A Synbiotic with Tumor Necrosis Factor-α Inhibitory Activity Ameliorates Experimental Jejunoileal Mucosal Injury.

Despite the recent development of biological modifiers for inflammatory bowel diseases (IBD), there continues to be considerable interest in fermented medicines because of its negligible adverse effects. We previously showed that the synbiotic Gut Working Tablet (GWT) alleviates experimental colitis. Here we show that GWT is capable of ameliorating jejunoileal mucosal injury, which is frequently seen with IBD. We created experimental jejunoileal mucositis in rats by injection of methotrexate (MTX) which increases intestinal permeability, a hallmark finding of IBD. Administering GWT to MTX-injected rats restored intestinal integrity by reversing villi shortening, crypt loss, and goblet cell depletion in the mucosa. Also GWT reduced activities of myeloperoxidase and lipid peroxidase and increased superoxide dismutase activity, which is critical for maintaining intestinal function. We further found that GWT suppressed mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) in macrophage and reduced TNF-α mRNA expression in specimens with experimental colitis, which is in contrast to VSL#3 that enhanced TNF-α production. Together, the current and previous animal studies clearly demonstrate the protective role of GWT in chemically induced enterocolitis. Crohn's disease, a well-known IBD, can affect any portion of the intestine, and these results suggest that GWT may be useful as a novel therapeutic or maintenance therapy for IBD.

Proteomic characterization of Helicobacter pylori CagA antigen recognized by child serum antibodies and its epitope mapping by peptide array.

Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against Helicobacter pylori, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for H. pylori infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 H. pylori (Hp)-positive and 2 Hp-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the Hp-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of H. pylori infection in children.

Colonization of an acid resistant Kingella denitrificans in the stomach may contribute to gastric dysbiosis by Helicobacter pylori.

In the stomach of a gastric ulcer patient who had been administered an anti-acid, a gram-negative and urease-negative bacillus similar in size to Helicobacter pylori was infected together with H. pylori. According to biochemical test and 16S rRNA gene analysis, the urease-negative bacterium was identified as Kingella denitrificans, a human nasopharyngeal commensal. In contrast to the standard strain of K. denitrificans, the isolate showed catalase activity, did not produce acid from glucose, and exhibited acid tolerance. Acid tolerance of H. pylori was increased by cocultivation with the K. denitrificans isolate, but not with other isolates of K. denitrificans. Disruption of physiological and immunological niche by dysbiotic colonization of bacterium may provide pathological attributes to human stomach. Collectively, a careful administration of anti-acids to the elderly, especially those with atrophic gastritis, is necessary to avoid repression of the gastric barrier to bacteria.

Genome sequences of 65 Helicobacter pylori strains isolated from asymptomatic individuals and patients with gastric cancer, peptic ulcer disease, or gastritis.

Helicobacter pylori, inhabitant of the gastric mucosa of over half of the world population, with decreasing prevalence in the U.S., has been associated with a variety of gastric pathologies. However, the majority of H. pylori-infected individuals remain asymptomatic, and negative correlations between H. pylori and allergic diseases have been reported. Comprehensive genome characterization of H. pylori populations from different human host backgrounds including healthy individuals provides the exciting potential to generate new insights into the open question whether human health outcome is associated with specific H. pylori genotypes or dependent on other environmental factors. We report the genome sequences of 65 H. pylori isolates from individuals with gastric cancer, preneoplastic lesions, peptic ulcer disease, gastritis, and from asymptomatic adults. Isolates were collected from multiple locations in North America (USA and Canada) as well as from Columbia and Japan. The availability of these H. pylori genome sequences from individuals with distinct clinical presentations provides the research community with a resource for detailed investigations into genetic elements that correlate either positively or negatively with the epidemiology, human host adaptation, and gastric pathogenesis and will aid in the characterization of strains that may favor the development of specific pathology, including gastric cancer.

Helicobacter pylori CagA inhibits endocytosis of cytotoxin VacA in host cells.

