Epigenetic silencing of miR-124 prevents spermine oxidase regulation: implications for Helicobacter pylori-induced gastric cancer.

Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3'-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.

Increased Helicobacter pylori-associated gastric cancer risk in the Andean region of Colombia is mediated by spermine oxidase.

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.

CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions.

The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3' region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.

Determinación de la eficacia analgésica de los bloqueos del ganglio estrellado en el síndrome doloroso regional complejo con dolor mediado por el sistema nervioso simpático: estudio preliminar / Study of the analgesic efficacy of stellate ganglion blockade in the management of the complex regional pain syndrome in patients with pain mediated by sympathetic nervous system: preliminary study

Objetivo: Este estudio fue realizado con el propósito de determinar la eficacia analgésica de los bloqueos del ganglio estrellado, en el alivio del dolor mediado por el sistema nervioso simpático, en pacientes con síndrome doloroso regional complejo. Pacientes y métodos: Se realizó un ensayo clínico controlado con asignación aleatoria y enmascaramiento simple. Treinta y nueve pacientes fueron tratados con una serie de bloqueos de ganglio estrellado, terapia física y tratamiento farmacológico, mientras que treinta y dos pacientes fueron tratados con fisioterapia y el mismo esquema farmacológico. Para determinar la asociación entre las variables se utilizó el riesgo relativo con sus respectivos intervalos de confianza. Resultados: En la evaluación clínica realizada un mes postratamiento se encontró alivio del dolor en 84,6% de los pacientes del grupo de intervención y en 78,1% de los controles (RR= 1,08; I.C. 95%=0,8-1,4; p=0.48), sin encontrarse diferencias estadísticamente significativas. No se encontró asociación entre la eficacia analgésica y tabaquismo, dominancia, género, tipo de SDRC, causa desencadenante y nivel educativo (AU)

Objective: The purpose of this study was to determine the analgesic efficacy of stellate ganglion blockade in pain mediated by the sympathetic nervous system in patients with Complex Regional Pain Syndrome (CRPS). Patients and methods: A randomized, simple-blinded controlled clinical trial was conducted. Thirty nine patients were randomly assigned to an intervention group which was treated with a series of stellate ganglion blockades, physical therapy and pharmacological treatment, and thirty two to a control group which was treated with physical therapy and the same pharmacological treatment. Risk ratio was used to evaluate outcome and determine association with predictor variables. Results: At the end of the first month post treatment, it was found that 84.6% of patients in the intervention group had alleviation of their pain while 78.1% of the control group had alleviation of their pain; there was not a statistically significant difference (RR=1.08; C.I. 95%=0.8-1.4; p=0.48). We found no association between analgesic efficacy, smoking, dominance, gender, and type of CRPS, unleashing cause or educational level (AU)

Long term follow up of patients treated for Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori infection induces progressive inflammatory changes in the gastric mucosa that may lead to gastric cancer. Understanding long term effects resulting from the cure of this infection is needed to design cancer prevention strategies. METHODS: A cohort of 795 adults with preneoplastic gastric lesions was randomised to receive anti-H pylori treatment and/or antioxidants. At the end of six years of intervention, those who did not receive anti-H pylori treatment were offered it. Gastric biopsies were obtained at baseline, and at 3, 6, and 12 years. A histopathology score was utilised to document changes in gastric lesions. Non-linear mixed models were used to estimate the cumulative effect of H pylori clearance on histopathology scores adjusted for follow up time, interventions, and confounders. RESULTS: Ninety seven per cent of subjects were H pylori positive at baseline, and 53% were positive at 12 years. Subjects accumulated 1703 person years free of infection. A multivariate model showed a significant regression in histopathology score as a function of the square of H pylori negative time. Subjects who were H pylori negative had 14.8% more regression and 13.7% less progression than patients who were positive at 12 years (p = 0.001). The rate of healing of gastric lesions occurred more rapidly as years free of infection accumulated, and was more pronounced in less advanced lesions. CONCLUSIONS: Preneoplastic gastric lesions regress at a rate equal to the square of time in patients rendered free of H pylori infection. Our findings suggest that patients with preneoplastic gastric lesions should be treated and cured of their H pylori infection.

