Essential oils as components of a diet-based approach to management of Helicobacter infection.

An increased density of Helicobacter pylori in the gastric mucosa can be associated with more severe gastritis and an increased incidence of peptic ulcers. Therefore, people with asymptomatic gastritis would certainly benefit from a nutritional approach to help them manage the infection and therefore decrease the risk of development of associated pathologies. We analyzed the activities of 60 essential oils against H. pylori P1 and identified 30 oils that affected growth, with in vitro inhibition zones ranging between 0.7 and 6.3 cm in diameter. We further analyzed the effects of 16 oils with different activities on H. pylori P1 viability. Fifteen showed strong bactericidal activities, with minimal bactericidal concentrations after 24 h ranging from 0.02 to 0.1 g/liter at pH 7.4. Even though slight variations in activities were observed, the essential oils that displayed the strongest bactericidal potentials against H. pylori P1 were also active against other Helicobacter strains tested. Among the pure constituents of different essential oils tested, carvacrol, isoeugenol, nerol, citral, and sabinene exhibited the strongest anti-H. pylori activities. Although oral treatment of H. pylori SS1-infected mice with carrot seed oil did not result in significant decreases in the bacterial loads in the treated animals compared to those in the control animals, in all experiments performed, the infection was cleared in 20 to 30% of carrot seed oil-treated animals. Our results indicate that essential oils are unlikely to be efficient anti-Helicobacter agents in vivo. However, their effects may not be irrelevant if one plans to use them as food additives to complement present therapies.

A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells.

The attenuated Salmonella typhimurium PhoPc strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoPc bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoPc was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoPc expressing green fluorescent protein at early time points. In addition, live PhoPc were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoPc is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo.

Urease-based mucosal immunization against Helicobacter heilmannii infection induces corpus atrophy in mice.

Mucosal immunization with Helicobacter heilmannii urease B or Helicobacter pylori urease, given nasally with cholera toxin, protects BALB/c mice against H. heilmannii infection and significantly reduces a preexisting infection. However, immunization aggravates gastric corpus atrophy. Our results underline the necessity of defining immunization regimens that do not enhance mucosal damage.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phoPc expressing urease A and B subunits.

Live Salmonella typhimurium phoPc bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens. The genes encoding the A and B subunits of H. pylori urease were introduced into S. typhimurium phoPc and expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB). Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7-ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer's patches of immunized mice, while the plasmid carrying tac-ureAB was rapidly lost. Spleen and Peyer's patch CD4+ lymphocytes from mice immunized with S. typhimurium phopc cT7-ureAB proliferated in vitro in response to urease, whereas cells from mice given S. typhimurium phoPc alone did not. Splenic CD4+ cells from mice immunized with phoPc cT7-ureAB secreted gamma interferon and interleukin 10, while Peyer's patch CD4+ cells did not secrete either cytokine. Specific H. pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized with S. typhimurium phoPc cT7-ureAB were found to be resistant to infection by H. pylori, while all mice immunized with phoPc tac-ureAB (15 of 15) or phoPc (15 of 15) were infected. Our data demonstrate that H. pylori urease delivered nasally by using a vaccine strain of S. typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection.

Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Perspectives of anti-H. pylori vaccination.

Mucosal vaccination using different antigens in conjunction with adjuvants has been used for the prevention and even cure of Helicobacter infection in animal models. A phase I-II trial was recently performed on infected volunteers with urease and the heat labile enterotoxin from E. coli (LT). A significant decrease in bacterial density but no cure of infection was observed in some patients. The immune effectors which prevent or cure infection with Helicobacter are not well understood and will need to be more clearly defined in order to improve vaccination strategies. Future developments will likely include the following: generation of new mucosal adjuvants without gastrointestinal toxicity; combination of two or three different antigens in order to ensure broader efficacy; use of different routes of administration such as nasal or rectal; coadministration of anti-Helicobacter treatment and vaccine; development of alternate vaccine methods which do not require a mucosal adjuvant, i.e. antigen expression by live carriers or by DNA vaccination; combination of different vaccination methods, for instance DNA vaccination followed by a mucosal boost.