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Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

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DFMO-treated H. pylori 60190 induce less IL-8 expression.Human gastric AGS cells were exposed to strain 60190 previously grown for 12 h with or without 1% DFMO and IL-8 expression was determined. (A) Real-time PCR was used to measure levels of IL-8 mRNA after 4 h of exposure. Bars indicate the mean level of gene expression relative to uninfected control cells (n = 3). (B) IL-8 protein levels in supernatants were measured by ELISA after 6 h of infection. Bars indicate the mean IL-8 concentration (n = 6). (C) After 2 h of infection adherent bacteria were quantified by serial dilution and plating. Each point is a single well of AGS cells and the means and standard errors are depicted (n = 8). **, p<0.0; ***, p<0.001 versus the control condition; §§, p<0.01; §§§, p<0.001 versus cells infected with untreated bacteria.
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pone-0017510-g007: DFMO-treated H. pylori 60190 induce less IL-8 expression.Human gastric AGS cells were exposed to strain 60190 previously grown for 12 h with or without 1% DFMO and IL-8 expression was determined. (A) Real-time PCR was used to measure levels of IL-8 mRNA after 4 h of exposure. Bars indicate the mean level of gene expression relative to uninfected control cells (n = 3). (B) IL-8 protein levels in supernatants were measured by ELISA after 6 h of infection. Bars indicate the mean IL-8 concentration (n = 6). (C) After 2 h of infection adherent bacteria were quantified by serial dilution and plating. Each point is a single well of AGS cells and the means and standard errors are depicted (n = 8). **, p<0.0; ***, p<0.001 versus the control condition; §§, p<0.01; §§§, p<0.001 versus cells infected with untreated bacteria.

Mentions: Acute active inflammation characterized by neutrophil infiltration is a central constituent of the immune response to H. pylori infection. Chemokines, which are secreted chemoattractant proteins, are responsible for the influx of these cells to the site of infection. IL-8 is one such chemokine, and its expression has been reported to be increased in the gastric mucosa of infected patients [16], [17], [40] and in gastric epithelial cells inoculated with H. pylori in vitro [41], [42]. We cocultured human gastric epithelial AGS cells with H. pylori 60190, a strain known to induce IL-8 in these cells [43] at an MOI of 200 for 4 h and then analyzed mRNA levels (Figure 7A). H. pylori 60190 induced a 145.1±9.5-fold increase in IL-8 mRNA expression compared with untreated cells (p<0.001) and this induction was nearly halved when bacteria cultured in the presence of 1% DFMO for 12 h prior to addition to host cells were used (73.4±9.4-fold, p<0.001). We also measured IL-8 protein levels in supernatants of AGS cells cocultured with H. pylori 60190 at an MOI of 100 for 6 h (Figure 7B). Cells infected with bacteria grown without DFMO produced 3316±392 pg/mL IL-8, a 7.2±2.5-fold increase over control cells (p<0.001). When H. pylori cultured in the presence of 1% DFMO were used, AGS cells generated only 2066±316 pg/mL IL-8, a 47.8±6.7% inhibition (p<0.01). These data suggest that the immunostimulatory effect of the bacteria is lessened by exposure to DFMO.


Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

DFMO-treated H. pylori 60190 induce less IL-8 expression.Human gastric AGS cells were exposed to strain 60190 previously grown for 12 h with or without 1% DFMO and IL-8 expression was determined. (A) Real-time PCR was used to measure levels of IL-8 mRNA after 4 h of exposure. Bars indicate the mean level of gene expression relative to uninfected control cells (n = 3). (B) IL-8 protein levels in supernatants were measured by ELISA after 6 h of infection. Bars indicate the mean IL-8 concentration (n = 6). (C) After 2 h of infection adherent bacteria were quantified by serial dilution and plating. Each point is a single well of AGS cells and the means and standard errors are depicted (n = 8). **, p<0.0; ***, p<0.001 versus the control condition; §§, p<0.01; §§§, p<0.001 versus cells infected with untreated bacteria.
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Related In: Results  -  Collection

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pone-0017510-g007: DFMO-treated H. pylori 60190 induce less IL-8 expression.Human gastric AGS cells were exposed to strain 60190 previously grown for 12 h with or without 1% DFMO and IL-8 expression was determined. (A) Real-time PCR was used to measure levels of IL-8 mRNA after 4 h of exposure. Bars indicate the mean level of gene expression relative to uninfected control cells (n = 3). (B) IL-8 protein levels in supernatants were measured by ELISA after 6 h of infection. Bars indicate the mean IL-8 concentration (n = 6). (C) After 2 h of infection adherent bacteria were quantified by serial dilution and plating. Each point is a single well of AGS cells and the means and standard errors are depicted (n = 8). **, p<0.0; ***, p<0.001 versus the control condition; §§, p<0.01; §§§, p<0.001 versus cells infected with untreated bacteria.
Mentions: Acute active inflammation characterized by neutrophil infiltration is a central constituent of the immune response to H. pylori infection. Chemokines, which are secreted chemoattractant proteins, are responsible for the influx of these cells to the site of infection. IL-8 is one such chemokine, and its expression has been reported to be increased in the gastric mucosa of infected patients [16], [17], [40] and in gastric epithelial cells inoculated with H. pylori in vitro [41], [42]. We cocultured human gastric epithelial AGS cells with H. pylori 60190, a strain known to induce IL-8 in these cells [43] at an MOI of 200 for 4 h and then analyzed mRNA levels (Figure 7A). H. pylori 60190 induced a 145.1±9.5-fold increase in IL-8 mRNA expression compared with untreated cells (p<0.001) and this induction was nearly halved when bacteria cultured in the presence of 1% DFMO for 12 h prior to addition to host cells were used (73.4±9.4-fold, p<0.001). We also measured IL-8 protein levels in supernatants of AGS cells cocultured with H. pylori 60190 at an MOI of 100 for 6 h (Figure 7B). Cells infected with bacteria grown without DFMO produced 3316±392 pg/mL IL-8, a 7.2±2.5-fold increase over control cells (p<0.001). When H. pylori cultured in the presence of 1% DFMO were used, AGS cells generated only 2066±316 pg/mL IL-8, a 47.8±6.7% inhibition (p<0.01). These data suggest that the immunostimulatory effect of the bacteria is lessened by exposure to DFMO.

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

Show MeSH
Related in: MedlinePlus