Interference of LPS H. pylori with IL-33-Driven Regeneration of Caviae porcellus Primary Gastric Epithelial Cells and Fibroblasts.

BACKGROUND: Lipopolysaccharide (LPS) of Helicobacter pylori (Hp) bacteria causes disintegration of gastric tissue cells in vitro. It has been suggested that interleukin (IL)-33 is involved in healing gastric injury. AIM: To elucidate whether Hp LPS affects regeneration of gastric barrier initiated by IL-33. METHODS: Primary gastric epithelial cells or fibroblasts from Caviae porcellus were transfected with siRNA IL-33. Such cells, not exposed or treated with LPS Hp, were sub-cultured in the medium with or without exogenous IL-33. Then cell migration was assessed in conjunction with oxidative stress and apoptosis, activation of extracellular signal-regulated kinase (Erk), production of collagen I and soluble ST2 (IL-33 decoy). RESULTS: Control cells not treated with LPS Hp migrated in the presence of IL-33. The pro-regenerative activity of IL-33 was related to stimulation of cells to collagen I production. Wound healing by cells exposed to LPS Hp was inhibited even in the presence of IL-33. This could be due to increased oxidative stress and apoptosis in conjunction with Erk activation, sST2 elevation and modulation of collagen I production. CONCLUSIONS: The recovery of gastric barrier cells during Hp infection potentially can be affected due to downregulation of pro-regenerative activity of IL-33 by LPS Hp.

The effect of Helicobacter pylori infection and different H. pylori components on the proliferation and apoptosis of gastric epithelial cells and fibroblasts.

BACKGROUND: Helicobacter pylori colonizes the human gastric mucosa, causing chronic inflammation, peptic ulcers and gastric cancer. A cascade of harmful processes results from the interaction of these bacteria with the gastric epithelium. AIM: To investigate these processes in terms of upregulation of oxidative stress and cell apoptosis and downregulation of the pro-regenerative activity of cells. METHODS: We employed an in vivo guinea pig model at 7 or 28 days postinoculation with H. pylori, corresponding to an acute or chronic stage of infection, respectively, and an in vitro model of guinea pig primary gastric epithelial cells and fibroblasts treated with bacterial components: glycine acid extract (GE), urease subunit A (UreA), cytotoxin-associated gene A protein (CagA) and lipopolysaccharide (LPS). Cells were evaluated for metabolic activity (MTT reduction), myeloperoxidase (MPO) and metalloproteinase (MMP-9) secretion, lipid peroxidation (4-hydroxynonenal (4HNE)), migration (wound healing), proliferation (Ki-67 antigen) and cell apoptosis (TUNEL assay; Bcl-xL, Bax, Bcl-2 expression; caspase 3 cleavage). RESULTS: Significant infiltration of the gastric mucosa by inflammatory cells in vivo in response to H. pylori was accompanied by oxidative stress and cell apoptosis, which were more intense 7 than 28 days after inoculation. The increase in cell proliferation was more intense in chronic than acute infection. H. pylori components GE, CagA, UreA, and LPS upregulated oxidative stress and apoptosis. Only H. pylori LPS inhibited cell migration and proliferation, which was accompanied by the upregulation of MMP-9. CONCLUSIONS: H. pylori infection induces cell apoptosis in conjunction with increased oxidative stress. Elevated apoptosis protects against deleterious inflammation and neoplasia; however, it reduces cell integrity. Upregulation of cell migration and proliferation in response to injury in the milieu of GE, CagA or UreA facilitates tissue regeneration but increases the risk of neoplasia. By comparison, downregulation of cell regeneration by H. pylori LPS may promote chronic inflammation.

Upregulation of MUC5AC production and deposition of LEWIS determinants by HELICOBACTER PYLORI facilitate gastric tissue colonization and the maintenance of infection.

