogen peroxide decreased to undetectable levels in the presence of fumarate. The specific activities of NADH-fumarate reductase and cytochrome c oxidase were significantly higher in mature proglottids than in immature and gravid proglottids. Isopycnic density-gradient centrifugation analyses and in situ electron microscopic histochemistry revealed that both adult and plerocercoid mitochondria were heterogeneous in terms of respiratory function and physicochemical properties. The physiological significance of adult and plerocercoid mitochondria is discussed in relation to the oxygen tension of their parasitic habitats.

Strongyloides venezuelensis-derived venestatin ameliorates asthma pathogenesis by suppressing receptor for advanced glycation end-products-mediated signaling.

INTRODUCTION: EF-hand Ca2+-binding proteins such as S100 protein family members are recognized by the receptor for advanced glycation end-products (RAGE) and are involved in the pathogenesis of asthma/allergic airway inflammation (AAI). Venestatin, an EF-hand Ca2+-binding protein, which is secreted by the parasitic helminth Strongyloides venezuelensis, binds with RAGE and suppresses RAGE-mediated inflammatory responses after parasite invasion. In this study, we evaluated the effect of venestatin on pathogenesis in a house dust mite (HDM) murine model of asthma/AAI. METHODS: Mice were intranasally treated with HDM, HDM with recombinant venestatin, or HDM with synthetic peptides, which were designed based on the EF-hand Ca2+-binding domain of venestatin. Pro-inflammatory responses in the lungs of mice were assessed. RESULTS: HDM treatment induced inflammatory cell infiltration, phosphorylation of the mitogen-activated protein kinase and inhibitor κB, and production of the cytokines tumor necrosis factor-α and interleukin-5 in the lungs. Co-administration of recombinant venestatin with HDM suppressed these pro-inflammatory responses. Treatment with synthetic peptides reduced inflammatory cell infiltration in a RAGE-dependent manner. CONCLUSION: The EF-hand domain of venestatin may have potential therapeutic benefits in asthma.

Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection.

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite's mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite's life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration.

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.

Characterization of the RAGE-binding protein, Strongyloides venestatin, produced by the silkworm-baculovirus expression system.

The receptor for advanced glycation end products (RAGE) recognizes Ca++-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca++-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca++ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca++-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes.

Positive phototropism is accelerated in Biomphalaria glabrata snails by infection with Schistosoma mansoni.

Parasite-induced behavioral changes in their hosts favor to complete the lifecycle of parasites. Schistosome infection is also known to cause physiological changes in infected freshwater snail intermediate hosts. Here, we report, a novel phenomenon in which Schistosoma mansoni, a highly debilitating worm affecting millions of people worldwide, alters the phototropic behavior of Biomphalaria glabrata, the vector snail. S. mansoni-infection enhanced positive phototropism of vector snails and infected snails spent significantly more time in light. Possibly, these behavioral changes help the parasite to be released efficiently from the infected intermediate hosts, and to infect mammalian hosts.

Interleukin-13/interleukin-4 receptor pathway is crucial for production of Sda-sialomucin in mouse small intestinal mucosa by Nippostrongylus brasiliensis infection.

Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.

Venestatin, a Ca++-binding protein from the parasitic nematode Strongyloides venezuelensis, is involved in the larval migration process.

The secretory EF-hand Ca++-binding proteins act as calcium signaling molecules for control of cell functions, but those proteins from parasitic helminths are poorly understood. Here, we have identified and characterized an EF-hand Ca++-binding protein from the rodent nematode, Strongyloides venezuelensis, termed 'venestatin', which is highly conserved in Strongyloides spp. Canonical two EF-hand domains and a signal peptide are present in venestatin. A gel mobility shift assay and Ruthenium red staining indicated that the recombinant venestatin possesses binding ability with Ca++ ions. Endogenous venestatin was seemingly localized in the hypodermis and gut of the worms and was found in the excretory-secretory products. Quantitative reverse transcription-PCR data showed that venestatin-specific transcript was upregulated in the parasitic stages of S. venezuelensis, and the upregulation occurred promptly after larval invasion through the host's skin, but not in the case of in vitro incubation. Immunization of mice with recombinant venestatin caused a 55% reduction in larval migration to the lungs, and lung hemorrhaging was mild compared with non-immunized groups, suggesting that anti-venestatin sera may interfere with larval migration from skin to lung. Our results suggest that venestatin is secreted from the hypodermis and gut of S. venezuelensis, and has pivotal roles in larval migration.

Correction: Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri.

[This corrects the article DOI: 10.1371/journal.pone.0083703.].

Initial development of Babesia ovata in the tick midgut.

The initial development of Babesia ovata in the midgut of the vector tick Haemaphysalis longicornis has been demonstrated through in vitro and in vivo studies. Although the research on the partial developmental cycles of B. ovata in the tick midgut was performed in our previous study by using ticks fed on experimentally B. ovata-infected cattle, detailed information on the developmental stages of B. ovata in H. longicornis was limited. This report describes the sequential development of stages of B. ovata in an in vitro study using B. ovata-infected erythrocytes and tick midgut contents. The in vivo study also confirmed the developmental stages in the midgut contents of artificially B. ovata-infected ticks. In this observation, we have recognized the distinct forms of B. ovata developmental stages in the tick midgut; the aggregation forms and ray bodies with shorter spikes and light-stained cytoplasm were shown by Giemsa staining. The similarities and differences of the stages as compared to previous reports have been discussed.

