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Following initial invasion of Schistosoma mansoni cercariae, schistosomula reside in the skin for several days during which they can interact with the dermal immune system. While murine experiments have indicated that exposure to radiation-attenuated (RA) cercariae can generate protective immunity which is initiated in the skin stage, contrasting non-attenuated cercariae, such data is missing for the human model. Since murine skin does not form a reliable marker for immune responses in human skin, we used human skin explants to study the interaction with non-attenuated and RA cercariae with dermal innate antigen presenting cells (APCs) and the subsequent immunological responses. We exposed human skin explants to cercariae and visualized their invasion in real time (initial 30 min) using novel imaging technologies. Subsequently, we studied dermal immune responses and found an enhanced production of regulatory cytokine interleukin (IL)-10, pro-inflammatory cytokine IL-6 and macrophage inflammatory protein (MIP)-1α within 3 days of exposure. Analysis of dermal dendritic cells (DDCs) for their phenotype revealed an increased expression of immune modulators programmed death ligand (PD-L) 1 and 2, and increased IL-10 production. Ex vivo primed DDCs suppress Th1 polarization of naive T-cells and increase T-cell IL-10 production, indicating their regulatory potential. These immune responses were absent or decreased after exposure to RA parasites. Using transwells, we show that direct contact between APCs and cercariae is required to induce their regulatory phenotype. To the best of our knowledge this is the first study that attempts to provide insight in the human dermal S. mansoni cercariae invasion and subsequent immune responses comparing non-attenuated with RA parasites. We reveal that cercariae induce a predominantly regulatory immune response whereas RA cercariae fail to achieve this. This initial understanding of the dermal immune suppressive capacity of S. mansoni cercariae in humans provides a first step toward the development of an effective schistosome vaccine.

Infection with schistosomes is accompanied by the induction of antibodies against the parasite. Despite having IgG against both protein and glycan antigens, infected individuals remain chronically infected until treated, and re-infection is common in endemic areas as immunity does not develop effectively. Parasite specific IgG subclasses may differ in functionality and effectivity with respect to effector functions that contribute to parasite killing and immunity. In this study, we investigated if specific IgG subclasses target specific antigenic schistosome glycan motifs during human infection. Sera from 41 S. mansoni infected individuals from an endemic area in Uganda were incubated on two glycan microarrays, one consisting of a large repertoire of schistosome glycoprotein- and glycolipid- derived glycans and the other consisting of chemically synthesized core xylosylated and fucosylated N-glycans also expressed by schistosomes. Our results show that highly antigenic glycan motifs, such as multi-fucosylated terminal GalNAc(β1-4)GlcNAc (LDN) can be recognized by all IgG subclasses of infection sera, however with highly variable intensities. Detailed examination of core-modified N-glycan targets revealed individual antibody responses specific for core-xylosylated and core α3-fucosylated glycan motifs that are life stage specifically expressed by schistosomes. IgG1 and IgG3 were detected against a range of N-glycan core structures, but IgG2 and IgG4, when present, were specific for the core α3-fucose and xylose motifs that were previously found to be IgE targets in schistosomiasis, and in allergies. This study is the first to address IgG subclass responses to defined helminth glycans.

Soluble egg antigen (SEA) and excretory-secretory (ES) products of the egg of Schistosoma mansoni contain a glycosylated T2 family ribonuclease termed omega-1 (ω1). Following release from the egg, ω1 instructs antigen presenting cells to induce naive CD4+ T cells to mature into T helper 2 effectors that, in turn, establish the Th2 immunological phenotype characteristic of schistosomiasis. Here, schistosome eggs were either transiently exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) of 20 nt complementary to exon 1 of i1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and this sgRNA. Subsequently, some eggs also were transduced with a single stranded oligodeoxynucleotide donor transgene that coded for a stop codon in all six frames, flanked by 50 nt-long 5’- and 3’-homology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the predicted DSB revealed ~4.5% of the reads (total reads >2×106 per treated sample) were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Messenger RNAs encoding ω1 were reduced >80%, and soluble egg antigen (SEA) from ω1-mutated eggs exhibited markedly reduced ribonuclease activity, indicative that programmed Cas9 cleavage mutated the ω1 gene. Following injection of schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas surrounding wild type eggs was 18-fold greater than ω-mutated eggs, 6.21×10−2 ± 1.61×10−3 versus 0.34×10−2 ± 0.12×10−4 mm3, respectively. In parallel, whereas wild-type SEA polarized Th2 cytokine responses including IL-4 and IL-5 in human monocyte/T cell co-cultures, significantly reduced levels of these cytokines followed the exposure to ω1-mutated SEA. In overview, programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end-joining, and mutation of ω1 impeded the capacity of schistosome eggs to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. These findings demonstrated for the first time the utility of CRISPR/Cas9-based genome editing for functional genomics for schistosomes.

