Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer. The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits. The present study describes the therapeutic vaccination of experimentally H. pyloriinfected beagle dogs, an animal model that reproduces several aspects of the human infection with H. pylori. The vaccine consisted of three recombinant H. pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 g each) with alum and administered intramuscularly either weekly or monthly. No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens. Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule. In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H. pylori colonization and the related gastric pathology.Helicobacter pylori is a spiral-shaped, gram-negative bacterium that infects the stomach of Ͼ50% of the population worldwide, with higher prevalence in the developing countries. H. pylori induces chronic inflammation of the stomach mucosa, causing chronic gastritis and peptic ulcer (9, 33); moreover, H. pylori infection is related to gastric mucosa-associated lymphoid tissue lymphoma (4) and to an increased risk of gastric cancer (36), as also proved in animal models (13,38).Current therapies, based on one antisecretory agent plus antibiotics, although effective in 80 to 90% of cases, face problems of patient compliance, increasing antibiotic resistance, and possible recurrence or reinfection; in spite of continuous effort to improve these treatments, no major breakthroughs have been achieved in the most recent years (30).To overcome the limits of antibiotic-based therapies, the vaccine approach has been undertaken since the last decade, leading us to identify some relevant bacterial antigens as candidates for vaccines (2). On the other hand, animal models of H. pylori infection have been developed to study the interaction between the bacterium and the host, the mechanisms of immune response to either infection or vaccination, and to determine the efficacy of both prophylactic and therapeutic vaccination (2,17,26,34). Among these animal models, that of the beagle dog reproduces several aspects of the human infection with H. pylori. In fact, in the beagle dog model, intragastric administration of H. pylori results in a long-term chronic infection, characterized by gastritis, epithelial alterations, superficial erosions, and the appearance of macroscopic follicles in the gastric mucosa, mainly in the antral region of the stomach (28,29).Most of the examples of vaccination against H. pylori in animal models reported in the...

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AIM:To investigate whether red wine and green tea could exert anti-H pylori or anti-VacA activity in vivo in a mouse model of experimental infection. METHODS:Ethanol-free red wine and green tea concentrates were administered orally as a mixture of the two beverages to H pylori infected mice, or separately to VacA-treated mice. Gastric colonization and gastric inflammation were quantified by microbiological, histopathological, and immunohistochemical analyses. RESULTS:In H pylori -infected mice, the red wine and green tea mixture significantly prevented gastritis and limited the localization of bacteria and VacA to the surface of the gastric epithelium. Similarly, both beverages significantly prevented gastric epithelium damage in VacA-treated mice; green tea, but not red wine, also altered the VacA localization in the gastric epithelium. CONCLUSION:Red wine and green tea are able to prevent H pylori -induced gastric epithelium damage, possibly involving VacA inhibition. This observation s u p p o r t s t h e p o s s i b l e r e l e va n c e o f d i e t o n t h e pathological outcome of H pylori infection.

SummaryHelicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor a a a a (TNF-a a a a ). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.

Helicobacter pylori colonizes the human gastric mucosa, causing inflammation that leads to atrophic gastritis, and it can cause peptic ulcer and gastric cancer. We show that polyphenol administration to mice experimentally infected by H. pylori or treated with VacA toxin can limit gastric epithelium damage, an effect that may be linked to VacA inhibition.Helicobacter pylori chronically infects the gastric mucosa of Ͼ50% of the human population, causing gastritis. The infection can lead to the development of peptic ulcer (27) and gastric mucosa-associated lymphoid tissue lymphoma (6) and increases the risk of gastric cancer in humans (8,11,31). It has been proven that H. pylori infection can cause gastric cancer in animals (9, 33). In both humans and animals, the gastric pathology depends on the virulence of the H. pylori infecting strain and on the genetic background of the host (4, 10, 22). In humans, lifestyle is also relevant (13, 26). The current antibiotic-based therapies are generally effective but can fail due to antibiotic resistance or lack of patient compliance. Thus, there is a continuous effort to develop new tools to fight against this pathogen. In particular, plant extracts or plant-derived substances have been investigated for anti-H. pylori activity in vitro (1,2,14,15,18,20,24) and in vivo (12,19,28).H. pylori expresses several factors that allow host stomach colonization and can play a role in pathogenesis. One of the most important factors is the vacuolating cytotoxin VacA (3, 29). We have already reported that polyphenols inhibit VacAinduced ion/urea conduction and cell vacuolation (30). Also, we have observed the ability of dealcoholized wine and green tea to reduce gastritis in mice experimentally infected by H. pylori (unpublished data). Hop bract extract has been recently reported to exert anti-VacA activity (35). Here, we investigated whether pure polyphenols could influence gastric colonization or gastritis in mice and/or counteract the effects of VacA in vivo.Animal experiments were done in compliance with current law. Data were evaluated by one-tailed Mann-Whitney U test, with P values of Ͻ0.05 considered significant.H. pylori strain SPM 326 type I, expressing the s1/m1 VacA isoform, was used to intragastrically infect specific-pathogenfree CD1 mice (Charles River, Calco, Italy), a well-established H. pylori infection model (17, 32). Noninfected controls received saline only. Starting 24 h before the infection, the animals had free access to drinking water containing 1% glucose and a mixture of 1.5 mg/ml each of tannic acid (average molecular weight, 1,701.20) and n-propyl gallate (Sigma-Aldrich, Milan, Italy) (TAϩNPG). TA and NPG were selected because of their previously shown anti-VacA activity in vitro (30), their water solubility, and the relative abundance of tannins and gallates in common beverages, such as wine and tea. Also, TA is a GRAS (generally regarded as safe) food additive. Based on previous results obtained with wine (unpublished data), the polyphenol concentration cho...

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

Helicobacter pylori has been widely recognized as an important human pathogen responsible for chronic gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Little is known about the natural history of this infection since patients are usually recognized as having the infection only after years or decades of chronic disease. Several animal models ofH. pylori infection, including those with different species of rodents, nonhuman primates, and germ-free animals, have been developed. Here we describe a new animal model in which the clinical, pathological, microbiological, and immunological aspects of human acute and chronic infection are mimicked and which allows us to monitor these aspects of infection within the same individuals. Conventional Beagle dogs were infected orally with a mouse-adapted strain of H. pylori and monitored for up to 24 weeks. Acute infection caused vomiting and diarrhea. The acute phase was followed by polymorphonuclear cell infiltration, interleukin 8 induction, mononuclear cell recruitment, and the appearance of a specific antibody response against H. pylori. The chronic phase was characterized by gastritis, epithelial alterations, superficial erosions, and the appearance of the typical macroscopic follicles that in humans are considered possible precursors of MALT lymphoma. In conclusion, infection in this model mimics closely human infection and allows us to study those phases that cannot be studied in humans. This new model can be a unique tool for learning more about the disease and for developing strategies for treatment and prevention.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.