Marilyn Owens

Can pre-neoplastic lesions be detected in gastric biopsies of children with Helicobacter pylori infection?

Background Active gastritis, gastric mucosal atrophy and intestinal metaplasia are lesions associated with Helicobacter pylori infection. Atrophy and intestinal metaplasia are only seen in adults. Objectives We describe pediatric patients with atrophy and metaplasia, and compare the inflammatory response in these patients to controls. Methods As part of a multicenter study of pediatric H. pylori infection, gastric biopsy specimens obtained during diagnostic upper endoscopy of 19 H. pylori-infected children and 45 uninfected controls were reviewed and graded by using the updated Sydney system. The inflammatory response was characterized using immunohistochemistry for T lymphocytes, B lymphocytes, and macrophages, and TUNEL assay for apoptosis. Results Histology of H. pylori-infected and control biopsy specimens showed active gastritis in 32% and 2% respectively (P = 0.002). Mild intestinal metaplasia was found in 4 H. pylori-infected children, in two of whom it appeared to be accompanied by atrophy. Specimens from patients with H. pylori infection contained increased numbers of B lymphocytes in lymphoid nodules, and apoptosis in the superficial epithelium and inflammatory cells. T lymphocytes and macrophages appeared in similar numbers in specimens from controls and infected patients. Conclusions We describe intestinal metaplasia associated with H. pylori infection in children. Since atrophy usually precedes intestinal metaplasia in adults, we suggest that atrophy exists in children. High numbers of B lymphocytes and apoptosis in the surface epithelium are seen in patients with H. pylori infection and may be related to the development of atrophy and intestinal metaplasia.

Genotypic, Clinical, and Demographic Characteristics of Children Infected with Helicobacter pylori

ABSTRACT Helicobacter pylori isolates vary between geographic regions. Certain H. pylori genotypes may be associated with disease outcome. Thirty-eight children underwent diagnostic upper endoscopy at four medical centers and were retrospectively analyzed to determine if H. pylori virulence genes were associated with endoscopic disease severity, histologic parameters, and host demographics. The H. pylori virulence genotype was analyzed by a reverse hybridization line probe assay and type-specific PCR. Endoscopic ulcers or erosions were found in 17 (45%) patients, with 13 (34%) of these patients having antral nodularity. Histological gastritis, of varying severity, was present in all children. Four patients harbored more than one H. pylori strain: one subject had both cagA+ andcagA-negative strains, while three patients harbored either two different cagA-negative strains (two children) or twocagA+ strains (one child). There were 28 (74%)cagA+ isolates; 19 were associated with thevacA s1b genotype, 7 were associated with thevacA s1a genotype, 1 was associated with thevacA s1c genotype, and 1 was associated with the s2 genotype. Of 14 cagA-negative isolates, 6 werevacA s2 genotype, 4 were vacA s1b, 3 werevacA s1a, and 1 was vacA s1c. Nine of ten (90%) Hispanics had similar H. pylori strains (vacA s1b,m1), and all Asian-Canadian children were infected by strains with vacA s1c genotype. No correlation between H. pylori strain and endoscopic or histopathologic abnormalities was found. This study provides a baseline framework of North American children and their H. pylori strains, serving as a powerful epidemiological tool for prospective investigations to better understand the transmission and evolution of diverse disease outcomes.

S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death.

Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2-24 h in the absence and presence of GSNO (dose range 0.1-5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration (P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2 consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2-24 h in the absence and presence of GSNO (dose range 0.1-5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration ( P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2 consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.

Polymorphisms in Cytokine Genes and Risk of Helicobacter pylori Infection among Jamaican Children

Background:  Infection by Helicobacter pylori is often acquired during childhood. Recent studies suggest that inflammatory cytokines may play a role in susceptibility to, and disease phenotype caused by, H. pylori infection, but the association of host genetic variability with risk of H. pylori infection has not been studied in children.

S-nitrosoglutathione inhibits TNF-α-induced NFκB activation in neutrophils

Abstract:Objective and Design: Cytokine expression is controlled by transcription factors including NFκB, which has recently been found to exist in human neutrophils. We previously showed that exogenous nitric oxide (NO) induces neutrophil apoptosis and hypothesized that this NO effect could be mediated by inhibition of NFκB activation.¶Materials and methods: Isolated human neutrophils were incubated with or without S-nitrosoglutathione (GSNO 0.1mM-5mM; Sigma) for 2 h. Neutrophils were either unstimulated or stimulated with TNFα or n-formyl methionyl leucine phenylalanine (fMLP). Viability was assessed by vital dye cytotoxicity assay. After nuclear extraction and measurement of protein concentration, NFκB binding was determined by electrophoretic mobility shift assay. Effects of GSNO on activation of IκBα, which inhibits intranuclear translocation of NFκB, were measured by Western immunoblot technique. For comparison, experiments were also performed in the presence of the NFκB inhibitor PDTC.¶Results: TNFα increased nuclear NFκB activity compared to unstimulated neutrophils (p<0.001, n=5). GSNO (500 μM) decreased TNFα-induced NFκB activity (p<0.05) and inhibited NFκB activity whether given prior to or during TNFα exposure. IκBα was significantly degraded at 30 and 120 min of TNFα exposure compared to control neutrophils (p<0.05). GSNO exposure (500 μM) inhibited IκBα degradation in the presence of TNFα. PDTC enhanced neutrophil cell death and DNA fragmentation, in association with decreased NFκB activity, similar to GSNO effects.¶Conclusion: Neutrophils possess NFκB activity that is increased by stimulation with TNFα. GSNO inhibits NFκB activity in association with inhibiting TNFα-induced degradation of IκBα. GSNO effects are similar to those seen with NFκB inhibition by PDTC. Inhibition of NFκB could represent a potential anti-inflammatory effect of GSNO.¶

