A total of 293 strains of Helicobacter pylori, including strains resistant to levofloxacin, clarithromycin, metronidazole, or amoxicillin, were examined for in vitro susceptibility to 10 antimicrobial agents. Among these agents, sitafloxacin (a fluoroquinolone) showed the greatest activity (MIC(90), 0.06 μg/ml), with high bactericidal activity and synergy in sitafloxacin-lansoprazole (a proton pump inhibitor) combination. In a Mongolian gerbil model with a H. pylori ATCC 43504 challenge, marked eradication effects were observed at ≥1 mg/kg for sitafloxacin, ≥10 mg/kg for levofloxacin, and ≥10 mg/kg for lansoprazole, reflecting MIC levels for each agent (0.008, 0.25, and 2 μg/ml, respectively). The therapeutic rates were 83.3% for the sitafloxacin (0.3 mg/kg)-lansoprazole (2.5 mg/kg) combination and 0% for either sitafloxacin or lansoprazole alone. The maximum serum concentration (C(max)) of sitafloxacin was 0.080 ± 0.054 μg/ml at 30 min, when orally administered at 1 mg/kg. The simultaneous administration of lansoprazole resulted in no difference. In the resistance development assay, MICs of levofloxacin increased 64- to 256-fold with gyrA mutations (Ala88Pro and Asn87Lys), while MICs of sitafloxacin only up to 16-fold with the Asn87Lys mutation. The data suggest that sitafloxacin exhibited superior anti-H. pylori activity with low rates of resistance development in vitro and that, reflecting high in vitro activities, sitafloxacin-lansoprazole combination exhibited strong therapeutic effects in Mongolian gerbils with a C(max) of sitafloxacin that was 10-fold higher than the MIC value at a 1-mg/kg administration.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that promotes the development of T-helper lymphocyte 1 (Th1). Chronic gastritis induced by Helicobacter pylori is considered a Th1-mediated process. IL-12 levels in gastric biopsy samples of H. pylori-infected patients are higher than in those of uninfected individuals, but the cellular source of IL-12 remains elusive. IL-12 staining was detected in mucosal epithelial cells, lymphocytes, and macrophages in specimens of patients with H. pylori-positive gastritis. Therefore, we investigated IL-12 p40 mRNA induction by H. pylori in gastric epithelial cells and T cells. Although cag pathogenicity island (PAI)-positive H. pylori induced IL-12 p40 mRNA expression, an isogenic mutant of the cag PAI failed to induce it in both cell types. Supernatants from H. pylori cultures and H. pylori VacA induced IL-12 p40 mRNA expression in T cells but not in epithelial cells. The activation of the IL-12 p40 promoter by H. pylori was mediated through NF-kappaB. The transfection of IkappaB kinase and NF-kappaB-inducing kinase dominant-negative mutants inhibited H. pylori-induced IL-12 p40 activation. Inhibitors of NF-kappaB, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and Hsp90 suppressed H. pylori- and VacA-induced IL-12 p40 mRNA expression. The results indicate that H. pylori induces IL-12 p40 expression by the activation of NF-kappaB, phosphatidylinositol 3-kinase, and p38 mitogen-activated protein kinase. Hsp90 is also a crucial regulator of H. pylori-induced IL-12 p40 expression. In addition to the cag PAI, VacA might be relevant in the induction of IL-12 expression and a Th1-polarized response only in T cells.

The objective of the study was to develop a stomach-specific drug delivery system for controlled release of clarithromycin for eradication of Helicobacter pylori (H. pylori). Floating-bioadhesive microspheres of clarithromycin (FBMC) were prepared by emulsification-solvent evaporation method using ethylcellulose as matrix polymer and Carbopol 934P as mucoadhesive polymer. The prepared microspheres were subjected to evaluation for particle size, incorporation efficiency, in vitro buoyancy, in vitro mucoadhesion and in vitro drug release characteristics. The prepared microspheres showed a strong mucoadhesive property with good buoyancy. The formulation variables like polymer concentration and drug concentration influenced the in vitro drug release significantly in simulated gastric fluid (pH. 2.0). The in vivo H. pylori clearance efficiency of prepared FBMC in reference to clarithromycin suspension following repeated oral administration to H. pylori infected Mongolian gerbils was examined by polymerase chain reaction (PCR) technique and by a microbial culture method. The FBMC showed a significant anti-H. pylori effect in the in vivo gerbil model. It was also noted that the required amount of clarithromycin for eradication of H. pylori was significantly less in FBMC than from corresponding clarithromycin suspension. The results further substantiated that FBMC improved the gastric stability of clarithromycin (due to entrapment within the microsphere) and eradicated H. pylori from the gastrointestinal tract more effectively than clarithromycin suspension because of the prolonged gastrointestinal residence time of the formulation.

