Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array, we explored the interactions of glycans with C-type lectins, C-reactive protein, and sera from T. suis-infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Trichuris trichiura egg extract proteome reveals potential diagnostic targets and immunomodulators.

Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae's surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.

Antihypertensive effects of Trichiurus lepturus myosin hydrolysate in spontaneously hypertensive rats.

In this study, the blood pressure-lowering effects of Trichiurus lepturus myosin hydrolysate (TMH) and its possible mechanism were investigated in spontaneously hypertensive rats (SHRs). After gavage administration of TMH for 4 h, systolic blood pressure (SBP) was significantly decreased in SHRs. Furthermore, the SBP of SHRs remained low at 1 month after daily TMH treatment at 400 mg kg-1. Meanwhile, plasma levels of angiotensin II, bradykinin and nitric oxide in SHRs were ameliorated by TMH. Western blotting also suggested that TMH down-regulated the expression of ICAM-1 and VCAM-1, indicating a strong anti-inflammatory effect. Additionally, nitrotyrosine and collagen I were down-regulated, revealing a significant anti-oxidant effect of TMH. No obvious side effects or toxicity were observed in normal Wistar rats given TMH. Various pathogenic factors related to hypertension were improved by TMH, which may explain the underlying mechanism by which TMH synergistically reduces blood pressure.

Effect of dewatering and composting on helminth eggs removal from lagooning sludge under semi-arid climate.

In this work, we assessed the drying and composting effectiveness of helminth eggs removal from sewage sludge of a lagoon wastewater treatment plant located in Chichaoua city. The composting was run after mixing sludge with green waste in different proportions: M1 (½ sludge + ½ green waste), M2 ([Formula: see text] sludge + [Formula: see text] green waste), and M3 ([Formula: see text] sludge + [Formula: see text] green waste) for 105 days. The analysis of the dewatered sewage sludge showed a load of 8-24 helminth eggs/g of fresh matter identified as Ascaris spp. eggs (5-19 eggs/g) followed by Toxocara spp. (0.2 to 2.4 eggs/g); Hookworm spp. and Capillaria spp. (0.4-1 egg/g); Trichuris spp., Taenia spp., and Shistosoma spp. (< 1 egg/g) in the untreated sludge. After 105 days of treatment by composting, we noted a total reduction of helminth eggs in the order of 97.5, 97.83, and 98.37% for mixtures M1, M2, and M3, respectively. The Ascaris spp. eggs were reduced by 98% for M1 and M3 treatments and by 97% for M2 Treatment. Toxocara spp., Hookworm spp., Trichuris spp., Capillaria spp., and Shistosoma spp. eggs were totally eliminated (100% decrease) and the Taenia spp. was absent from the first stage of composting. These results confirm the effectiveness of both dehydrating and composting processes on the removal of helminth eggs.

Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

Helminths have strong immunoregulatory properties that may be exploited in treatment of chronic immune disorders, such as multiple sclerosis and inflammatory bowel disease. Essential players in the pathogenesis of these diseases are proinflammatory macrophages. We present evidence that helminths modulate the function and phenotype of these innate immune cells. We found that soluble products derived from the Trichuris suis (TsSP) significantly affect the differentiation of monocytes into macrophages and their subsequent polarization. TsSPs reduce the expression and production of inflammatory cytokines, including IL-6 and TNF, in human proinflammatory M1 macrophages. TsSPs induce a concomitant anti-inflammatory M2 signature, with increased IL-10 production. Furthermore, they suppress CHIT activity and enhance secretion of matrix metalloproteinase 9. Short-term triggering of monocytes with TsSPs early during monocyte-to-macrophage differentiation imprinted these phenotypic alterations, suggesting long-lasting epigenetic changes. The TsSP-induced effects in M1 macrophages were completely reversed by inhibiting histone deacetylases, which corresponded with decreased histone acetylation at the TNF and IL6 promoters. These results demonstrate that TsSPs have a potent and sustained immunomodulatory effect on human macrophage differentiation and polarization through epigenetic remodeling and provide new insights into the mechanisms by which helminths modulate human immune responses.-Hoeksema, M. A., Laan, L. C., Postma, J. J., Cummings, R. D., de Winther, M. P. J., Dijkstra, C. D., van Die, I., Kooij, G. Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

Sweet secrets of a therapeutic worm: mass-spectrometric N-glycomic analysis of Trichuris suis.