BACKGROUND: Helicobacter pylori bacteria colonize human gastric mucosa, cause chronic inflammation, peptic ulcers and gastric cancer. Colonization is mediated by H. pylori adhesins, which preferentially bind mucin 5 (MUC5AC) and Lewis (Le) determinants. The aim of this study was to evaluate the influence of H. pylori and their components on MUC5AC production and deposition of LeX/LeY in gastric epithelial cells in relation to bacterial adhesion using Caviae porcellus primary gastric epithelial cells and an in vivo model of experimental H. pylori infection in these animals. METHODS: MUCA5C and LeX/LeY were induced in vitro by live H. pylori reference strain CCUG 17874 (2 × 107 CFU/ml), H. pylori glycine acid extract (GE), 10 µg/ml; cytotoxin associated gene A (CagA) protein, 1 µl/ml; UreA urease subunit, 5 µg/ml; lipopolysaccharide (LPS) 25 ng/ml and imaged by fluorescence microscopy after anti-MUC5AC or anti-LeX/LeY FITC antibody staining. Bacterial adhesion was imaged by using anti-H. pylori FITC antibodies. The animals were inoculated per os with H. pylori (3 times in 2 days intervals, 1 × 1010 CFU/ml). After 7 or 28 days an infection and inflammation were assessed by histological, serological and molecular methods. Gastric tissue sections of infected and control animals were screend for MUCA5C and LeX, and H. pylori adhesion as above. RESULTS: MUC5AC production and deposition of Lewis determinants, especially LeX were upregulated in the milieu of live H. pylori as well as GE, CagA, UreA or LPS in vitro and in vivo during infection, more effectively in the acute (7 days) than in the chronic (28 days) phase of infection. This was related to enhanced adhesion of H. pylori, which was abrogated by anti-MUC5AC and anti-LeX or anti-LeY antibody treatment. CONCLUSIONS: Modulation of MUCA5C production and LeX/LeY deposition in the gastric mucosa by H. pylori can significantly increase gastric tissue colonization during H. pylori infection.

Impact of Helicobacter pylori on the healing process of the gastric barrier.

AIM: To determine the impact of selected well defined Helicobacter pylori (H. pylori) antigens on gastric barrier cell turnover. METHODS: In this study, using two cellular models of gastric epithelial cells and fibroblasts, we have focused on exploring the effects of well defined H. pylori soluble components such as glycine acid extract antigenic complex (GE), subunit A of urease (UreA), cytotoxin associated gene A protein (CagA) and lipopolysaccharide (LPS) on cell turnover by comparing the wound healing capacity of the cells in terms of their proliferative and metabolic activity as well as cell cycle distribution. Toxic effects of H. pylori components have been assessed in an association with damage to cell nuclei and inhibition of signal transducer and activator of transcription 3 (STAT3) phosphorylation. RESULTS: We showed that H. pylori GE, CagA and UreA promoted regeneration of epithelial cells and fibroblasts, which is necessary for effective tissue healing. However, in vivo increased proliferative activity of these cells may constitute an increased risk of gastric neoplasia. In contrast, H. pylori LPS showed a dose-dependent influence on the process of wound healing. At a low concentration (1 ng/mL) H. pylori LPS accelerated of healing epithelial cells, which was linked to significantly enhanced cell proliferation and MTT reduction as well as lack of alterations in cell cycle and downregulation of epidermal growth factor (EGF) production as well as cell nuclei destruction. By comparison, H. pylori LPS at a high concentration (25 ng/mL) inhibited the process of wound repair, which was related to diminished proliferative activity of the cells, cell cycle arrest, destruction of cell nuclei and downregulation of the EGF/STAT3 signalling pathway. CONCLUSION: In vivo H. pylori LPS driven effects might lead to the maintenance of chronic inflammatory response and pathological disorders on the level of the gastric mucosal barrier.

Helicobacter pylori antigens, acetylsalicylic acid, LDL and 7-ketocholesterol - their potential role in destabilizing the gastric epithelial cell barrier. An in vitro model of Kato III cells.

Colonization of gastric tissue in humans by H. pylori Gram-negative bacteria initiates gastric and duodenal ulcers and even gastric cancers. Infections promote inflammation and damage to gastric epithelium which might be followed by the impairment of its barrier function. The role of H. pylori components in these processes has not been specified. H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs including acetylsalicylic acid (ASA). The lipid transport-associated molecule such as low density lipoprotein (LDL), which is a classic risk factor of coronary heart disease (CHD) and 7-ketocholesterol (7-kCh) a product of cholesterol oxidation, which may occur during the oxidative stress in LDL could also be considered as pro-inflammatory. The aim of this study was to evaluate the cytotoxicity of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III gastric epithelial cells, on the basis of the cell ability to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Kato III cells were stimulated for 24 h, at 37°C and 5% CO2, with H. pylori antigens: cytotoxin associated gene A (CagA) protein, the urease A subunit (UreA), lipopolysaccharide (LPS) and ASA, LDL or 7-kCh. H. pylori LPS, ASA, LDL and 7-kCh, but not H. pylori glycine acid extract (GE), demonstrated cytotoxicity against Kato III cells, which was related to a diminished percentage of MTT reducing cells and to an increased cell population with the signs of DNA damage. The results suggest that damage to gastric epithelial cells can be induced independently by H. pylori antigens, ASA and endogenous lipid transport-associated molecules. During H. pylori infection in vivo, especially in CHD patients, synergistic or antagonistic interactions between these factors might possibly influence the disease course. Further study is necessary to explain these potential effects.