Helicobacter pylori, a common pathogen that causes chronic gastritis and cancer, has evolved to establish persistent infections in the human stomach. Epidemiological evidence suggests that H. pylori with both highly active vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), the major virulence factors, has an advantage in adapting to the host environment. However, the mechanistic relationship between VacA and CagA remains obscure. Here, we report that CagA interferes with eukaryotic endocytosis, as revealed by genome-wide screening in yeast. Moreover, CagA suppresses pinocytic endocytosis and the cytotoxicity of VacA in gastric epithelial cells without affecting clathrin-dependent endocytosis. Our data suggest that H. pylori secretes VacA to attack distant host cells while injecting CagA into the gastric epithelial cells to which the bacteria are directly attached, thereby protecting these attached host cells from the cytotoxicity of VacA and creating a local ecological niche. This mechanism might allow H. pylori to balance damage to one population of host cells with the preservation of another, allowing for persistent infection.

Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

Aspirin inhibits Chlamydia pneumoniae-induced NF-kappa B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth.

Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study was designed to investigate the mechanisms of the anti-chlamydial activity of aspirin. A reporter gene assay for NF-kappa B activity, immunoblot analysis for cyclo-oxygenase (COX)-2 and radioimmunoassay for prostaglandin E(2) (PGE(2)) were performed. Following infection of HEp-2 cells with C. pneumoniae, NF-kappa B was activated, COX-2 was induced and PGE(2) was elevated. Aspirin inhibited NF-kappa B activation at a concentration of 0.1 mM, partially inhibited COX-2 induction and blocked PGE(2) synthesis completely. In addition, high doses of aspirin (1 and 2 mM) inhibited chlamydial growth in HEp-2 cells, decreasing the number and size of inclusion bodies; this effect could be overcome by adding tryptophan to the culture. Indomethacin also blocked the synthesis of PGE(2), but had no effect on COX-2 expression or chlamydial growth. These results indicate that aspirin not only has an anti-inflammatory activity through prevention of NF-kappa B activation but also has anti-chlamydial activity at high doses, possibly through depletion of tryptophan in HEp-2 cells.

Growth cycle of Helicobacter pylori in gastric mucous layer.

Helicobacter pylori bacterium is characterized by its strong urease activity. Our studies on the role of H. pylori urease revealed; (i) it is essential for colonization, (ii) exogenous urea is required for acid resistance, (iii) the bacteria have the ability to move toward urea and sodium bicarbonate, (iv) urea hydrolysis accelerates chemotactic locomotion, and (v) decay of urease mRNA to accomplish the active center is pH-regulated; i.e., the mRNA is stabilized and destabilized under acidic and neutral conditions, respectively. Based on the above results, I propose the growth cycle of H. pylori in gastric mucous layer. H. pylori bacteria proliferate on the epithelial cell surface by utilizing nutrients derived from degraded cells. Proliferated bacteria leave the cell surface to pH-variable region where they encounter strong acid. Urease is activated with simultaneous opening of UreI channel so that urea is hydrolyzed to neutralize acid. Chemotaxis of H. pylori toward urea and sodium bicarbonate that are abundant on the cell surface is accelerated by urea hydrolysis so that the bacteria go back to the cell surface for the next round of proliferation. This growth cycle may allow the bacteria to infect persistently in the stomach.

Accumulation of polyphosphate granules in Helicobacter pylori cells under anaerobic conditions.

Helicobacter pylori is known to transform to coccoid forms which might be involved in faecal-oral transmission. When the bacteria enter the intestine, they encounter anaerobiosis that is unfavourable for growth. The effect of anaerobiosis was investigated to determine whether H. pylori is viable under such conditions. H. pylori in the late logarithmic growth phase transformed from spiral to coccoid forms when transferred to and incubated anaerobically in fresh medium. Acridine orange staining indicated that the viability of coccoid forms was significantly reduced, but still measurable even at day 5 or 7 of anaerobic culture. The cells retained low but significant levels of the major sigma factor RpoD at day 5 or 7 of anaerobic culture. The cellular structures of coccoid forms contained polyphosphate granules at day 1 and even at day 7 when incubated anaerobically, whereas only a few granules were observed under micro-aerobic conditions. Poor formation of polyphosphate granules in micro-aerobic cultures correlated particularly well with lower levels of acridine orange staining. These results suggest that acridine orange-positive anaerobic coccoid forms are viable to a certain extent and that polyphosphate may support this viability.