Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.

Oxidative stress is linked to carcinogenesis due to its ability to damage DNA. The human gastric pathogen Helicobacter pylori exerts much of its pathogenicity by inducing apoptosis and DNA damage in host gastric epithelial cells. Polyamines are abundant in epithelial cells, and when oxidized by the inducible spermine oxidase SMO(PAOh1) H 2 O 2 is generated. Here, we report that H. pylori up-regulates mRNA expression, promoter activity, and enzyme activity of SMO (PAOh1)

Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the upregulation of iNOS mRNA levels but in a concentrationdependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.Helicobacter pylori is a Gram-negative, microaerophilic bacterium that selectively colonizes the human stomach. Current prevalence of H. pylori is ϳ30 -40% of the population in the United States (1) and substantially higher in underdeveloped regions. H. pylori infection induces a vigorous mucosal immune response that fails to eradicate the organism and results in chronic gastritis that can lead to peptic ulcers and gastric cancer. In addition to a chronic lymphocytic response, H. pylori infection induces activation of an innate immune response in neutrophils, monocytes, and macrophages (2-8). Inducible NO synthase (iNOS) 1 -derived NO is a central effector molecule in the innate immune response to pathogens, with essential antimicrobial functions in host defense. We have reported that H. pylori induces iNOS expression and activity in macrophages (4 -7). H. pylori is considered a noninvasive pathogen, but it can disrupt epithelial integrity, and its antigens are present in the lamina propria (3). H. pylori can induce iNOS and other innate immune response genes in macrophages even when separated by filter supports or when water extracts are used (6). Although H. pylori-induced NO production can kill the bacterium in vitro (7, 9), it survives in the stomach, despite detection of iNOS in infected gastric mucosa (10).Production of NO by macrophages can be limited by H. pylori arginase that competes with iNOS for the same substrate, L-arginine (7) under conditions ...

Using an assay with a lower functional sensitivity may give an earlier indication of the presence of Tg in the serum on L-T4 treatment and may be used to study the trend in serum Tg without performing any TSH stimulation. Serum Tg determination obtained after TSH stimulation still permits a more reliable assessment of cure and patient's reassurance.

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