Helicobacter pylori-driven modulation of NK cell expansion, intracellular cytokine expression and cytotoxic activity.

During Helicobacter pylori (Hp) infections, innate immune cells may be positively or negatively modulated by Hp compounds or by Hp-induced cytokines. We have shown previously that the natural cytotoxic activity of PBMC was lower in Hp-infected [Hp(+)] than Hp-uninfected individuals [Hp(-)]. Here, we asked whether the Hp-modulated cytotoxic amplitude is associated with changes in the number of NK cells, their activation or intracellular cytokine expression. Flow cytometry immunophenotyping of PBMC was performed with regard to the surface receptors CD3, CD56 and CD25, and intracellular cytokine expression of IL-2, IFN-γ and IL-10 after in vitro stimulation with Hp glycine acid extract (GE), Hp LPS or standard Escherichia coli LPS. Hp GE-driven enhancement of lymphocyte cytotoxic activity was associated with the expansion of CD3(-)CD56(+)CD25(+) NK cells and the up-regulation of IFN-γ and/or IL-2 synthesis, up to the higher level in Hp(-) than in Hp(+), while Hp LPS-mediated decrease in lymphocyte cytotoxicity was accompanied by the lack of CD3(-)CD56(+)CD25(+) NK propagation, the inhibition of pro-inflammatory cytokine expression and intense expansion of IL-10-producing NK cells. Thus, the cytotoxic and cytokine activities of NK cells were dependent on the type of antigenic challenge and the Hp status, that is, NK cells could be modulated positively by Hp GE Ags and negatively by Hp LPS.

Campylobacter jejuni strain discrimination and temperature-dependent glycome expression profiling by lectin microarray.

Gram-negative Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans worldwide and the most frequently identified infectious trigger in patients developing Guillain-Barré syndrome (GBS). While C. jejuni is pathogenic in humans, it is a commensal in avian hosts. Bacterial cell surface carbohydrates are important virulence factors and play roles in adherence, colonisation and infection. The mechanisms leading to infection or persistent colonisation of C. jejuni are not well understood but host temperature may provide an important stimulus for specific adaptation. Thus, examination of the modulation of the total surface glycome of C. jejuni in response to temperature may help shed light on commensal and pathogenic mechanisms for this species. C. jejuni strains 81116 and 81-176 were cultured at 37 and 42°C to simulate human and avian host conditions, respectively, and whole cells were profiled on lectin microarrays constructed to include a wide range of binding specificities. C. jejuni 81116 profiles indicated that the previously characterised lipopolysaccharide (LPS)-like molecule and N-linked glycans were the predominantly recognised cell surface structures while capsular polysaccharide (CPS), lipooligosaccharides (LOS) and N-linked glycosylation were best recognised for strain 81-176 at 37°C. The profiles of both strains varied and were distinguishable at both temperatures. At the higher temperature, reduced dominance of the LPS-like structure was associated with strain 81116 and a change in the relative distribution of CPS and LOS structures was indicated for strain 81-176. This change in LOS molecular mass species distribution between temperatures was confirmed by SDS-PAGE analysis. Additionally, opposite behaviour of certain lectins was noted between the plate agglutination assay and the microarray platform. Insights into the important glycosylation involved in C. jejuni host cell tropism at different growth temperatures were gained using the lectin microarray platform.

Invariant Natural Killer T-cell anergy to endogenous myelin acetyl-glycolipids in multiple sclerosis.