A cysteine protease from Spirometra erinaceieuropaei plerocercoid is a critical factor for host tissue invasion and migration.

Sparganosis in humans caused by the plerocercoid larvae of Spirometra erinaceieuropaei is found worldwide, especially in Eastern Asia and the Far East. Previous studies have suggested that dissolution of plerocercoid body, plerocercoid invasion of host tissue, and migration are important processes for sparganosis progression. However, the mechanisms underlying these processes have yet to be determined. Here, we demonstrated the enzymatic property and involvement of a native 23kDa cysteine protease (Se23kCP), purified from plerocercoids, in sparganosis pathogenesis. Se23kCP is mature protease consisting of 216 amino acids and has a high sequence similarity with cathepsin L in various organisms. Se23kCP conjugated with N-glycans, which have a core fucose residue. Both cysteine and serine protease-specific activities were determined in Se23kCP and their optimal pHs were found to be different, indicating that Se23kCP has a wide range of substrate specificity. Se23kCP was secreted from tegumental vacuoles of the plerocercoid to host subcutaneous tissues and degraded human structural proteins, such as collagen and fibronectin. In addition, the plerocercoid body was lysed by Se23kCP, which facilitated larval invasion of host tissue. Our findings suggest that Se23kCP induces host tissue invasion and migration, and might be an essential molecule for sparganosis onset and progression.

Establishment of a novel tick-Babesia experimental infection model.

Ticks are potent vectors of many deadly human and animal pathogens. Tick-borne babesiosis is a well-recognized malaria-like disease that occurs worldwide and recently has attracted increased attention as an emerging zoonosis. Although the proliferation of Babesia organisms is essential in the vectors, their detailed lifecycle with time information for migration in ticks remains unknown. A novel study model for the elucidation of the migration speed of Babesia parasites in their vector tick, Haemaphysalis longicornis, has been developed using an artificial feeding system with quantitative PCR method. The detectable DNA of Babesia parasites gradually disappeared in the tick midgut at 1 day post engorgement (DPE), and in contrary increased in other organs. The results indicated that the Babesia parasite passed the H. longicornis midgut within 24 hours post engorgement, migrated to the hemolymph, and then proliferated in the organs except the midgut. This time point may be an important curfew for Babesia parasites to migrate in the tick lumen. We also visualized the Babesia parasites in the experimentally infected ticks and in their eggs using IFAT for detecting their cytoskeletal structure, which suggested the successful tick infection and transovarial transmission of the parasite. This model will shed light on the further understanding of tick-Babesia interactions.

Collection methods of trematode eggs using experimental animal models.

Although observing the eggs of human parasitic helminth is essential for medical education in parasitology, opportunities for collection of the eggs are limited. Collection of the eggs using experimental animal models is needed for a sustainable supply. The metacercariae of three trematode species, Paragonimus westermani, Clonorchis sinensis and Metagonimus yokogawai, were collected from the second intermediate hosts: freshwater crabs and fishes, which were obtained using online shopping in Japan, and inoculated to experimental animal rat and dog. Consequently, eggs of the three trematode species were obtained abundantly from the feces of the animals. The eggs are being used for student training in several Japanese universities. In this article, we introduce the collection procedures for trematode eggs.

Induction of Sd(a)-sialomucin and sulfated H-sulfomucin in mouse small intestinal mucosa by infection with parasitic helminth.

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, and sulphated H type 2 antigen, Fucα1-2Galß1-4GlcNAc(6SO3H)ß-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N. brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H. polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V. nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins.

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes.

Identification and characterization of sulfated carbohydrate-binding protein from Lactobacillus reuteri.

We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6 × Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.

Rapid and specific alterations of goblet cell mucin in rat airway and small intestine associated with resistance against Nippostrongylus brasiliensis reinfection.

The intestinal parasitic nematode Nippostrongylus brasiliensis is expelled rapidly from the rat in reinfection challenge compared with that of the primary infection owing to the host defense mechanisms raised against the pre-intestinal- and intestinal-stage larvae. We examined the relationship between the mucin alterations in airway and jejunal mucosae and the worm expulsion after third-stage larva reinfection. When rats had been inoculated with fourth-stage larvae and immunized with only the intestinal-stage worms for more than 8 days, the challenge larvae were expelled during the intestinal stage along with a rapid increase of the specific sialomucin in jejunal mucosa, without any effect on the bronchial mucus. When rats had been infected with third-stage larvae and immunized with only the pre-intestinal stage larvae by killing with antihelminthic, the challenge larvae were rejected during the pre-intestinal stage along with marked goblet cell hyperplasia and Muc5AC mucin hyperproduction on the bronchial mucosa, but not as a result of jejunal mucin alteration. Taking these finding together, immunization with pre-intestinal- and intestinal-stage worms independently increases the airway and intestinal goblet cell mucins, respectively, and in both cases, the mucin alterations may contribute to rapid worm expulsion upon reinfection.

The monoclonal antibody HCM31 specifically recognises the Sd(a) tetrasaccharide in goblet cell mucin.

Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis (N.b). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide-alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b-infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß- (Sd(a) blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd(a) antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd(a) tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd(a) antigen.