Anti-inflammatory and tissue-protectant drug effects: results from a randomized placebo-controlled trial of gastritis patients at high risk for gastric cancer.

BACKGROUND: The inflammatory process involving Helicobacter pylori-associated gastritis is thought to lead to epithelial damage and contribute to the development of gastric cancer. Evidence exists from animal and in vitro studies suggesting that tetracyclines have both anti-inflammatory and tissue-protectant effects unrelated to their antimicrobial activity. We attempted to modulate components of H. pylori's inflammatory process by: (i) eliminating the infection; (ii) using tetracycline to alter the host's reaction to the infection without reducing the bacterial load; and (iii) using calcium to counteract the effect of excessive dietary salt. METHODS: We conducted a 16-week placebo-controlled clinical trial with 374 H. pylori-associated gastritis patients randomly assigned to one of five groups: (1) triple therapy consisting of metronidazole, amoxicillin and bismuth subsalicylate for 2 weeks, followed by bismuth alone for 14 weeks; (2) calcium carbonate; (3) triple therapy and calcium carbonate; (4) tetracycline; or (5) placebo. RESULTS: Subjects in the tetracycline and triple therapy groups, but not the calcium carbonate only group, showed a reduction in inflammation and epithelial damage vs. those in the placebo group, independent of a change in H. pylori density and other factors. Our results also indicate that epithelial damage may be affected by mechanisms independent of H. pylori density or inflammation. CONCLUSION: The results are consistent with the hypothesis that tetracycline can decrease inflammation independent of a reduction in the bacterial load. More research is needed to investigate mechanisms leading to epithelial damage which are independent of H. pylori density and inflammation.

Helicobacter pylori genotypes may determine gastric histopathology.

The outcome of Helicobacter pylori infection has been associated with specific virulence-associated bacterial genotypes. The present study aimed to investigate the gastric histopathology in Portuguese and Colombian patients infected with H. pylori and to assess its relationship with bacterial virulence-associated vacA, cagA, and iceA genotypes. A total of 370 patients from Portugal (n = 192) and Colombia (n = 178) were studied. Corpus and antrum biopsy specimens were collected from each individual. Histopathological features were recorded and graded according to the updated Sydney system. H. pylori vacA, cagA, and iceA genes were directly genotyped in the gastric biopsy specimens by polymerase chain reaction and reverse hybridization. Despite the significant differences between the Portuguese and Colombian patient groups, highly similar results were observed with respect to the relation between H. pylori genotypes and histopathology. H. pylori vacA s1, vacA m1, cagA+ genotypes were significantly associated with a higher H. pylori density, higher degrees of lymphocytic and neutrophilic infiltrates, atrophy, the type of intestinal metaplasia, and presence of epithelial damage. The iceA1 genotype was only associated with epithelial damage in Portuguese patients. These findings show that distinct H. pylori genotypes are strongly associated with histopathological findings in the stomach, confirming their relevance for the development of H. pylori-associated gastric pathology.

Morphometric evaluation of gastric antral atrophy: improvement after cure of Helicobacter pylori infection.

OBJECTIVE: Our purpose was to find out if morphometric techniques can document long term changes in gastric antral atrophy after curing Helicobacter pylori infection with or without dietary supplementation with antioxidant micronutrients. METHODS: Study subjects were 132 adult volunteers from a Colombian region with high gastric cancer rates. Participants were randomly assigned to ascorbic acid, beta-carotene, and anti-H. pylori treatment, following a factorial design. Gastric biopsies were obtained at baseline and after 72 months of intervention. Atrophy was evaluated by a standard visual analog scale and by morphometry. RESULTS: Statistically significant changes in antral atrophy were detected with morphometric techniques after intervention in subjects who received anti-H. pylori treatment. A nonsignificant trend was also observed with visual scores. This effect was greater among those who were free of infection at the end of the trial. After accounting for the effect of anti-H. pylori treatment, no significant effect was noted for dietary supplementation with ascorbic acid and/or beta-carotene. CONCLUSIONS: We conclude that gastric atrophy improves significantly after long term control of H. pylori infection. This effect can be demonstrated both by conventional histological grading and by morphometry.