To extend our studies on glycolipid-reactive invariant Natural Killer T-cell (iNKT-cell) function in multiple sclerosis (MS), we investigated the stimulatory activities of two myelin-derived glycolipids that are poly-acetylated derivatives of ß-galactosylceramide designated as fast-migrating cerebrosides (FMC) by thin-layer chromatography. In healthy subjects, FMC stimulation of peripheral blood cells significantly expanded iNKT-cells similar to α-GalCer and induced significant increases in Th1, Th2 and Th17 cytokines. In marked contrast, MS patients failed to respond to FMCs or to α-GalCer stimulation indicating an anergic response. We propose that myelin-derived FMC glycolipids stimulate iNKT-cell responses in vivo and this is blocked in MS.

Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

Interaction of Helicobacter pylori with C-type lectin dendritic cell-specific ICAM grabbing nonintegrin.

In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori Le(X/Y) positive whole cells and H. pylori LPS of Le(X/Y) type was fucose dependent, whereas in Le(XY) negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic Le(X) and Le(Y) to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the Le(XY) dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by Le(X) and Le(Y) determinants.

Helicobacter pylori antigens as potential modulators of lymphocytes' cytotoxic activity.

Helicobacter pylori (H.p) colonizes human gastric mucosa and causes gastric and duodenal ulcer disease or gastric cancer. Various H.p compounds may modulate the host immune response in regards to tolerance of the infection or disease development. The aim of this study was to determine whether H.p lipopolysaccharide (LPS) and glycine acid extract antigens (GE) or E. coli LPS influence the cytotoxic activity of peripheral blood lymphocytes from H.p infected - H.p (+) or uninfected - H.p (-) individuals, in the presence or absence of exogenous interleukin (IL)12. Individual H.p status was defined by the urea breath test. Lymphocytes, stimulated or not with H.p, and control antigens, with or without IL-12, were used as effector cells and epithelial HeLa cells as targets. The cytotoxicity of lymphocytes was expressed as the percentage of dead target cells unable to reduce tetrazolium salt. The supernatants from HeLa/lymphocyte cultures were used for detection of the cellular cytotoxicity markers granzyme B and caspase 8. The natural cytotoxic activity of lymphocytes from H.p (+) was less than that of H.p (-) donors. This may have been due to fewer natural killer cells of CD3(-) CD56(+) Nkp46(+) phenotype in H.p (+) in comparison to H.p (-) subjects. H.p GE and standard E. coli LPS enhanced the cytotoxicity of lymphocytes towards target cells whereas H.p LPS downregulated this activity. The decrease in lymphocyte cytotoxicity in response to H.p LPS correlated with a lack of IL-2 and IL-12 production, inhibition of interferon-γ production, and low IL-10 secretion by mononuclear leukocytes. IL-12 significantly enhanced the natural as well as H.p LPS and H.p GE driven cytotoxic capacity of lymphocytes. In conclusion, H.p LPS may negatively modulate natural cytotoxic activity and cytokine secretion by immunocompetent cells and thus be involved in the maintenance of infection and development of gastric pathologies.

Lectin typing of Campylobacter jejuni using a novel quartz crystal microbalance technique.

Seven Campylobacter jejuni strains were characterised by a lectin typing assay. The typing system was based on a quartz crystal microbalance technique (QCM) with four commercially available lectins (wheat germ agglutinin, Maackia amurensis lectin, Lens culinaris agglutinin, and Concanavalin A), which were chosen for their differing carbohydrate specificities. Initially, the gold surfaces of the quartz crystals were modified with 11-mercaptoundecanoic acid followed by lectin immobilisation using a conventional amine-coupling technique. Bacterial cells were applied for lectin typing without preliminary treatment, and resonant frequency and dissipation responses were recorded. The adhesion of microorganisms on lectin surfaces was confirmed by atomic force microscopy. Scanning was performed in the tapping mode and the presence of bacteria on lectin-coated surfaces was successfully demonstrated. A significant difference in the dissipation response was observed for different C. jejuni strains which made it possible to use this parameter for discriminating between bacterial strains. In summary, the QCM technique proved a powerful tool for the recognition and discrimination of C. jejuni strains. The approach may also prove applicable to strain discrimination of other bacterial species, particularly pathogens.

Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.

