Antigenicity of Helicobacter pylori lipopolysaccharides.

An investigation of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of lipopolysaccharides (LPSs) extracted from seven strains of Helicobacter pylori revealed that these molecules were silver stainable and exhibited a high degree of variability in their patterns. Two strains synthesized a variety of sizes of LPS molecules such that fractionation by SDS-PAGE resulted in a stepwise gradation of bands which extended from the top to the bottom of the silver-stained gel. The LPSs from the remaining five strains were made up of molecules which were more homogeneous in size and clustered around two separate areas of the gel. Antigenic analyses of phenol-water-extracted LPSs by immunoblotting and the passive hemagglutination assay suggested that, in addition to strain-specific antigens, all of the LPSs carried a common antigen. Antibodies to this common antigen could be removed from antisera by absorption, and the resulting antisera were used to differentiate strains on the basis of their O antigens by the passive hemagglutination assay technique. The finding that LPSs from 3 of 10 clinical isolates reacted specifically in one or two of the typing antisera suggested that the development of a scheme for differentiating H. pylori on the basis of O antigens is feasible.     

Effect of purified lipopolysaccharides from strains of Helicobacter pylori and Helicobacter felis on acid secretion in mouse gastric glands in vitro

As a bacterial product, Helicobacter pylori lipopolysaccharide (LPS) can originate in close proximity to parietal cells, but the role of this uniquely structured endotoxin on acid secretion has not been fully investigated and remains unclear. The purpose of this study was to test the direct effect of purified LPS (tested range, 0.1 to 100 microg/ml) from various strains of H. pylori and from one Helicobacter felis strain on histamine- and carbachol-stimulated acid secretion in vitro using mouse gastric glands and the accumulation of [(14)C]aminopyrine. In addition, we investigated whether H. pylori LPS can interfere with two native antisecretory substances, prostaglandin E(2) (PGE(2)) and somatostatin, which may contribute to bacterial pathogenicity. Except for the LPS from H. pylori SS1 (Sydney strain), which gave a statistically significant increase in both histamine- and carbachol-stimulated acid output (38 and 24%, respectively; P < 0.05), no effect of the tested LPS was observed on acid secretion. H. pylori LPS purified from a patient isolate did not affect the potency or the efficacy of the inhibitory dose response curve to PGE(2) or somatostatin. Bacterial interstrain variation in the direct stimulatory effect of Helicobacter-derived LPS on acid secretion was observed, which probably reflects the molecular structure of LPS and the potential to contribute to virulence. Importantly, the data showed that H. pylori LPS did not have any direct antisecretory properties. It can be speculated that the acid stimulatory properties of LPS from H. pylori SS1 may contribute to the gastric damage observed in the mouse model of H. pylori infection.     

Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin.

The ability of hemagglutinating and poorly hemagglutinating strains of the gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled laminin was quantitated in a liquid phase assay. Although all strains bound laminin, some hemagglutinating strains were good binders of laminin (maximum of 31% binding), whereas poorly hemagglutinating strains bound intermediate to small amounts of laminin (minimum of 6% binding). Since a hydrophobic component of the bacterium has been reported to be involved in binding of laminin (T. J. Trust, P. Doig, L. Emödy, Z. Kienle, T. Wadström, and P. O'Toole, Infect. Immun. 59:4398-4404, 1991), we investigated the role of lipopolysaccharide (LPS) in the interaction of both types of strains with laminin. Although the extent of inhibition varied among strains, laminin binding to hemagglutinating and poorly hemagglutinating strains was inhibited with homologous and heterologous smooth-form LPS. The ability of heterologous rough-form LPS to produce inhibition comparable to that shown by smooth-form LPS indicated that the O side chain of H. pylori LPS was not involved in the interaction. Further inhibition experiments with dephosphorylated LPS, isolated core oligosaccharide, and free lipid A suggested that a phosphorylated structure in the core oligosaccharide mediates the interaction of a hemagglutinating strain of H. pylori with laminin, whereas a conserved nonphosphorylated structure in the core oligosaccharide mediates the interaction of a poorly hemagglutinating strain. Furthermore, we showed that the interaction of H. pylori LPS with 125I-radiolabelled laminin in a solid phase assay was saturable, specific, and inhibitable with unlabelled laminin. It was postulated that the initial recognition and binding of laminin by H. pylori may occur through LPS and that subsequently a more specific interaction with a lectin-like adhesin on the bacterial surface occurs.     

Helicobacter pylori lipopolysaccharides preferentially induce CXC chemokine production in human monocytes

Helicobacter pylori infection can cause duodenal ulcers and may also induce gastric adenocarcinoma. The bacteria colonize the gastric mucosa and areas of gastric metaplasia in the duodenum for decades, resulting in active chronic inflammation in the infected areas. A characteristic feature of the infection is the ongoing recruitment of neutrophils to the infected sites. To evaluate the role of H. pylori lipopolysaccharides (LPS) in the recruitment of leukocytes to the gastric mucosa, we have examined the cytokine and chemokine production from human monocytes stimulated with LPS isolated from different H. pylori strains, as well as from several other gram-negative bacteria. Our results show that H. pylori LPS induce a large production of neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related oncogene alpha) from purified human monocytes, to almost the same extent as Escherichia coli LPS. However, and in agreement with previous studies, H. pylori LPS was much less potent in inducing production of proinflammatory cytokines by purified human monocytes and was also a weak inducer of the CC chemokine RANTES. There was no difference between LPS preparations from different H. pylori strains in their ability to induce cytokines and chemokines. The preferential production of CXC chemokines after stimulation with H. pylori LPS indicates an important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.     

Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides are poorly transferred to recombinant soluble CD14.

Helicobacter pylori and Porphyromonas gingivalis are gram-negative bacteria associated with chronic inflammatory diseases. These bacteria possess lipopolysaccharides (LPSs) that are able to activate human monocytes to produce tumor necrosis factor alpha but fail to activate human endothelial cells to express E-selectin. With Escherichia coli LPS, tumor necrosis factor alpha activation requires membrane-bound CD14 and E-selectin expression requires soluble CD14 (sCD14). Therefore, the ability of H. pylori and P. gingivalis LPSs to transfer to and bind sCD14 was examined by using immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP). H. pylori and P. gingivalis LPSs were transferred to sCD14 when serum or LBP was present. However, the transfer of these LPSs to CD14 in serum was significantly slower than the transfer of E. coli LPS. Quantitation of the transfer rates by Michaelis-Menten kinetics yielded K(m) values of 6 and 0.1 nM for H. pylori and E. coli LPSs, respectively. The amount of P. gingivalis LPS required to obtain half-maximum binding to CD14 was approximately 10-fold greater than the amount of E. coli LPS required. The slower transfer rates displayed by these LPSs can be explained by the poor binding to LBP observed in direct binding assays. These results are consistent with the proportionately lower ability of these LPSs to activate monocytes compared with E. coli LPS. However, the ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 demonstrates that the lack of endothelial cell CD14-dependent cell activation by these LPSs occurs distal to sCD14 binding.     

Unsaturated fatty acids and viability of Helicobacter (Campylobacter) pylori.

Helicobacter (Campylobacter) pylori was found to be sensitive to the toxic effects of an unsaturated fatty acid (arachidonic acid). Data are presented that support the hypothesis that exogenous catalase added to basal media enhances the growth of H. pylori by preventing the formation of toxic peroxidation products from long-chain unsaturated fatty acids.     

Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori has been found in the stomachs of humans in all parts of the world. In developing countries, 70 to 90% of the population carries H. pylori. In developed countries, the prevalence of infection is lower. There appears to be no substantial reservoir of H. pylori aside from the human stomach. Transmission can occur by iatrogenic, fecal-oral, and oral-oral routes. H. pylori is able to colonize and persist in a unique biological niche within the gastric lumen. All fresh isolates of H. pylori express significant urease activity, which appears essential to the survival and pathogenesis of the bacterium. A variety of tests to diagnose H. pylori infection are now available. Histological examination of gastric tissue, culture, rapid urease testing, DNA probes, and PCR analysis, when used to test gastric tissue, all require endoscopy. In contrast, breath tests, serology, gastric juice PCR, and urinary excretion of [15N]ammonia are noninvasive tests that do not require endoscopy. In this review, we highlight advances in the detection of the presence of the organism and methods of differentiating among types of H. pylori, and we provide a background for appropriate chemotherapy of the infection.     

Molecular mimicry in Campylobacter jejuni and Helicobacter pylori lipopolysaccharides: contribution of gastrointestinal infections to autoimmunity

Molecular mimicry of host structures by the saccharide portion of lipopolysaccharides (LPS) of the gastrointestinal pathogens Campylobacter jejuni and Helicobacter pylori is thought to be associated with the development of autoimmune sequelae. C. jejuni, a leading cause of gastroenteritis, is the most common antecedent infection in Guillain-Barré syndrome (GBS), an inflammatory neuropathy. Chemical analyses of the core oligosaccharides of neuropathy-associated C. jejuni strains have revealed structural homology with human gangliosides. Serum antibodies against gangliosides are found in one third of GBS patients but are generally absent in enteritis cases. Collective data suggest that the antibodies are induced by antecedent infection with C. jejuni, and subsequently react with nerve tissue causing damage. The O-chains of most H. pylori strains express Lewis blood group antigens which are thought to have a role in camouflage of the bacterium as these antigens are also present on human gastric epithelial cells. In chronic H. pylori infections, bacterial expression of Lewis antigens is suggested to be involved in the induction of autoantibodies against the Lewis antigen-expressing gastric proton pump. Many aspects of the autoimmune mechanisms in C. jejuni -associated GBS and H. pylori -induced atrophic gastritis remain unclear, such as the involvement of T cells and the role of host factors.     

非酒精性脂肪性肝病与幽门螺杆菌感染的相关性

非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)本质上是一种肝代谢性疾病,其发病机制复杂,目前尚未完全明确.目前各项资料显示幽门螺杆菌感染(Helicobacter pylori,Hp)与NAFLD的发生和发展有密切的关系,但也有研究得出了相反的结论.对于存在争议的部分至今尚未解决,但如证实Hp感染可作为NAFLD的一种独立危险因素存在,则对NAFLD发病预测以及治疗提供帮助,因此研究两者相关性的工作仍然具有临床意义.     

