Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Interleukin-1ß affects the phospholipid biosynthesis of fibroblast-like synoviocytes from human osteoarthritic knee joints.

OBJECTIVE: Phospholipids (PLs), together with hyaluronan and lubricin, are involved in boundary lubrication within human articular joints. Levels of lubricants in synovial fluid (SF) have been found to be associated with the health status of the joint. However, the biosynthesis and release of PLs within human joints remains poorly understood. This study contributes to our understanding of the effects of cytokines on the biosynthesis of PLs using cultured fibroblast-like synoviocytes (FLS) from human osteoarthritic knee joints. METHODS: Cultured FLS were stimulated with IL-1ß, TNFα, IL-6, or inhibitors of cell signaling pathways such as QNZ, SB203580 and SP600125 in the presence of stable isotope-labeled precursors of PLs. Lipids were extracted and quantified using electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Our analyses provide for the first time a detailed overview of PL species being synthesized by FLS. IL-1ß increased the biosynthesis of both phosphatidylethanolamine (PE) and PE-based plasmalogens. We show here that the NF-κB, p38 MAPK and JNK signaling pathways are all involved in IL-1ß-induced PL biosynthesis. IL-6 had no impact on PLs, whereas TNFα increased the biosynthesis of all PL classes. CONCLUSION: The biosynthesis of various PLs is controlled by IL-1ß and TNFα. Our detailed PL species analysis revealed that FLS can partly contribute to the elevated PL levels found in human osteoarthritis (OA) SF. IL-1ß in particular stimulates PE and PE-based plasmalogens which can act as cell-protective antioxidants. These results suggest that during OA progression, FLS undergo alterations in their PL composition to adapt to the new diseased environment.

Genetic variants of bovine ß- and κ-casein result in different immunoglobulin E-binding epitopes after in vitro gastrointestinal digestion.

Immunoglobulin E-mediated allergy to cow milk is a common allergy in industrialized countries, mainly affecting young children and infants. ß-Casein (CN) and κ-CN belong to the major allergens in cow milk. Within these milk proteins, genetic polymorphisms occur, which are characterized by substitutions or deletions of AA, resulting in different variants for each protein. Until now, these variants have not been considered when discussing the allergenic potential of bovine milk. In this study, the focus was placed on the arising peptide pattern after in vitro gastrointestinal digestion of several ß- and κ-CN variants to determine resistant fragments containing IgE-binding epitopes and to identify potential differences between these variants. ß-Casein A(1), A(2), and B, as well as κ-CN A, B, and E, were separated and isolated from milk of cows homozygous for these variants and digested with an in vitro gastrointestinal digestion model. The resulting peptides were identified using mass spectrometry and compared with previously determined epitopes. Seven ß-CN and 4 κ-CN peptides, common in all ß- or κ-CN variants, remained of sufficient size to harbor IgE-binding epitopes. In addition, some peptides and, consequently, epitopes differ from each other due to the AA substitution occurring in the individual variants. The distinct peptides AA 108 to 129 of ß-CN A(1) and A(2), AA 103 to 123 of ß-CN B, as well as AA 59 to 72, AA 59 to 80, and AA 58 to 80 of all 3 ß-CN variants correspond to the IgE-binding epitopes AA 107 to 120 and AA 55 to 70, respectively. In κ-CN, the 2 variant-specific peptides AA 136 to 149 (κ-CN A, E) and AA 134 to 150 (κ-CN B) are congruent with the IgE-binding epitope AA 137 to 148. The present study shows that genetic polymorphisms affected the arising peptide pattern of the caseins and thus modifications in the IgE-binding epitopes occurred. As a consequence, the casein variants could show differences in their allergenicity. Studies investigating the allergenic potential of these different peptides are currently in progress.

Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

N6-Methyladenosine detected in RNA of testicular germ cell tumors is controlled by METTL3, ALKBH5, YTHDC1/F1/F2, and HNRNPC as writers, erasers, and readers.