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of inflammatory cells such as neutrophils and eosinophils. However, little information is available on the relationship between H. pylori virulence factors and chemokine expression in eosinophils. This study investigates the role of vacuolating cytotoxin (VacA) in chemokine expression from human eosinophils. Eosinophils were isolated from the peripheral blood of healthy volunteers using a magnetic cell separation system. VacA(+) H. pylori water-soluble proteins (WSP) induced higher expression of interleukin-8, growth-related oncogene alpha, monocyte chemotactic protein 1, and RANTES (regulated on activation, normal, T-cell expressed and secreted) than Vac(-) WSP in human eosinophils, as assessed by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Purified VacA not only increased chemokine expression but also activated p65/p50 NF-kappaB heterodimers and phosphorylated IkappaB kinase (IKK) alpha/beta signals in human eosinophils. Inhibition of NF-kappaB and IKK significantly decreased the chemokine expression in VacA-stimulated eosinophils. Furthermore, VacA-induced NF-kappaB activation and chemokine release from eosinophils were dependent on Ca(2+) influx and mitochondrial generation of reactive oxygen intermediates (ROI). These results suggest that NF-kappaB and IKK signals via Ca(2+) influx and mitochondrial ROI play a role in the up-regulation of chemokine expression in eosinophils stimulated with H. pylori VacA.

The aim of this study was to develop a new intra-gastric floating in situ gelling system for controlled delivery of amoxicillin for the treatment of peptic ulcer disease caused by Helicobacter pylori (H. pylori). Gellan based amoxicillin floating in situ gelling systems (AFIG) were prepared by dissolving varying concentrations of gellan gum in deionized water containing sodium citrate, to which varying concentrations of drug and calcium carbonate, as gas-forming agent, was added and dissolved by stirring. The formulation variables like concentration of gellan gum and calcium carbonate significantly affected the in vitro drug release from the prepared AFIG. The in vivo H. pylori clearance efficacy of prepared AFIG in reference to amoxicillin suspension following repeated oral administration to H. pylori infected Mongolian gerbils was examined by polymerase chain reaction (PCR) technique and by a microbial culture method. AFIG showed a significant anti-H. pylori effect in the in vivo gerbil model. It was noted that the required amount of amoxicillin for eradication of H. pylori was 10 times less in AFIG than from the corresponding amoxicillin suspension. The results further substantiated that the prepared AFIG has feasibility of forming rigid gels in the gastric environment and eradicated H. pylori from the gastrointestinal tract more effectively than amoxicillin suspension because of the prolonged gastrointestinal residence time of the formulation.

To investigate the effectiveness of 4 d' anti-Helicobacter pylori therapy on the H pylori-infected Mongolian gerbils based on physiological and pathological changes.

We used 6-wk-old male gerbils orally inoculated with H pylori (ATCC43504, 2X10(8) CFU/mL). Seven weeks after H pylori inoculation, the animals of study group received 4 d' anti-H pylori triple therapy (H pylori-eradicated group). Seven days later, all animals of the H pylori-eradicated and control groups (H pylori-infected and H pylori-uninfected groups) were sacrificed. We examined gastric mucosal lesions macroscopically, studied gastritis microscopically and determined the stomach weight ratio, myeloperoxidase (MPO) activity and prostaglandin (PG) E(2) level.

The results showed that both macroscopic and histological gastric damages were significantly less in H pylori-eradicated group than H pylori-infected group. Stomach weight ratio, MPO activity and PGE(2) levels were significantly higher in H pylori-infected group than those in the other two groups.

Four days' anti-H pylori therapy was effective in the improvement of H pylori-induced gastric lesions in Mongolian gerbils.

Urease has been suggested to be essential for colonization and pathogenesis of Helicobacter pylori infection. In the present study, we evaluated the effects of urease inhibitors [acetohydroxamic acid (AHA) and flurofamide (FFA)] on H. pylori-induced gastritis in Mongolian gerbils. Animals were orally inoculated with H. pylori, and given urease inhibitors in their diet throughout the experimental period of six weeks or four weeks, starting from two weeks after H. pylori inoculation. With the administration of AHA at doses of 100, 500, and 2500 ppm throughout the experimental period, H. pylori-induced gastritis in animals was decreased in a dose-dependent manner, significantly so at 2500 ppm. Suppression of gastric lesions was also evident in animals administered 2500 ppm AHA after the H. pylori infection. Bacterial infection rates were reduced to 40-50% of the control value of 100%, by the highest dose of AHA. The potent urease inhibitor, FFA, also caused marked amelioration of H. pylori-associated gastritis on administration at 100 ppm throughout the six-week experimental period or for four weeks after H. pylori infection. Animals treated with FFA had few visible gastric lesions, and the proportion infected with H. pylori was reduced to less than 10%. Since antibiotic-resistant strains of H. pylori have become a serious problem, nonantibiotic urease inhibitors may be very useful to control H. pylori-associated gastroduodenal disease.

We previously reported the effect of Helicobacter pylori eradication on hyperammonaemia in patients with liver cirrhosis. However, the role of H pylori as a cause of hyperammonaemia is controversial. We developed an animal model with liver cirrhosis and investigated the effect of H pylori infection on hyperammonaemia.