Biochemical studies of Helicobacter mustelae fatty acid composition and flagella.

The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0, 3-OH-C14:0, C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.     

Helicobacter heilmannii gastritis: association with acid peptic diseases and comparison with Helicobacter pylori gastritis.

We analyzed 2 antral and 1 corpus full-thickness random endoscopic gastric mucosal samples obtained from 946 patients with duodenal ulcers (6077 biopsies) and from 281 patients with nonsteroidal anti-inflammatory drug-associated gastric ulcers (1794 biopsies). We stained tissue sections with hematoxylin and eosin and Warthin-Starry silver stain and immunostained them with polyclonal antibodies against Helicobacter pylori. Hematoxylin- and eosin-stained sections from 6 patients with Helicobacter heilmannii (18 biopsies) and 23 randomly selected patients with H. pylori (68 biopsies) were evaluated and semiquantitated for the presence of acute inflammation, chronic inflammation, glandular atrophy, intestinal metaplasia, H. pylori, H. heilmannii, lymphoid follicles, or vasodilatation. Additional specimens were obtained for H. pylori culture, a CLO test, and serologic examination. H. heilmannii was detected in 6 (0.49%) of 1227 patients (14 [0.18%] of 7871 biopsies). Of these, 4 (0.42%) of 946 were patients with duodenal ulcers (9 [0.15%] of 6077 biopsies), and 2 (0.71%) of 281 were patients with nonsteroidal anti-inflammatory drug-associated gastric ulcers (5 [0.28%] of 1794 biopsies). We found H. heilmannii with hematoxylin and eosin stain, Warthin-Starry stain, and immunoperoxidase stain for H. pylori. Culture for H. pylori was negative in the four patients with duodenal ulcers. The CLO and serologic tests were positive in three of five and five of five patients, respectively. Our results indicate that H. heilmannii, like H. pylori, is associated with peptic ulcer disease (both active and inactive gastritis) and that it preferentially colonizes the gastric antrum. The severity of the H. heilmannii-associated gastritis is less intense and lymphoid aggregates are less common than in H. pylori-associated gastritis. Morphologic detection seems to be the method of choice for detecting H. heilmanni. Immunoperoxidase stain specific for H. pylori also stains H. heilmannii, indicating cross-reacting antigenic epitopes between H. heilmannii and H. pylori.     

Influence of culture conditions on the fatty acid profiles of laboratory-adapted and freshly isolated strains of Helicobacter pylori

Cellular fatty acids of Helicobacter pylori have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions. H. pylori is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9c), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9c and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9c and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.     

Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D

We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.     

Lipid A in Helicobacter pylori.

Free lipid A of Helicobacter pylori was characterized with regard to chemical composition, reactivity with anti-lipid A antibodies, and activity in a Limulus lysate assay. The predominant fatty acids of H. pylori lipid A were 3-OH-18:0, 18:0, 3-OH-16:0, 16:0, and 14:0. Hexosamine was present in amounts similar to those in Campylobacter jejuni or Salmonella typhimurium lipid A. The lipopolysaccharide of H. pylori contained 2-keto-3-deoxyoctonic acid, a common constituent of enterobacterial and C. jejuni lipopolysaccharides. In the enzyme-linked immunosorbent assay, the doses of lipid A required to inhibit anti-lipid A by 50% (EI50 values) by absorption of the immune (rabbit) serum were 7.9, 1.2, and 1.4 micrograms of O-deacylated lipid A's from H. pylori, C. jejuni, and S. typhimurium per ml, respectively. The lower reactivity of H. pylori lipid A compared with those of the other two lipid A preparations (as shown by the higher EI50 value) was underscored by the use of a murine monoclonal anti-lipid A antibody in the inhibition assay. An EI50 value was not obtained at the concentrations tested for H. pylori lipid A; the corresponding figures for C. jejuni and S. typhimurium lipid A's were 13 and 14 micrograms/ml, respectively. No inhibition was obtained with H. pylori lipopolysaccharide, which showed a low-molecular-weight profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of H. pylori lipid A in the Limulus assay was approximately 71 and 650 times lower than those of C. jejuni and S. typhimurium lipid A's, respectively. These findings suggest that lipid A is an integral part of the outer cell wall of H. pylori. The lower reactivity of H. pylori lipid A with anti-lipid A antibodies and in the Limulus assay compared with that of C. jejuni or S. typhimurium lipid A may be explained by a different composition of the fatty acids, especially the 3-hydroxy fatty acids, and a possible deviating phosphorylation pattern.     

Comparison of isolates of Helicobacter pylori and Helicobacter mustelae.

On the basis of analysis of protein profiles, isolates of Helicobacter pylori and Helicobacter mustelae were less than 40% similar. Cytotoxin produced by H. pylori was not detected in isolates of H. mustelae. Both bacterial species agglutinated human erythrocytes. These results substantiate a taxonomic difference between H. pylori and H. mustelae.