Trichuris suis, a nematode parasite of pigs, has attracted attention as its eggs have been administered to human patients as a potential therapy for inflammatory diseases. The immunomodulatory factors remain molecularly uncharacterised, but in vitro studies suggest that glycans on the parasite's excretory/secretory proteins may play a role. Using an off-line LC-MS approach in combination with chemical and enzymatic treatments, we have examined the N-linked oligosaccharides of T. suis. In addition to the paucimannosidic and oligomannosidic N-glycans typical of many invertebrates, a number of glycans carry N,N'-diacetyllactosamine (LacdiNAc) modified by fucose and/or phosphorylcholine. Such antennal epitopes are similar to ones previously associated with immunomodulation by helminths; here we propose phosphorylcholine modifications predominantly of terminal N-acetylgalactosamine but also of subterminal α1,3-fucosylated N-acetylglucosamine. Exact knowledge of the glycome of T. suis will facilitate more targeted studies on glycan receptors in the host as well as the engineering of cell lines to produce correctly glycosylated recombinant forms of candidate proteins for future studies on immunomodulation.

The helminth Trichuris suis suppresses TLR4-induced inflammatory responses in human macrophages.

Recent clinical trials in patients with inflammatory diseases like multiple sclerosis (MS) or inflammatory bowel disease (IBD) have shown the beneficial effects of probiotic helminth administration, although the underlying mechanism of action remains largely unknown. Potential cellular targets may include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation, leading to reduced IL-1ß secretion. In conclusion, we show that T. suis SPs suppress a broad range of inflammatory pathways in GM-CSF-differentiated macrophages in a TLR4-dependent manner, thereby providing enhanced mechanistic insight into the therapeutic potential of this helminth for patients with inflammatory diseases.

A proteomic approach to identify proteins from Trichuris trichiura extract with immunomodulatory effects.

Infections with Trichuris trichiura and other trichurid nematodes have been reported to display protective effects against atopy, allergic and autoimmune diseases. The aims of the present study were to investigate the immunomodulatory properties of T. trichiura adult worm extract (TtE) and its fractions (TtEFs) on the production of cytokines by peripheral blood mononuclear cells and to identify their proteinaceous components. Fourteen TtEFs were obtained by ion exchange chromatography and tested for effects on cytokine production by peripheral blood mononuclear cells. The molecular constituents of the six most active fractions were evaluated using nano-LC/mass spectrometry. The homology between T. trichiura and the related nematode Trichinella spiralis was used to identify 12 proteins in TtEFs. Among those identified, fructose biphosphate aldolase, a homologue of macrophage migration inhibitory factor and heat-shock protein 70 may contribute to the immunomodulatory effects of TtEFs. The identification of such proteins could lead to the development of novel drugs for the therapy of allergic and other inflammatory diseases.

Trichuris suis-induced modulation of human dendritic cell function is glycan-mediated.

Human monocyte-derived dendritic cells (DCs) show remarkable phenotypic changes upon direct contact with soluble products (SPs) of Trichuris suis, a pig whipworm that is experimentally used in therapies to ameliorate inflammation in patients with Crohn's disease and multiple sclerosis. These changes may contribute to the observed induction of a T helper 2 (Th2) response and the suppression of Toll-like receptor (TLR)-induced Th1 and Th17 responses by human DCs primed with T. suis SPs. Here it is demonstrated that glycans of T. suis SPs contribute significantly to the suppression of the lipopolysaccharide (LPS)-induced expression in DCs of a broad variety of cytokines and chemokines, including important pro-inflammatory mediators such as TNF-α, IL-6, IL-12, lymphotoxin α (LTA), C-C Motif Ligand (CCL)2, C-X-C Motif Ligands (CXCL)9 and CXCL10. In addition, the data show that human DCs strongly bind T. suis SP-glycans via the C-type lectin receptors (CLRs) mannose receptor (MR) and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN). The interaction of DCs with T. suis glycans likely involves mannose-type glycans, rather than fucosylated glycans, which differs from DC binding to soluble egg antigens of the human worm parasite, Schistosoma mansoni. In addition, macrophage galactose-type lectin (MGL) recognises T. suis SPs, which may contribute to the interaction with immature DCs or other MGL-expressing immune cells such as macrophages. The interaction of T. suis glycans with CLRs of human DCs may be essential for the ability of T. suis to suppress a pro-inflammatory phenotype of human DCs. The finding that the T. suis-induced modulation of human DC function is glycan-mediated is novel and indicates that helminth glycans contribute to the dampening of inflammation in a wide range of human inflammatory diseases.