BACKGROUND: Type II testicular germ cell tumors (GCTs) arise from a common precursor lesion (germ cell neoplasia in situ) and are stratified into seminomas and non-seminomas, which differ considerably in morphology, gene expression, and epigenetic landscape. The N6-methyladenosine (6mA) epigenetic modification is the most abundant modification in mRNA and is also detectable in eukaryotic DNA. The functional role of 6mA is not fully understood, but 6mA residues may influence transcription by affecting splicing, miRNA processing, and mRNA stability. Additionally, the methyl group of 6mA destabilizes Watson-Crick base-pairing affecting RNA structure and protein binding. OBJECTIVES: Here, we analyzed the presence of the 6mA epigenetic modification in germ cells and GCT tissues and cell lines. MATERIALS AND METHODS: We screened for the presence of 6mA in DNA and RNA by immunohistochemistry, mass spectrometry or ELISA-based quantification assays. Additionally, expression of 6mA writer-, eraser- and reader-factors was analyzed by microarrays, qRT-PCR, western blotting and screening of public databases. RESULTS: We demonstrate that 6mA is detectable in RNA, but not DNA, of GCT cell lines and tissues, fibroblasts, and Sertoli cells as well as germ cells of different developmental stages. Based on expression analyses, our results suggest METTL3, ALKBH5, YTHDC1, YTHDF1, YTHDF2 and HNRNPC as main writers, erasers, and readers of the 6mA modification in GCTs. DISCUSSION: Owing to the lack of 6mA in DNA of GCTs, a functional role in regulating DNA transcription can be excluded. Interestingly, expression levels of 6mA regulators are comparable between tumor and normal tissues/cells, suggesting a similar mechanism of 6mA regulation in RNA. Finally, we demonstrate that 6mA levels in RNA increase upon differentiation of GCT cell lines, suggesting a role of 6mA in cell fate decisions. CONCLUSION: In summary, our data provide the starting point for further experiments deciphering the role of 6mA in the RNA of GCTs.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Structural analysis of glycolipids from Borrelia burgdorferi.

In this study the lipids of Borrelia burgdorferi, the causative agent of Lyme disease, were analyzed. Lipids comprise about 25-30% of the cell dry weight. The lipid fraction could be separated by HPTLC into 11 components. Staining of these components revealed two glycolipids and two phospholipids. The glycolipids represented about 50% of the total lipids and comprised only galactose as monosaccharide constituents. By means of mass spectrometric and gas chromatographic analysis both glycolipids could be identified as alpha-galactosyl-diacylglycerolipids with different fatty acid compositions. The phospholipids were identified as phosphatidylcholine and phosphatidylglycerol. Immunoassays with sera from patients with Lyme disease showed antibody reactivity only to the glycolipids, which was present in all stages of the disease. Other lipid components seemed to be non-immunogenic in Lyme disease. The glycolipids of B. burgdorferi may be, thus, considered promising candidates for diagnosis and possibly also for vaccination.

Immunochemical characterisation of Schistosoma mansoni glycolipid antigens.

The aim of this study was to investigate the occurrence, distribution and immunochemical properties of antibody-defined carbohydrate epitopes in neutral glycolipid fractions of Schistosoma mansoni eggs, cercariae and adults. The amount of extractable, antigenic, neutral glycolipids was lowest in adult worms, increasing consecutively in cercariae and eggs. The immunoreactivity of the glycolipids resided in the carbohydrate moiety in that it was periodate-sensitive. Serological reactivity, and monosaccharide component analysis, anomeric configuration and methylation-linkage analyses indicated that there were two dominant epitopes, which could be partially defined immunologically. The first epitope was detected on egg, cercarial and adult glycolipids. It was strongly recognised by mouse chronic infection sera and rabbit hyperimmune sera raised against specific egg antigens, and was defined by the monoclonal antibody M2D3H (Bickle QD, Andrews BJ. Characterisation of Schistosoma mansoni monoclonal antibodies which block in-vitro killing: failure to demonstrate blockage of immunity in vivo. Parasite Immunol 1988;10:151-168). M2D3H appeared to have the same epitope specificity as monoclonal antibody 128C3/3 (Weiss J, Magnani JL, Strand M. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J Immunol. 1986;136:4275-82). The internal epitope was defined structurally by the presence of fucose 3-linked to 3,4-disubstituted N-acetylglucosamine, which was itself partially substituted by a second fucose residue, to yield the determinant -4[Fucalpha1,2Fucalpha3]GlcNAcbeta1-. The second epitope was defined by the anti-LewisX monoclonal antibody 4D1 and was found primarily on cercarial glycolipids. It was chemically characterised as the LewisX epitope of Galbeta1,4[Fucalpha1,3]GlcNAcbeta1- in a terminal position. The removal of fucose greatly diminished the binding of the anti-LewisX and M2D3H monoclonal antibodies, as well as the polyclonal chronic infection sera, to glycolipids of all three life-cycle stages and thus revealed the epitopic importance of fucose.

RNase1 prevents the damaging interplay between extracellular RNA and tumour necrosis factor-α in cardiac ischaemia/reperfusion injury.

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial "ischaemia/reperfusion (I/R) injury" remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Surfactant release from hydrophilized vinylpolysiloxanes.