Helicobacter pylori causes chronic gastritis, peptic ulcers, and gastric carcinoma. Gastric epithelial cells provide the first point of contact between H. pylori and the host. TLRs present on these cells recognize various microbial products, resulting in the initiation of innate immunity. Although previous reports investigated TLR signaling in response to intact H. pylori, the specific contribution of H. pylori LPS with regard to functional genomics and cell-signaling events has not been defined. This study set out to define downstream signaling components and altered gene expression triggered by H. pylori LPS and to investigate the role of the signaling protein tribbles 3 (TRIB3) during the TLR-mediated response to H. pylori LPS. Cotransfections using small interfering RNA and dominant-negative constructs demonstrated that H. pylori LPS functions as a classic TLR2 ligand by signaling through pathways involving the key TLR signaling components MyD88 adaptor-like, MyD88, IRAK1, IRAK4, TNFR-associated factor 6, IκB kinase ß, and IκBα. Microarray analysis, real-time PCR, and ELISA revealed the induction of a discrete pattern of chemokines as a direct effect of LPS:TLR2 signaling. H. pylori infection was associated with decreased expression of TRIB3 in human gastric epithelial cell lines and tissue samples. Additionally, H. pylori decreased expression of C/EBP homologous protein and activating transcription factor 4, the transcription factors involved in the induction of TRIB3 expression. Furthermore, knockdown of TRIB3 and C/EBP homologous protein enhanced TLR2-mediated NF-κB activation and chemokine induction in response to H. pylori LPS. Thus, modulation of TRIB3 by H. pylori and/or its products may be an important mechanism during H. pylori-associated pathogenesis.

Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2.

Lipopolysaccharide (LPS), which generally activates Toll-like receptor 4 (TLR4), is expressed on commensal colonic bacteria. In a number of tissues, LPS can act directly on epithelial cells to increase paracellular permeability. Such an effect in the colon would have an important impact on the understanding of normal homeostasis and of pathology. Our aim was to use a novel primary culture of colonic epithelial cells grown on Transwells to investigate whether LPS, or Pam(3)CSK( 4), an activator of TLR2, affected paracellular permeability. Consequently, [(14)C]-mannitol transfer and transepithelial electrical resistance (TEER) were measured. The preparation consisted primarily of cytokeratin-18 positive epithelial cells that produced superoxide, stained for mucus with periodic acid-Schiff reagent, exhibited alkaline phosphatase activity and expressed TLR2 and TLR4. Tight junctions and desmosomes were visible by transmission electron microscopy. Basally, but not apically, applied LPS from Escherichia coli increased the permeability to mannitol and to a 10-kDa dextran, and reduced TEER. The LPS from Helicobacter pylori increased paracellular permeability of gastric cells when applied either apically or basally, in contrast to colon cells, where this LPS was active only from the basal aspect. A pan-caspase inhibitor prevented the increase in caspase activity caused by basal E. coli LPS, and reduced the effects of LPS on paracellular permeability. Synthetic Pam(3)CSK(4) in the basal compartment prevented all effects of basal E. coli LPS. In conclusion, LPS applied to the base of the colonic epithelial cells increased paracellular permeability by a mechanism involving caspase activation, suggesting a process by which perturbation of the gut barrier could be exacerbated. Moreover, activation of TLR2 ameliorated such effects.

Temperature-dependent phenotypic variation of Campylobacter jejuni lipooligosaccharides.

BACKGROUND: Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry. RESULTS: Herein we describe the existence and heterogeneity of multiple LOS forms in C. jejuni strains of human and chicken origin grown at 37 °C and 42 °C, respectively, as determined on sodium dodecyl sulphate-polyacrylamide electrophoresis gels with carbohydrate-specific silver staining and blotting with anti-ganglioside ligands, and confirmed by nuclear magnetic resonance (NMR) spectroscopy. The C. jejuni NCTC 11168 original isolate (11168-O) was compared to its genome-sequenced variant (11168-GS), and both were found to have a lower-M(r) LOS form, which was different in size and structure to the previously characterized higher-M(r) form bearing GM1 mimicry. The lower-M(r) form production was found to be dependent on the growth temperature as the production of this form increased from ~5%, observed at 37 °C to ~35% at 42 °C. The structure of the lower-M(r) form contained a ß-D-Gal-(1→3)-ß-D-GalNAc disaccharide moiety which is consistent with the termini of the GM1, asialo-GM1, GD1, GT1 and GQ1 gangliosides, however, it did not display GM1 mimicry as assessed in blotting studies but was shown in NMR to resemble asialo-GM1. The production of multiple LOS forms and lack of GM1 mimicry was not a result of phase variation in the genes tested of NCTC 11168 and was also observed in most of the human and chicken isolates of C. jejuni tested. CONCLUSION: The presence of differing amounts of LOS forms at 37 and 42 °C, and the variety of forms observed in different strains, indicate that LOS form variation may play a role in an adaptive mechanism or a stress response of the bacterium during the colonization of different hosts.