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeX monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.

Summary While antigens from Schistosoma schistosomula have been suggested as potential vaccine candidates, the association between antibody responses with schistosomula antigens and infection intensity at reinfection is not well known. Schistosoma mansoni‐infected individuals were recruited from a schistosomiasis endemic area in Uganda (n = 372), treated with 40 mg/kg praziquantel (PZQ) and followed up at five weeks and at one year post‐treatment. Pre‐treatment and five weeks post‐treatment immunoglobulin (Ig) E, IgG1 and IgG4 levels against recombinant schistosomula antigens rSmKK7, rSmLy6A, rSmLy6B and rSmTSP7 were measured using ELISA. Factors associated with detectable pre‐treatment or post‐treatment antibody response against the schistosomula antigens and the association between five‐week antibody responses and one year post‐treatment reinfection intensity among antibody responders were examined. Being male was associated with higher pre‐treatment IgG1 to rSmKK7, rSmLy6a and AWA. Five weeks post‐treatment antibody responses against schistosomula antigens were not associated with one year post‐treatment reinfection intensity among antibody responders’ antibody levels against rSmKK7, rSmLy6B and rSmTSP7 dropped, but increased against rSmLy6A, AWA and SEA at five weeks post‐treatment among antibody responders. S. mansoni‐infected individuals exhibit detectable antibody responses to schistosomula antigens that are affected by treatment. These findings indicate that schistosomula antigens induce highly varied antibody responses and could have implications for vaccine development.

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

To accelerate the development of novel vaccines for schistosomiasis, we set out to develop a human model for Schistosoma mansoni infection in healthy volunteers. During natural infections, female schistosomes produce eggs that give rise to morbidity. Therefore, we produced single-sex, male Schistosoma mansoni cercariae for human infection without egg production and associated pathology. Cercariae were produced in their intermediate snail hosts in accordance with the principles of good manufacturing practice (GMP). The application of GMP principles to an unconventional production process is a showcase for the controlled production of complex live challenge material in the European Union or under Food and Drug Administration guidance.

Teichoic acids (TAs) are key components of the Gram‐positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well‐defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid‐phase synthesis approach for the rapid generation of well‐defined glycosylated, glycerol‐based TA oligomers. Building on the use of a “universal” linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA‐microarray, which is used to reveal, for the first time, the binding preferences of anti‐LTA (lipoteichoic acids) antibodies at the molecular level.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (K(m) = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

There is currently no vaccine against schistosomiasis. With few Schistosoma vaccine candidates in clinical trials, unexplored antigens from the vulnerable schistosomulum should be considered as possible vaccine candidates. In addition, we suggest developing synthetic vesicles as a new delivery vehicle and adjuvant for immunoprophylactic schistosomula vaccine candidates.

Glycoproteins and glycolipids of parasitic helminths play important roles in biology and host-parasite interaction. This review discusses recent helminth glycomics studies that have been expanding our insights into the glycan repertoire of helminths. Structural data are integrated with biological and immunological observations to highlight how glycomics advances our understanding of the critical roles that glycans and glycan motifs play in helminth infection biology. Prospects and challenges in helminth glycomics and glycobiology are discussed.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Deficiency of α-galactosidase A (α-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal α-N-acetyl-galactosaminidase (α-NAGAL) with increased α-galactosidase activity (α-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional α-GAL, α-NAGAL, and α-NAGALEL enzymes for research purposes. All enzymes could be visualized with activity-based probes covalently binding in their catalytic pocket. Characterization of purified proteins indicated that α-NAGALEL is improved in activity toward artificial 4MU-α-galactopyranoside. Recombinant α-NAGALEL and α-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than α-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of α-NAGALEL, and to a lesser extent that of α-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified α-NAGALEL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in Nicotiana benthamiana and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.