Chemoprevention of gastric dysplasia: randomized trial of antioxidant supplements and anti-helicobacter pylori therapy.

BACKGROUND: Previous research has identified a high risk of gastric carcinoma as well as a high prevalence of cancer precursor lesions in rural populations living in the province of Nariño, Colombia, in the Andes Mountains. METHODS: A randomized, controlled chemoprevention trial was conducted in subjects with confirmed histologic diagnoses of multifocal nonmetaplastic atrophy and/or intestinal metaplasia, two precancerous lesions. Individuals were assigned to receive anti-Helicobacter pylori triple therapy and/or dietary supplementation with ascorbic acid, beta-carotene, or their corresponding placebos. Gastric biopsy specimens taken at baseline were compared with those taken at 72 months. Relative risks of progression, no change, and regression from multifocal nonmetaplastic atrophy and intestinal metaplasia were analyzed with multivariate polytomous logistic regression models to estimate treatment effects. All statistical tests were two-sided. RESULTS: All three basic interventions resulted in statistically significant increases in the rates of regression: Relative risks were 4.8 (95% confidence interval [CI] = 1.6-14.2) for anti-H. pylori treatment, 5. 1 (95% CI = 1.7-15.0) for beta-carotene treatment, and 5.0 (95% CI = 1.7-14.4) for ascorbic acid treatment in subjects with atrophy. Corresponding relative risks of regression in subjects with intestinal metaplasia were 3.1 (95% CI = 1.0-9.3), 3.4 (95% CI = 1.1-9.8), and 3.3 (95% CI = 1.1-9.5). Combinations of treatments did not statistically significantly increase the regression rates. Curing the H. pylori infection (which occurred in 74% of the treated subjects) produced a marked and statistically significant increase in the rate of regression of the precursor lesions (relative risks = 8.7 [95% CI = 2.7-28.2] for subjects with atrophy and 5.4 [95% CI = 1.7-17.6] for subjects with intestinal metaplasia). CONCLUSIONS: In the very high-risk population studied, effective anti-H. pylori treatment and dietary supplementation with antioxidant micronutrients may interfere with the precancerous process, mostly by increasing the rate of regression of cancer precursor lesions, and may be an effective strategy to prevent gastric carcinoma.

Effects of acid suppression and bismuth medications on the performance of diagnostic tests for Helicobacter pylori infection.

OBJECTIVE: This study was designed to investigate whether acid suppression and bismuth medications interfere with the performance of diagnostic tests for Helicobacter pylori (H. pylori) infection. METHODS: Sixty patients with previous diagnoses of atrophic gastritis and H. pylori infection made in gastric biopsies taken at Hospital Departmental, Pasto, Colombia, were enrolled in the study. 13C breath urea test (UBT) and stool antigen test (HpSA) were performed simultaneously. Two baseline tests were performed: one 7 days before and another the day before starting medications. A total of 20 patients received for 2 wk one of the following treatments: a) ranitidine; b) lansoprazole; or c) bismuth subsalicylate. The tests were repeated while the patients were on the prescribed medication on days 7 and 14 and then 2 wk after finishing the medication. RESULTS AND CONCLUSIONS: Utilizing standard cut-off values for the tests, our results indicate that in the case of the 13C UBT test, ranitidine does not interfere with the results, whereas lansoprazole and bismuth may be expected to yield a significant proportion of false negative results (30-40% for lansoprazole and 45-55% for bismuth). In the case of the HpSA test, ranitidine does not interfere, whereas lansoprazole and bismuth may be expected to yield a nonsignificant proportion of false negative results (15-25% for lansoprazole and 10-15% for bismuth). Absolute values for both tests may be used to study the effects of the pharmacological agents on the characteristics of the infection.

Eradication of Helicobacter pylori infection with proton pump-based triple therapy in patients in whom bismuth-based triple therapy failed.

