Microbiology

BACKGROUND & AIMS-Helicobacter pylori-induced immune responses fail to eradicate the bacterium. Nitric oxide (NO) can kill H. pylori. However, translation of inducible NO synthase (iNOS) and NO generation by H. pylori-stimulated macrophages is inhibited by the polyamine spermine derived from ornithine decarboxylase (ODC), and is dependent on availability of the iNOS substrate L-arginine (L-Arg). We determined if spermine inhibits iNOS-mediated immunity by reducing L-Arg uptake into macrophages.

BACKGROUND & AIMS-Helicobacter pylori-induced gastric carcinogenesis has been linked to the microbial oncoprotein CagA. Spermine oxidase (SMO) metabolizes the polyamine spermine into spermidine and generates H 2 O 2 that causes apoptosis and DNA damage. We determined if pathogenic effects of CagA are attributable to SMO.

Background and Aim: Dysregulated epithelial secretory function can lead to the clinical manifestation of diarrhea. Intestinal fluid secretion is driven by active Clion secretion and is dependent on the availability of O 2 for generation of cellular energy. Hydroxylases are the primary intracellular sensors of O 2 availability, and we have previously shown that hydroxylase inhibition attenuates colonic epithelial secretory capacity. Here, we sought to investigate the molecular mechanisms involved and to test the efficacy of hydroxylase inhibitors in preventing diarrhea In Vivo. Methods: The pan-hydroxylase inhibitor dimethyloxallyl glycine (DMOG) was used to inhibit hydroxylase activity. Clsecretion was measured as changes in short circuit current across monolayers of T 84 cells or muscle-stripped segments of mouse colon. Western blotting and biotinylation techniques were used to assess protein expression. DMOG effects were also tested In Vivo in a mouse allergic (ovalbumin) model of diarrhea. Results: As previously reported treatment of T 84 cells with DMOG (1 mM) reduced Clsecretory responses to the Ca 2+ and cAMP-dependent agonists, carbachol (CCh) and forskolin (FSK), to 20.2 ± 2.6% and 38.8 ± 4.8% of controls, respectively (n=16; p<0.001). To determine molecular mechanisms involved we analysed the activity of transport proteins comprising the Clsecretory pathway. Hydroxylase inhibition did not alter apical Clor basolateral K + conductances but significantly reduced the activity of the Na + /K + ATPase, the energy-dependent step of Clsecretion, to 42.7% ± 5.5% of controls (n=5; p≤0.01). However, DMOG did not alter total cellular or surface expression of the ATPase. Cellular ATP, required to drive pump activity, was reduced to 67% ± 8.5% of controls (n=6; p≤0.05) by DMOG. Intraperitoneal injection of mice with DMOG (320 mg/kg; 24 hrs) attenuated colonic secretory responses to CCh and FSK to 54.8 ± 6.1% (n=11; p≤0.001) and 72.4 ± 11.5% (n=11; p≤0.05) of those of controls, respectively. Furthermore, in an In Vivo mouse model of allergic diarrhea, DMOG pretreatment reduced the occurrence of diarrhea by 80% compared to controls (n=10). Conclusion: Our studies show that hydroxylase inhibition in colonic epithelial cells inhibits secretory function. This antisecretory effect is mediated by attenuation of Na + /K + -ATPase pump activity, either directly, or secondary to the depletion of cellular ATP. Our data suggest that by virtue of their ability to regulate epithelial transport In Vitro and In Vivo, hydroxylases present a promising target for the development of novel anti-diarrheal agents that act by directly modulating transport protein function.

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2 2/2 mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2 2/2 mice but not in WT mice. When mice infected with H. pylori were compared, Arg2 2/2 mice had more gastric macrophages, more of these cells were iNOS + , and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2 2/2 versus WT mice, indicating increased NO generation. Infected Arg2 2/2 mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-g, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.

Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of Larginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2 2/2 mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2 2/2 mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2 2/2 mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection. Citation: Barry DP, Asim M, Scull BP, Piazuelo MB, de Sablet T, et al. (2011) Cationic Amino Acid Transporter 2 Enhances Innate Immunity during Helicobacter pylori Infection. PLoS ONE 6(12): e29046.

who achieved clinical response at wk8 were randomized to IFX 5mg/kg q8wks through wk46 or q12wks through wk42. Non-responders were discontinued from study agent. Results: 60 patients were enrolled. Baseline demographics and disease characteristics were generally comparable across the grps:53% female, median age 14.5yrs, weight 50.8kg, disease duration 1.4yrs, CRP 0.3mg/dL, median Mayo score 8.0, and median pediatric UC activity index (PUCAI) score 55. IFX induced clinical response at wk8 in 73.3%(44/60) patients [95% CI: 62.1%, 84.5%] and met the criteria for a positive study. At wk8, 40.0%(24/60) were in clinical remission by the Mayo score and 33.3%(17/51) were in remission by the PUCAI. At wk8, 68.3%(41/60) patients achieved mucosal healing (endoscopy subscore of 0/1). At wk54, among patients who continued treatment, a numerically greater proportion of patients were in remission in the 5mg/kg q8wk grp(38.1%, [8/21]) vs 5mg/kg q12wk grp(18.2% [4/22]) though this did not reach statistical significance (p=0.146). More patients on corticosteroids at baseline were in remission and off corticosteroids at wk54 in the q8wk maintenance grp (38.5% [5/13]) than in the q12 wk maintenance grp (0.0% [0/13]). IFX was generally well tolerated. The proportions of patients experiencing serious AEs and infusion reactions were similar across maintenance grps. No deaths, malignancies, opportunistic infections, TB or delayed hypersensitivity reactions were reported. Conclusions: IFX is effective and safe in the treatment of pediatric patients with moderately to severely active UC with results comparable to those of the ACT trials. At wk54, twice as many patients were in remission following q8wk vs q12w therapy.

Helicobacter pylori incites a futile inflammatory response, which is the key feature of its immunopathogenesis. This leads to the ability of this bacterial pathogen to survive in the stomach and cause peptic ulcers and gastric cancer. Myeloid cells recruited to the gastric mucosa during H. pylori infection have been directly implicated in the modulation of host defense against the bacterium and gastric inflammation. Heme oxygenase-1 (HO-1) is an inducible enzyme that exhibits anti-inflammatory functions. Our aim was to analyze the induction and role of HO-1 in macrophages during H. pylori infection. We now show that phosphorylation of the H. pylori virulence factor cytotoxin-associated gene A (CagA) in macrophages results in expression of hmox-1, the gene encoding HO-1, through p38/NF (erythroid-derived 2)-like 2 signaling. Blocking phagocytosis prevented CagA phosphorylation and HO-1 induction. The expression of HO-1 was also increased in gastric mononuclear cells of human patients...