Background Human immunity to Schistosoma infection requires many years of exposure, and multiple infections and treatments to develop. Unlike humans, rhesus macaques clear an established schistosome infection naturally at the same time acquiring immunity towards re-infection. In macaques, schistosome egg production decreases after 8 weeks post-infection and by week 22, physiological impairment of the worm caused by unclarified antibody-mediated processes is observed. Since strong antibody responses have been observed against schistosome glycan antigens in human and animal infections, we here investigate if anti-glycan antibodies are associated with immunity against schistosome infections in macaques. Methods We used a microarray containing a large repertoire of glycoprotein- and glycolipid-derived glycans from different schistosome life stages to analyse anti-glycan serum IgG and IgM from S. japonicum-infected macaques during the course of infection and self-cure. We also used an in vitro schistosomula assay to investigate whether macaque sera containing anti-glycan antibodies can kill schistosomula. Conclusions/significance Antibody responses towards schistosome glycans at week 4 post-infection were dominated by IgM while IgG was high at week 8. The profound increase in IgG was observed mainly for antibodies towards a large subset of glycans that contain (multi-)fucosylated terminal GalNAcβ1-4GlcNAc (LDN), and Galβ1-4(Fucα1–3)GlcNAc (LeX) motifs. In general, glycans with a higher degree of fucosylation gave rise to stronger antibody responses than non-fucosylated glycans. Interestingly, even though many IgG and IgM responses had declined by week 22 post-infection, IgG towards O-glycans with highly fucosylated LDN motifs remained. When incubating macaque serum with schistosomula in vitro, schistosomula death was positively correlated with the duration of infection of macaques; macaque serum taken 22 weeks post-infection caused most schistosomula to die, suggesting the presence of potentially protective antibodies. We hypothesize that IgGs against highly fucosylated LDN motifs that remain when the worms deteriorate are associated with infection clearance and the resistance to re-infection in macaques., Author summary Schistosomes express many glycan antigens to which antibodies are raised by the infected host. These glycans may therefore form potential vaccine targets. Unlike humans where the disease persists chronically if not treated, schistosome-infected rhesus macaques are able to elicit a self-cure process naturally. To find out if anti-glycan responses could contribute to the natural clearance process, we followed the dynamics of anti-glycan serum antibodies in Schistosoma-infected macaques in a longitudinal study starting from the onset of infection until 22 weeks post-infection, when the macaques had eliminated most of the parasites. We found that sera of macaques taken after 22 weeks of infection contained high IgG titres towards specific schistosome glycan epitopes highly abundant on schistosome larvae. Moreover, infected macaque serum at week 22 was able to kill schistosomula in vitro. Our results suggest that anti-glycan antibodies play an important role in the self-cure process and the acquired resistance to re-infection in Schistosoma infected macaques.

Biofunctionalized gold nanoparticles (AuNPs) enable innovative translational research and development in biomedicine. Biomolecules such as peptides, proteins, lipids, and carbohydrates can be assembled onto AuNPs to yield nanomaterials with unique properties for applications in imaging, photothermal therapy, vaccination strategies, and drug delivery. The characterization of functionalized AuNPs still remains an analytical challenge that normally requires the combination of multiple techniques. Laser desorption/ionization (LDI) and matrix-assisted LDI (MALDI) have been applied successfully in combination with time-of-flight (TOF) mass spectrometry (MS) for the analysis of the surface chemistry of AuNPs functionalized with synthetic ligands, however only for ligands with a molecular mass limited to 1000 Da. TOF-MS-based approaches in addition exhibit limited performance in terms of mass resolution and MS/MS possibilities. To overcome these limitations, we designed an approach for the analysis of AuNPs based on ultrahigh resolution Fourier transform ion cyclotron resonance (FTICR) MS and a combination of LDI and MALDI. To illustrate the performance of the method, we present a comprehensive characterization of the surface chemistry of AuNPs conjugated via a thiol-ending linker to either the ovalbumin peptide (OVA 323-339), the Lewis X antigen (Galβ1-4[Fucα1-3]GlcNAcβ1) trisaccharide, the tetramannoside Manα1-2Manα1-2Manα1-3Manα1, or a mixture of both carbohydrates. Collision-induced dissociation (CID) was used to characterize the structure of pseudomolecular ions generated by LDI/MALDI in-depth. These included [M + H]+ and [M + Na]+, and importantly also [M + Au]+ and [M + 2Au–H]+ ions. This first observation of gold-containing pseudomolecular ions provides direct evidence for the Au-conjugation of ligands. In addition, we show the applicability of the method to monitor proteolytic cleavage of peptides that are conjugated to the AuNP surface. The presented LDI/MALDI–FTICR–MS and MS/MS approach will be applicable to the characterization of a wide range of functionalized AuNPs.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of Nglycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg., Author Summary Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. These worms infect the liver and can survive for many years in its animal or human host because they supress the host’s immune system that is important in clearing worm infection. Worms are similar to humans in that they are made of proteins, fats and sugars, and while there are many studies on worm proteins, few studies have examined the sugars. We are interested in the sugars because we believe that they help the parasite survive for many years within its host. To examine this, we have used a technique called mass spectrometric analysis to characterise the sugars present in F. hepatica. We also have developed systems in the laboratory to test if these sugars can suppress the host’s immune system. We conclude that F. hepatica sugars are crucial in suppressing its host’s immune system; however, the exact way the sugars can do this requires further studies. These studies are important for the development of worm vaccines or therapies.