Trichuris suis: detection of antibacterial activity in excretory-secretory products from adults.

Antibacterial activity was detected in excretory-secretory products (ESP) of adult Trichuris suis cultured in vitro in serum-free media. Gram-negative bacteria (Campylobacter jejuni, Campylobacter coli, and Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were sensitive to ESP. Susceptibility was dependent on the concentration of ESP but not on the inoculum size. Preliminary assessment of the mode of action suggests a bacteriocidal mechanism. This antibacterial activity was heat stable and resistant to digestion with pronase E and trypsin. Based on ultrafiltration experiments, the activity is less than 10,000 MW. This excreted/secreted antibacterial activity from T. suis is likely a component of a humoral defense system for this helminth.

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator.

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.

Trichuris suis: A secretory serine protease inhibitor.

A trypsin inhibitor was identified in extracts of adult Trichuris suis and culture fluids from 24-h in vitro cultivation of adult parasites. The inhibitor was isolated by acid precipitation, affinity chromatography (trypsin-agarose), and reverse phase HPLC as a single polypeptide with a molecular weight estimated at 6.6 kDa by laser desorption mass spectrometry. The purified inhibitor associated strongly with trypsin (equilibrium dissociation inhibitory constant (K(j)) of 3.07 nM) and chymotrypsin (K(j) = 24.5 nM) and was termed TsTCI. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular mass of 6.687 kDa. The N-terminal region of TsTCI (46 residues) showed limited homology (36%) to a protease inhibitor from the hemolymph of the honeybee Apis mellifera, which is considered to be a member of the Ascaris inhibitor family. However, TsTCI did not display sequence homology with other members of this family or the distinctive cysteine residue pattern which distinguishes this family. However, similarity of a region of TsTCI (11 residues) with the reactive site regions of inhibitors from the nematodes Ascaris suum, Anisakis simplex, and Ancylostoma caninum was apparent.

The major secreted product of the whipworm, Trichuris, is a pore-forming protein.

The data presented here describe the first unequivocable characterization of a pore-forming protein in any helminth parasite. The excretory/secretory (E/S) material of the human whipworm T. trichiura contains a highly abundant protein of molecular mass 47 kDa (TT47); the murine model parasite, T. muris, contains a similarly abundant protein of molecular mass 43 kDa (TM43). When purified to homogeneity, these proteins induce ion-conducting pores in lipid bilayers. Antibodies raised against TM43 abolish channel activity. Pore formation in epithelial cell membranes may facilitate invasion of the host gut by Trichuris and enable the parasite to maintain its syncytial environment in the caecal epithelium. The observation that this activity may be inhibited by antibody opens a possible avenue for drug and vaccine development.

Trichuris chilensis: physicochemical and immunological characterization of the different antigenic components.

The protein pattern of Trichuris chilensis obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was analyzed. Complex protein band patterns covering a wide range of molecular weights were obtained. The molecular weights of the major proteins present in different tissue homogenates were estimated. Antisera raised in rabbits against homogenates of T. chilensis and sera from naturally infected Ctenomys australis were used in Western blotting, immunoelectrophoresis and passive hemagglutination to compare the antigenicity of the adult male, adult female, eggs, oocytes, stichosome and cuticle of this parasite. Specific antibodies to parasite antigens were also detected in faecal preparations and caecum mucosal extracts of C. australis naturally infected with T. chilensis.