Vinylpolysiloxane impression materials (VPS) exhibit an apolar (hydrophobic) backbone chemistry. Hence, surfactants are added to improve their hydrophilicity for impression-taking in moist environments. However, the mechanisms at the liquid-VPS-interface regarding the surfactant are unknown. We hypothesized that surfactant is leached from the VPS. Four experimental VPS formulations were fabricated containing 0 (control), 1.5, 3, and 5 wt% non-ionic surfactant. Samples were prepared (n = 6) and contact angles determined 30 min after mixing. After 60 sec, droplets were transferred onto the control. Mass spectrometry was used to analyze the droplets. Contact angles were inversely correlated with the surfactant concentration (p < 0.05). Droplets transferred from hydrophilized specimens onto the control showed similar contact angles. Surfactant could be clearly identified inside the droplets from the hydrophilized samples, however, not inside the control. Surfactants reduced the surface tension of the liquid in contact and did not change the surface properties of the VPS itself.

Dissecting Ascaris glycosphingolipids for immunomodulatory moieties--the use of synthetic structural glycosphingolipid analogues.

We have previously shown glycosphingolipids of Ascaris suum to have phosphorylcholine (PC) and non-PC immunomodulatory moieties. In the present study we further investigated the nature of the immunomodulatory moieties by employing three synthetic glycosphingolipids each possessing features of the original molecule to examine effects on macrophage and dendritic cell (DC) cytokine production and surface co-stimulatory molecule expression. Compound 2, which lacked PC but contained ceramide, had no effect on either macrophages or DCs. Surprisingly however, Compound 1, which contained PC and hence arguably most resembled the native material, had, with the exception of a small increase in surface antigen expression, no immunomodulatory properties. Conversely, Compound 3, which contained PC but was otherwise least like the native molecule, demonstrated a number of effects on both macrophages and DCs, including induction of Th-1/pro-inflammatory cytokines, inhibition of such cytokines induced by IFN-gamma/LPS and increased expression of co-stimulatory molecules. Taken together these results indicate: (i) that although PC is an immunomodulatory component of the native molecule other structural feature are necessary to allow it to act; (ii) that carbohydrate rather than ceramide is likely to represent a non-PC immunomodulatory moiety; and (iii) that synthetic PC-containing molecules have the potential to act as immunomodulatory drugs.

Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

Carbohydrate structure analysis of batroxobin, a thrombin-like serine protease from Bothrops moojeni venom.

The carbohydrate side chains of batroxobin were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, pyridylaminated and separated by two-dimensional HPLC. Neutral oligosaccharide derivatives obtained after desialylation were characterized by methylation analysis, liquid secondary-ion mass spectrometry, digestion with exoglycosidases and endoglycosidases and, in part, by acetolysis, whereas sialic acid constituents were identified by reverse-phase HPLC after conjugation with 1,2-diamino-4,5-methylene-dioxybenzene. The overall glycosylation status of the protein was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results revealed that batroxobin is heterogeneously glycosylated carrying predominantly diantennary, partially incomplete complex-type glycans in addition to hybrid-type species. Most glycans were core-fucosylated at C6 of the innermost GlcNAc. As a characteristic feature, galactose was completely replaced by GalNAc beta 4-substituents in complex-type antennae, the GlcNAc-residues of which were, in part, fucosylated at C3. Furthermore, evidence was obtained that suggested the presence of a novel type of glycoprotein-N-glycan comprising two GalNAc beta 4GlcNAc beta 4GlcNAc beta 2Man-antennae. Sialic acid residues represented a mixture of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), which were exclusively linked to C3 of subterminal GalNAc. A precise assignment of these sialic acid derivatives to distinct oligosaccharide structures or antennae, however, was not carried out. Finally, MALDI-TOF-MS demonstrated that both potential N-glycosylation sites of batroxobin are substituted by carbohydrate chains. In conclusion, our studies revealed that this snake venom glycoprotein is characterized by a unique oligosaccharide pattern partly comprising novel structural elements.

Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda:Rhabditida).

The free-living nematode, Caenorhabditis elegans, has been proposed and analyzed as a prototypic model for parasitic nematodes. In order to study whether there is a structural basis for the proposed analogy with respect to nematode glycoconjugates, we have analyzed Caenorhabditis elegans glycosphingolipids. Three, simple neutral glycosphingolipid components of the neutral glycolipid fraction were isolated by high-performance liquid chromatography. Structural analysis was performed by methylation analysis, exoglycosidase cleavage, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, and ceramide analysis. The chemical structures have been determined as Glc beta 1Cer, Man beta 4Glc beta 1Cer and GlcNAc beta 3Man beta 4Glc beta 1Cer; that are characterized as belonging to the arthroseries of protostomial glycosphingolipids. The ceramide moiety of the parent glycosphingolipid-ceramide mono-hexoside was dominated by 2-hydroxy fatty acids, and a d17:1 spingoid-base with an iso- or anteiso-branched chain. The chemical composition of the three glycosphingolipids from Caenorhabditis elegans displayed close structural coincidence with the equivalent structures from the porcine parasitic nematode, Ascaris suum (G.Lochnit, R.D. Dennis, U.Zähringer, and R.Geyer, Glycoconjugate J., 1997), in support of this organism as a prototypic glycosphingolipid model for parasitic nematodes.