Background Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA). Methodology/Principal findings We developed and evaluated an ELISA to detect human IgG4 antibodies to AsHb. We tested 1066 plasma samples collected at different times from 599 subjects who lived in a village in rural Indonesia that was highly endemic for ascariasis. The community received 6 rounds of MDA for lymphatic filariasis with albendazole plus diethylcarbamazine between 2002 and 2007. While the AsHb antibody assay was not sensitive for detecting all individuals with Ascaris eggs in their stools, the percentage of seropositive individuals decreased rapidly following MDA. Reductions in antibody rates reflected decreased mean egg output per person both at the community level and in different age groups. Two years after the last round of MDA the community egg output and antibody prevalence rate were reduced by 81.6% and 78.9% respectively compared to baseline levels. Conclusion/Significance IgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission., Author Summary Ascariasis is a neglected tropical disease caused by the intestinal nematode Ascaris lumbricoides that affects hundreds of millions of people in the developing world. Current methods for diagnosis of this infection are based on detecting eggs in the stool that are excreted by adult Ascaris worms. However, these methods have limited sensitivity for recent infections, and they do not detect infections with immature parasite stages that do not always result in the establishment of adult worms in the human intestine. We have previously shown that an assay for antibodies to Ascaris hemoglobin in pig serum is useful for assessing transmission of Ascaris infections on pig farms. In this study, we developed and evaluated a similar antibody assay that is based on the detection of human IgG4 antibodies to Ascaris haemoglobin (AsHb). Community antibody rates decreased rapidly following mass drug administration of the anthelmintic drug albendazole, and this decrease reflected reduced Ascaris egg excretion at the community level. This antibody test may be a useful tool for assessing the impact of control measures on the transmission of new Ascaris infections in endemic populations.

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite's eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/α1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/α1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/α1 is bound via the fucosylated Galβ1-4(Fucα1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells via all three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors. Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcβ1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcβ1-4(Fucα1-3)GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcβ1-4(Fucα1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcβ1-4(Fucα1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response. © 2012 Australian Society for Parasitology Inc..

Schistosome ribonuclease Omega-1 primes DCs to generate Th2 responses by binding and internalization by the mannose receptor and by subsequently impairing protein synthesis., Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynolds layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins. (C) 2010 Elsevier Inc. All rights reserved.

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered. Molecular & Cellular Proteomics 9: 667-681, 2010.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1–depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1–mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.

Lewis X trisaccharides normally function as essential cell–cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54-5C10-A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 A crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291-2G3-A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54-5C10-A. Proteins 2009. © 2008 Wiley-Liss, Inc.

Many diseases are associated with changes in the glycosylation of plasma proteins. To search for glycan biomarkers, large sample sets have to be investigated for which high-throughput sample preparation and analysis methods are required. We here describe a 96 well plate-based high-throughput procedure for the rapid preparation of 2-aminobenzoic acid (2-AA) labeled N-glycans from 10 microL of human plasma. During this procedure, N-glycans are released from glycoproteins and subsequently labeled with 2-AA without prior purification. A hydrophilic interaction chromatography (HILIC)-based solid phase extraction method is then applied to isolate the 2-AA labeled N-glycans, which can be analyzed by MALDI-TOF-MS, HPLC with fluorescence detection, and CE-MS. The relative standard deviation for the intrabatch repeatability and the interbatch repeatability of the sample preparation method remained below 7% and below 9%, respectively, for all peaks observed by HPLC. Similar results were obtained with MALDI-TOF-MS, where 47 N-glycans could be measured consistently. The 2-AA labeled N-glycans were additionally analyzed by a CE-ESI-Q-TOF-MS method, which featured high resolution and mass accuracy, allowing the unambiguous determination of the N-glycan compositions. Up to four times, 96 human plasma samples can be handled in parallel, which, together with the versatility of the 2-AA label, makes this procedure very attractive for glycomics analysis of larger sample cohorts.