The role of endotoxin in infection: Helicobacter pylori and Campylobacter jejuni.

Both Helicobacter pylori and Campylobacter jejuni are highly prevalent Gram-negative microaerophilic bacteria which are gastrointestinal pathogens of humans; H. pylori colonizes the gastroduodenal compartment and C. jejuni the intestinal mucosa. Although H. pylori causes chronic gastric infection leading to gastritis, peptic ulcers and eventually gastric cancer while C. jejuni causes acute infection inducing diarrhoeal disease, the endotoxin molecules of both bacterial species contrastingly contribute to their pathogenesis and the autoimmune sequelae each induces. Compared with enterobacterial endotoxin, that of H. pylori has significantly lower endotoxic and immuno-activities, the molecular basis for which is the underphosphorylation and underacylation of the lipid A component that interacts with immune receptors. This induction of low immunological responsiveness by endotoxin may aid the prolongation of H. pylori infection and therefore infection chronicity. On the other hand, this contrasts with acute infection-causing C. jejuni where overt inflammation contributes to pathology and diarrhoea production, and whose endotoxin is immunologically and endotoxically active. Futhermore, both H. pylori and C. jejuni exhibit molecular mimicry in the saccharide components of their endotoxins which can induce autoreactive antibodies; H. pylori expresses mimicry of Lewis and some ABO blood group antigens, C. jejuni mimicry of gangliosides. The former has been implicated in influencing the development of inflammation and gastric atrophy (a precursor of gastic cancer), the latter is central to the development of the neurological disorder Guillain-Barré syndrome. Both diseases raise important questions concerning infection-induced autoimmunity awaiting to be addressed.

O-linked oligosaccharides from salivary agglutinin: Helicobacter pylori binding sialyl-Lewis x and Lewis b are terminating moieties on hyperfucosylated oligo-N-acetyllactosamine.

Salivary agglutinin plays a vital biological role modulating the protective effect in the oral cavity by interacting with a broad range of oral pathogens. Here, we describe the first characterization of the O-linked oligosaccharides of salivary agglutinin identified by negative ion liquid chromatography-mass spectrometry. The dominating structures were neutral or monosialylated core 1 (Galbeta1-3GalNAcalpha1-Ser/Thr) and core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-Ser/Thr) structures extended by fucosylated oligo-N-acetyllactosamine units. Oligosaccharides detected as [M-H](-) or [M-2H](2)(-) ions ranged from the disaccharide Galbeta1-3GalNAcol up to structures of almost 4000 Da, corresponding to core 1/2 structures with five N-acetyllactosamine units and 11 fucoses. Fucose was found either as terminal or internal blood group H structures in type 1 (Galbeta1-3GlcNAcbeta1-R), type 2 (Galbeta1-4GlcNAcbeta1-R) and type 3 (Galbeta1-3GalNAcalpha1-Ser/Thr) units, where the chains also could be fucosylated on GlcNAc yielding repeated Lewis a/b or Lewis x/y structures. Sialylation was located either at the non-reducing end of the N-acetyllactosamine chains as sialyl-Lewis x or as sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAcalpha1-Ser/Thr) type structures with or without further extension of the C-6 branch of GalNAc with neutral fucosylated N-acetyllactosamine chains. The data indicated that sialylation, fucosylation and type 1 N-acetyllactosamine termination are important regulatory elements for controlling the oligosaccharide chain length. Furthermore, it was shown that these regulatory oligosaccharide elements could be utilized by the pathogen Helicobacter pylori to colonize the oral cavity, reside in dental plaque and serve as a reservoir for reinfection after successful clearance of H. pylori gastric infection.

Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid.

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.

Assessment of the duration of protection in Campylobacter jejuni experimental infection in humans.

A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 10(5), 10(7), or 10(9) CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 10(9)-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 10(9)-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.