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda:Ascaridida).

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(beta 1-1)ceramide, Man(beta 1-4)Glc(beta 1-1)ceramide, GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)ceramide and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine.

Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine, parasitic nematode, Ascaris suum.

The acidic glycolipid fraction (AF) of the porcine, parasitic nematode, Ascaris suum , consisted of two subfractions. The major component AF II reacted with orcinol-sulfuric acid and molybdate, while the minor component AF I gave a positive reaction with azure-A, a cationic dye specific for sulfatides. Sugar constituent analysis, methanolysis, methylation analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary-ion mass spectrometry, and gas-liquid chromatography/mass spectrometry specified AF II to be an unusual phosphoinositolglycosphingolipid (Galalpha1-Ins-P-1ceramide) and the minor component AF I to be a 3-sulfogalactosylcerebroside (HSO3-3Galss1-1ceramide). The ceramide moiety of both components consisted of lignoceric (C24:0) and cerebronic (C24h:0) acids and mainly C17 iso-branched sphingosine. Immunohistochemical localization studies of the glycolipid-bound antigenic determinants with a polyclonal antiserum against AF II and an anti-sulfatide monoclonal antibody against AF I revealed the presence of the AF II-epitope in the intestine, whereas the AF I-epitope was found in the hypodermis, contractile zone of somatic muscle cells and the external musculature of the uterus. To our knowledge, this is the first report of the presence of a sulfatide in an invertebrate.

Phosphorylcholine substituents in nematodes: structures, occurrence and biological implications.

Phosphorylcholine (PC), a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes, as well as in fungi. Linked to parasite-specific glycoprotein glycans or glycolipids, it is assumed to be responsible for a variety of immunological effects, including invasion mechanisms and long-term persistence of parasites within the host. Numerous reports have indicated various effects of PC-substituted molecules derived from parasitic nematodes on signal transduction pathways in B and T lymphocytes, displaying a highly adapted and profound modulation of the immune system by these parasites. The Nematoda, comprising parasitic and free-living species, can be regarded as promising prototypic systems for structural analyses, immunological studies and biosynthetic investigations. In this context, Ascaris suum, the pig parasitic nematode, is an ideal organism for immunological studies and an excellent source for obtaining large amounts of PC-substituted (macro)molecules. Caenorhabditis elegans, as a completely genome-sequenced species and expressing parasite analogous PC-substituted structures, together with the possibility for easy in vitro cultivation, represents a conceptual model for biosynthetic studies, whereas filarial parasites represent important model systems for human pathogens, especially in developing countries. This review summarises current knowledge on the tissue-specific expression of PC epitopes, structural data of glycoprotein glycans and glycosphingolipids bearing this substituent and biological implications for the immune systems of the respective hosts.

Structural elucidation and monokine-inducing activity of two biologically active zwitterionic glycosphingolipids derived from the porcine parasitic nematode Ascaris suum.

The isolated neutral glycosphingolipid fraction from the pig parasitic nematode, Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor alpha, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below), [structure: see text] The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.

Immunohistochemical localization and differentiation of phosphocholine-containing antigens of the porcine, parasitic nematode, Ascaris suum.

The glycolipids of Ascaris suum represent either neutral, zwitterionic or acidic structures. The acidic fraction comprises a sulphatide and an unusual phosphoinositolglycosphingolipid (Lochnit et al. 1998b). The sulphatide was previously localized to the hypodermis, contractile zone of somatic muscle cells and the external musculature of the female uterus, whereas the presence of the phosphoinositolglycosphingolipid species was restricted to the intestine. The neutral and zwitterionic components belong to the arthro-carbohydrate series, which are substituted in their zwitterionic structures by phosphocholine (PC) and in one glycolipid by an additional phosphoethanolamine residue. In previous immunohistochemical localization studies, however, the chemical nature of the PC-substituted biomolecules has not been investigated in detail. Here, we report on the immunohistochemical localization and differentiation of phosphocholine-containing structures into lipid- and protein-bound species in adult A. suum. The patterns of immunostaining, obtained with a PC-specific monoclonal antibody and anti-zwitterionic glycolipid hyperimmune serum in the female worm, indicated a parallel organ distribution for glycolipid- and protein-bound PC-epitopes. Immunoreactivity was localized to specific tissues of the body wall, intestine and reproductive tract. This is the first report of surface-located PC-epitopes for ascarids. The patterns of immunolabelling obtained with antibodies directed against the unsubstituted arthro-carbohydrate series backbone suggested that the glycolipid-bound epitope was restricted to the hypodermis, whilst the protein-bound antigenic determinant resembled that for PC.