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

Publication of the transcriptomes of Schistosoma mansoni and Schistosoma japonicum, in conjunction with the sequencing and assembly of their genomes, has generated a comprehensive picture of Schistosoma transcriptional and genomic diversity. Subsequently, researchers who study conjugal and developmental biology, tegumental composition and larval or egg, secretory and excretory products have used these data, in combination with the latest '-omics' technologies, to extend large-scale screens of the schistosome transcriptome, proteome and glycome. In this article, we review these postgenomic investigations and contend that the generated datasets provide a plethora of novel drug, vaccine and immunomodulatory targets that might be useful for developing new antischistosome agents.

In infections with Schistosoma mansoni the paired adult worms produce hundreds of eggs daily, of which many get trapped in various organs of the human host. The eggs produce complex and unique protein- and lipid-linked glycans, which are important activators and modulators of the host's immune response. The same parasite-derived glycoconjugates are also attractive immunodiagnostic targets in enzyme-linked immunosorbent assays (ELISAs), which detect circulating antigens in serum or urine of the host. Here, we report for the first time that in addition to glycoprotein and glycolipid antigens, schistosome eggs also excrete unique unconjugated oligosaccharides. Employing the schistosome-specific anti-carbohydrate monoclonal antibody 114-4D12 in an affinity purification approach, a specific set of free oligosaccharides was detected by matrix-assisted laser-desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in human S. mansoni infection urine as well as in egg-incubation medium, but not in worm-culture medium. Nano-scale reverse-phase liquid chromatography-mass spectrometry (nano-RP-LC-MS) analysis of the purified egg-derived oligosaccharides indicated that the captured compounds form a series of multi-fucosylated multimeric N-acetylhexosamine chains with a non-reducing terminal Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1- (DF-LDN-DF) sequence which forms the epitope of mAb 114-4D12. Since fucosylated (egg) glycoconjugates have been shown to harbour immunogenic properties, we anticipate that these unconjugated oligosaccharides also play a role in the immunobiology associated with schistosome eggs. Moreover, our data indicate that mass spectrometric detection of a set of signature molecules in urine has potential as a new approach for the diagnosis of schistosomiasis and possibly other helminth infections.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reverse-phase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Background Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. Methodology/Principal Findings In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. Conclusion This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack., Author Summary Schistosomiasis is one of the major parasitic diseases in developing countries and still causes 200,000 deaths per year. Mass drug administration programs with praziquantel, the drug of choice against schistosomiasis, are currently undertaken in Sub-Saharan Africa. Praziquantel, although efficient against adult worms, fails to cure early infection. The complex developmental stages of schistosomes and migration through varying host compartments with different local drug concentration are a challenge for drug treatment. After infecting their mammalian host, schistosomula traverse through skin and the vasculature of lung, liver and intestines. During their migration, they develop from larvae to paired adults in approximately 4 to 5 weeks. So far, drug effects have been analyzed on adult worms or freshly transformed schistosomula only. Information about the effects on the larval stages is lacking. We were able to transfer the larval development of the first three weeks into the culture dish. This tool can be used for the analysis of drug effects against schistosomula and for investigation of the accessibility, expression and localization of antigens. Rendering the parasite’s larvae vulnerable to the host’s immune system by increasing antigen presentation is an important aspect of drug activity. We demonstrate on in vitro-cultured Schistosoma mansoni larvae, that SmCD59a and SmKK7, as examples for hidden antigens, become accessible to antibodies following drug treatment.

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.