To study the effects of treatment of Helicobacter pylori infection in a hyperendemic population, 143 infected patients from the region of Nariño, Colombia, were treated for 2 weeks with clarithromycin (500 mg twice a day), amoxicillin (1 g twice a day), and either lansoprazole (30 mg twice a day) or omeprazole (30 mg twice a day). All patients belong to a low socioeconomic strata, had multifocal atrophic gastritis documented by gastric biopsies, and had been treated previously and unsuccessfully for 2 weeks with bismuth subsalicylate (262 mg four times a day), amoxicillin (500 mg three times a day), and metronidazole (400 mg three times a day). 13C-urea breath tests were performed 6, 12, 24, and 60 weeks after completing therapy. The 13C-urea breath test was negative in 79.7% of patients 1 month after finishing therapy, and in 69.2% of patients 1 year after finishing treatment. There were no differences in eradication rates between patients treated with omeprazole versus lansoprazole. Dyspepsia symptoms decreased from 74% in patients at baseline to 19% at the time of finishing treatment. In low-socioeconomic status populations with hyperendemic infection, triple therapy using omeprazole or lansoprazole plus clarithromycin and amoxicillin is an effective alternative when previous standard bismuth-based triple therapy has failed.

Review article: Antioxidant micronutrients and gastric cancer.

A review of the literature reveals a very consistent association between gastric cancer risk and low intake of fruits and vegetables. This observation has been documented in many countries with different epidemiological techniques: interpopulation correlations, case-control studies and follow up of several cohorts. Low serum levels of beta-carotene and alpha-tocopherol, but not vitamin C, have been reported in patients with gastric dysplasia. Helicobacter pylori infection has been associated with lower concentrations of vitamin C in the gastric juice. Detailed studies in Colombia and New Orleans have shown a gradient towards lower concentration in the gastric juice and lower ratios of gastric juice to serum concentration of vitamin C in the following comparisons: i) lower vs. higher gastric cancer risk; ii) mild vs. advanced gastric precancerous histopathologic lesions; iii) mild vs. advanced degree of atrophy; iv) mild vs. advanced damage to the surface gastric epithelium; v) lower vs. higher gastric pH. Such a gradient is not observed for serum levels of vitamin C. The role of infection with H. pylori in the metabolism of ascorbic acid is discussed, as well as the possible role of ascorbic acid in inhibiting cell damage by reactive oxygen species.

Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants.

Helicobacter pylori infection is a known risk factor for gastric cancer. We hypothesized that H. pylori infection would lead to the sustained production of the reactive nitrogen species nitric oxide and peroxynitrite as part of the host immune response. We further hypothesized that H. pylori infection would lead to increased apoptosis of gastric epithelial cells, possibly in response to free radical-mediated DNA damage. Using immunohistochemistry, we stained and scored gastric antral biopsies from 84 Colombian patients with nonatrophic gastritis before and after treatment for H. pylori infection. We examined expression of inducible nitric oxide synthase (iNOS); nitrotyrosine, a marker for peroxynitrite; and DNA fragmentation, a marker for apoptosis. Patients were treated with triple therapy (amoxicillin, 500 mg three times a day for 2 weeks; metronidazole, 400 mg three times a day for 2 weeks; and bismuth subsalicylate, 262 mg four times a day for 2 weeks, followed by 262 mg every day for 4-12 months). Eradication of H. pylori infection resulted in a significant reduction in iNOS and nitrotyrosine staining and a marginally significant reduction in apoptosis. Dietary supplementation with beta-carotene (30 mg every day for 4-12 months) resulted in a significant decrease in iNOS staining. Supplementation with ascorbic acid (1 g twice a day for 4-12 months) led to a significant reduction in nitrotyrosine staining. In patients supplemented with either ascorbic acid or beta-carotene, there was a trend toward a reduction in apoptosis, but this was not statistically significant. We conclude that H. pylori infection is accompanied by the formation of endogenous reactive nitrogen intermediates, which may contribute to DNA damage and apoptosis. In addition to antimicrobial therapy, dietary supplementation with beta-carotene and ascorbic acid may prevent the formation of these potential carcinogens.