Five week old male Mongolian gerbils were inoculated orally with broth culture of H pylori. Forty eight gerbils were divided into four groups. Gerbils not inoculated with H pylori were fed a commercial rodent diet (group A) or a choline deficient diet (group C). Gerbils inoculated with H pylori were fed the commercial rodent diet (group B) or the choline deficient diet (group D). Blood ammonia levels of the femoral vein and portal vein were measured 30 weeks later.

All gerbils fed the choline deficient diet developed liver cirrhosis with fatty metamorphosis. The survival rate of group D was significantly lower than that of the other groups. Systemic and portal blood ammonia levels in group D were significantly higher than those in the other groups.

H pylori infection induces hyperammonaemia in gerbils with liver cirrhosis.

Motility of Helicobacter pylori is essential for colonization. H. pylori has been shown to exhibit chemotactic activity toward urea and sodium and bicarbonate ions, which are secreted from the gastric epithelia. The importance of urease activity for chemotactic motility of H. pylori in a viscous environment has also been shown. Consequently, application of drugs inhibiting chemotactic motility has been proposed as a strategy for H. pylori eradication. This inhibitory effect can be evaluated through assay of chemotaxis and swarming.

H. pylori CPY3401 and ATCC43504 were grown on brucella agar plates/broth supplemented with 3% horse serum under microaerobic conditions (N2, 85%; O2, 5%; CO2, 10%). For motility assay, H. pylori cells grown on brucella-serum agar were stabbed into motility agar containing 0.35% refined agar in brucella-serum broth and the swarming zone was measured. For the chemotaxis assay, cells were suspended in 10 mM potassium phosphate buffer, pH 7.0, with 3% polyvinyl-pyrrolidone and assayed as described previously. Bacterial swimming in the fluid environment was observed under dark-field microscopy.

Numbers of bacteria attracted toward 1 microM flurofamide were reduced with increasing concentrations of sofalcone (0.2-222 microM). In addition, the size of the swarming zone was reduced in motility agar containing 22 and 222 microM sofalcone. On the other hand, 22 microM sofalcone did not inhibit bacterial growth on day 3. Bacterial swimming speed in brucella broth was slower in the presence of 22 and 222 microM sofalcone than in its absence.

Sofalcone was found to inhibit chemotactic motility of H. pylori. This drug may be useful for inhibiting the bacterium's ability to colonize the human stomach.

Animal models have played a significant role in research that aims to understand peptic ulceration. Firstly, they have helped define basic mechanisms of gastric mucosal defence and repair. The basis for gastric injury following NSAID administration was facilitated by animal models that correlated well with disease in humans. In early studies, ulceration was induced by grossly damaging insults to the gastric mucosa that were unphysiological. With refinement these models provided a clearer appreciation of stress ulceration. The discovery of Helicobacter pylori (H. pylori), as the cause of most ulcers, resulted in a need to re-evaluate the early literature and to look for new models. To date, these have contributed little to our understanding of the pathogenesis of H. pylori-induced ulcer. A major aim of this chapter is to suggest that thorough understanding of the animal models of Helicobacter infection may provide important new insights, in particular the factors controlling gastritis, the essential precursor lesion of ulceration. Available models include primates, cats, guinea pigs, ferrets and pigs. The mouse models provide opportunity for identifying both essential bacterial and host factors. The most severe pathologies are seen in the H. pylori-infected Mongolian gerbil with ulcers being formed in most animals. This is likely to become the standard animal model for investigation of peptic ulcer disease.

Helicobacter pylori can be eradicated by administration of antimicrobials, but resistant strains have emerged, and there is a need for novel therapeutic approaches against this infection. This study aimed to determine the safety and efficacy of 3'-sialyllactose sodium salt (3'SL), an oligosaccharide that occurs naturally in human and bovine milk and that can inhibit the adhesion of H. pylori to human epithelial cells in vitro.

Twelve H. pylori-positive rhesus monkeys were given 3'SL, either alone (regimens 1 and 2; n = 6) or in combination with omeprazole (regimen 3; n = 4), or bismuth subsalicylate (regimen 4; n = 6). Videogastroscopies were performed before, during, and after treatment, and gastric biopsy specimens were obtained for quantitative cultures and histology. The H. pylori strains colonizing the animals were genotyped.

After regimen 1 or 2, 2 of 6 animals were cured permanently, and a third animal was transiently cleared. The 3 other animals remained persistently colonized and did not respond to regimen 3. Regimen 4 resulted in transient decreases in colony counts in 3 of 6 other animals. Gastritis was suppressed only in the 2 animals who became persistently H. pylori negative. There was no apparent relation between 3'SL efficacy and any of the H. pylori tested genotypes. No side effects were observed in any of the animals receiving 3'SL.

Antiadhesive therapy is safe; it can cure or decrease H. pylori colonization in some rhesus monkeys, but the addition of a proton pump inhibitor or bismuth subsalicylate does not increase cure rate.