Schistosomiasis is characterised by periovular granuloma formation within the portal tract and presinusoidal venules. As inflammation wanes, continued attempts to wall off and repair hepatic injury, lead to the development of extensive fibrosis. The codependence of chronic inflammation and angiogenesis is a well-known phenomenon. Neovascularisation is a complex process of endothelial cell proliferation and remodelling of the extracellular matrix. Previous studies demonstrated the ability of schistosome soluble egg antigens (SEAs) to stimulate endothelial cell activation in vitro. In the present study, we investigated the angiogenic potential of SEA in Swiss and BALb/c mice, after infection with Schistosoma mansoni or S. haematobium and by implanting SEA-coated beads into the murine liver. Anti-CD34 and anti-Ki-67 immunohistochemical stainings demonstrated newly formed blood vessels within and at the periphery of the granulomas. However, in one third of the granulomas the pre-existing portal stroma was not destroyed by the granulomatous inflammation, angiogenesis was minimal or absent and further growth of the granuloma was prevented. In C57BL/6J and C3H/HeN inbred mice, this polarisation was even more pronounced. In 91% of the granulomas in C57BL6/J mice the portal stroma was preserved. These mice had significantly smaller granulomas, less fibrosis and less mortality as compared to the high pathology C3H/HeN mice, where 87% of the granulomas were of the angiogenic type with destruction of the pre-existing stroma, leading to more severe chronic pathology. Thus, host's genetic mechanisms regulating the degree of angiogenesis and fibrosis, determine the severity of schistosome-induced pathology.

Fragmentation behavior of fucosylated N-glycans in both protonated and sodiated form was studied by low-energy collision-induced dissociation with an ion trap mass spectrometer as well as by laser-induced dissociation with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Diantennary, core-(α1–6)-fucosylated N-glycans with Lewis X (Gal(β1–4)[Fuc(α1–3)]GlcNAcβ1-) and/or fucosylated LacdiNAc antennae (GalNAc(β1–4)[Fuc(α1–3)]GlcNAcβ1-) were obtained from the human parasite Schistosoma mansoni and used as model substances, after labeling with 2-aminobenzamide, or as native reducing glycans. While fragment spectra of sodiated as well as protonated species obtained in both mass spectrometers resulted in B- and Y-type ions, fragmentation of proton adducts additionally gave rise to various fragment ions which had acquired fucose residues from other parts of the molecule. In particular, fucose was transferred efficiently to the Lewis X antennae suggesting the occurrence of difucosylated antennae, which could erroneously be interpreted as Lewis Y epitopes. By studying two additional model substances, this fucose gain was shown to occur by transfer of fucose between the antennae, but not by transfer of a core-(α1–6)-fucose. Despite the drastically different lifetimes of the ions, protonated species analyzed on the ion trap (millisecond range) and by MALDI-TOF/TOF-MS (microsecond range) showed similar rearrangement patterns, suggesting that the fucose mobility goes hand in hand with decomposition. Notably, permethylation of the model N-glycans seemed to completely preclude fucose migration. This study indicates that caution should be applied with the interpretation of tandem mass spectrometric (MS/MS) data of protonated glycoconjugates, including glycopeptides, because of the potential occurrence of fucose rearrangements. Copyright © 2006 John Wiley & Sons, Ltd.

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.

Schistosomes, major parasitic helminths, express numerous glycoconjugates that provoke humoral and cellular immune responses in the infected human host. The main pathology in schistosomiasis is due to the formation of granulomas around tissue-trapped eggs and the resulting organ damage. By using a mouse model of induction of granulomas by hepatic implantation of antigen-coated beads, it has been determined that the glycan part of schistosomal soluble egg antigens (SEA) initiates granulomogenesis. To identify which individual glycan elements in this complex SEA mixture are granulomogenic, we have tested in the same mouse model conjugates of various synthetic oligosaccharides characteristic for schistosome eggs, including GalNAcbeta1-4GlcNAc (LacdiNAc, LDN), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewisx), Fucalpha1-2Fucalpha1-3GlcNAc (DF-Gn), and Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F). Ribonuclease (RNase) A and B, and different fetuin glycoforms were included as controls. Only beads that carry glycoconjugates with terminal LacdiNAc or Galbeta1-4GlcNAc (LacNAc, LN) elements gave rise to granulomas, with macrophage, lymphocyte, and eosinophil levels similar to the granulomatous lesions caused by schistosome eggs in a natural infection. Uncoated beads, and beads coated with fucosylated glycoconjugates or glycoconjugates lacking terminally exposed Gal or GalNAc, only attracted a monolayer of macrophages. These results indicate that the formation of hepatic granulomas is triggered specifically by glycoconjugates which carry terminal LacNAc or LacdiNAc, both constituents of the schistosome egg.

Fitzsimmons, C. M., Schramm, G., Jones, F. M., Chalmers, I. W., Hoffmann, K. F., Grevelding, C. G., Wuhrer, M., Hokke, C. H., Haas, H., Doenhoff, M. J., Dunne, D. W. (2005). Molecular characterization of omega-1: A hepatotoxic ribonuclease from Schistosoma mansoni eggs. Molecular & Biochemical Parasitology, 144(1), pp. 123-7.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 μl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver–operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6–BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Conventional normal-phase high performance liquid chromatography of fluorescently labeled oligosaccharides with or without on-line mass spectrometry is an established tool for the structure characterization of protein and lipid derived glycans. Here we describe the miniaturization of such a system to the nano-scale using a 75 μm internal diameter normal-phase amide column on-line with electrospray ionization ion-trap mass spectrometry. 2-Aminobenzamide-labeled oligosaccharides have a predictable retention on this normal-phase column that can be expressed as glucose units by comparison to the retention of a standard 2-aminobenzamide glucose polymer mixture. Isobaric compounds are separated on the basis of their structural differences, and by on-line electrospray ionization ion-trap mass spectrometry, the sequence of the monosaccharides can be deduced. The major improvement of the on-line nano-LC-MS system in comparison to conventional systems is the gain in sensitivity with detection of low femtomole amounts of glycans. This implies that LC-MS of 2-aminobenzamide-labeled oligosaccharides can now be performed at higher sensitivity than their analysis with fluorescence detection.

We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures. Separation of oligosaccharide mixtures over a 1-h range permits the detailed characterization of the different species by multiple ion selection and fragmentation steps using ion trap MS. The here presented miniaturization of the online-LC system to the nanoscale in combination with ion trap MS allows the detection of oligosaccharide species in a mixture at low-femtomole sensitivity. Online normal-phase nano-LC-MS of complex oligosaccharide mixtures further facilitates the sensitive and detailed structural analysis of oligosaccharides by overcoming the need for cumbersome and time-consuming derivatization procedures such as reductive amination for labeling with hydrophobic fluorophores or labeling with tritium. The method should be useful for the sensitive and quick analysis of glycosylation patterns and individual oligosaccharides from biotechnologically produced glycoproteins as well as scarcely available biological samples.

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucα1-3GalNAcβ1-4GlcNAcβ1-3Galα1), LDN-DF [GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAcβ1], and LDNF [GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Galα1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, whereSchistosoma mansoniis endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls. Associations with responses toS. mansoniworm, egg (SEA), and cercarial (CERC) antigens were examined. In addition, we measured responses to keyhole limpet hemocyanin, a glycoprotein which carries glycan epitopes that are also expressed by schistosomes. Specific IgG1 responses were most pronounced against FLDN and LDN-DF and strongly associated with those previously measured to SEA and CERC. Similarly to previously published age profiles of IgG1 and IgG2 responses to SEA, levels of IgG1 against LDN-DF decreased with age. In contrast, specific IgM responses against the three schistosome-derived carbohydrate structures were most marked against LDNF. Our results indicate that, of the three glycan structures tested, the acute response against schistosome glycoconjugate antigens in young children is mainly directed against the LDN-DF epitope. The response to LDN-DF in older individuals and the responses to the two other epitopes were similar in the two cohorts, suggesting that these antigens are recognized in the early stages of infection and that the immune response persists. The biological significance of these observations needs further elucidation.

Background : Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos. Results : Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30–100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5′ tsRNAs and fourteen 3′ tsRNAs). Conclusions : The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions. Keywords: extracellular vesicles; small non-coding RNAs; miRNAs; tsRNAs; proteome; Schistosoma mansoni; platyhelminth (Published: 5 October 2015) Citation: Journal of Extracellular Vesicles 2015, 4: 28665 - http://dx.doi.org/10.3402/jev.v4.28665

Contains fulltext : 155377.pdf (Publisher’s version ) (Open Access) Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galbeta1-4(Fucalpha1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcbeta1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with alpha3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galbeta1-3(Galbeta1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1¿ and IgG1¿ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-a, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1¿-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1¿- and IgG1¿-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1¿-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core a1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core a1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets.

Graphical abstract, Highlights • Schistosoma mansoni VAL9 (SmVAL9) is a secreted N-linked glycoprotein containing a unique, difucosyl modification. • SmVAL9 is found throughout miracidia/sporocyst parenchymal cell inclusions/vesicles and germinal cells. • SmVAL9 differentially regulates murine and snail matrix metalloproteinases., The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.