Helicobacter pylori Prevalence among Indigenous Peoples of South America

The seroprevalence of Helicobacter pylori among secluded Indian populations of South America was determined to gain insight into the evolutionary history and possible transmission patterns of the organism. Serum samples obtained from 1024 donors in 22 different villages were tested by enzyme-linked immunosorbent assay for immunoglobulin G antibodies, and the results were confirmed by Western blot. The overall seroprevalence was 92%: >80% of children tested positive by 3 years of age, the highest prevalence in populations studied to date. Comparison of H. pylori prevalence with that of herpes simplex virus type 1, which is known to be transmitted orally, demonstrated a linear correlation in their prevalence rates, suggesting that these pathogens share risk factors. However, H. pylori seroprevalence was consistently higher, indicating that additional routes of transmission exist and/or that the organism is more transmissible. Seroprevalence did not correlate with the length of contact with the outside world. These results suggest that H. pylori was indigenous to the South American Indians and was not introduced by contact with outsiders.

Characteristics of Helicobacter pylori infection in Jamaican adults with gastrointestinal symptoms.

ABSTRACT Helicobacter pylori infection is common in Jamaica. Describing its epidemiology in a population-based study depends largely on serology, but serologic assays have not been validated in this population. To address this issue, we examined the presence ofH. pylori infection in 30 sequential adult patients with gastroduodenal symptoms by three biopsy-based methods (rapid urease test, histology, and culture) as well as by one research and two commercial enzyme-linked immunosorbent assays (ELISAs). A patient was considered H. pylori positive if the organism was detected by at least one biopsy-based method. Eighteen (60%) of the 30 patients were H. pylori positive by these criteria, whereas 21 (70%) were seropositive for H. pyloriimmunoglobulin G by our research ELISA. The presence of H. pylori infection in patients with gastric cancer and those with chronic gastritis was missed by biopsy-based methods but was detected by serologic assays. This observation indicates that serologic assays may be better suited for the detection of this infection in a population in which H. pylori-associated pathology is prevalent. The performance of our research ELISA in detecting biopsy-based H. pylori-positive cases was excellent, with a sensitivity and specificity of 100% and 75%, respectively. Molecular genotyping of the isolates revealed that the predominant H. pylori genotypes in this cohort of Jamaicans were cagA+ vacA slb-m1, andiceA2. The validated serologic assay enables us to interpret epidemiologic data from population-based studies in Jamaica by comparison to those from other populations.

S-NITROSOGLUTATHIONE INHIBITS ACTIVATION OF TRANSCRIPTION FACTOR NF|[kappa]|B IN HUMAN NEUTROPHILS (PMNS) |[bull]| 182

PMNs produce direct lung injury from release of oxygen radicals, lipid peroxides, and proteases, but also serve a paracrine role by releasing proinflammatory cytokines. NFκB is an important gene transcription factor regulating cytokine expression in many cell types. PMN NFκB activity has not been evaluated. Exogenous nitric oxide (NO) inhibits PMN function (Daher et al., AJRCMB, in press, 1997). We hypothesized that PMNs express NFκB, and that exogenous NO could limit PMN function by inhibiting NFκB activation. Methods: Isolated human PMNs(2×108/sample) were incubated with or without S-nitrosoglutathione (GSNO 0.1mM-5mM; Sigma) for 2 hours. PMNs were either unstimulated or stimulated with TNFα (10 ng/ml) or n-formyl methionyl leucine phenylalanine (fMLP; 10 nM). Viability was assessed by vital dye cytotoxicity assay. After PMN nuclear extraction, protein concentration was determined by Bradford assay to provide equivalent loading. NFκB binding was determined by electrophoretic mobility shift assay. Western immunoblot technique was used to determine the effects of GSNO on degradation of IκB-α, a cytoplasmic inhibitor of NFκB nuclear translocation and activation. Results: TNFα and fMLP induced PMN nuclear NFκB activity compared to unstimulated PMNs (n=5). PMN viability was not decreased by GSNO exposure. GSNO decreased NFκB activity in a concentration-dependent fashion. GSNO alone did not affect NFκB activity in the absence of PMN stimulation. GSNO (0.5 mM) inhibited NFκB activity whether given before or during TNFα exposure. IκB-α was maximally degraded at 90 minutes of TNFα exposure. GSNO exposure (500μM) inhibited IκB-α degradation in the presence of TNFα. Conclusion: PMNs manifest NFκB activity which is increased by stimulation with TNFα and fMLP. GSNO inhibits NFκB activation, possibly by inhibiting degradation of IκB-α, a potential anti-inflammatory effect. Funded by a grant from the American Lung Association.

EXOGENOUS NITRIC OXIDE (NO) ENHANCES APOPTOTIC NEUTROPHIL CELL DEATH.|[utrif]| 266

Inhaled NO may modulate immune response at clinically relevant concentrations (20 ppm) by depressing neutrophil (PMN) oxidative function1. NO could affect function by causing cell necrosis or by inducing apoptosis, an “anti-inflammatory” form of programmed cell death by which aging PMNs are terminated2. We hypothesize that NO exposure hastens PMN death by enhancing apoptosis.

ALBUTEROL TREATMENT BLOCKS EXERCISE-INDUCED RETENTION OF RESPIRABLE H2O2 AND BRONCHOSPASM IN CHILDREN WITH ASTHMA. |[utrif]| 2330

ALBUTEROL TREATMENT BLOCKS EXERCISE-INDUCED RETENTION OF RESPIRABLE H 2 O 2 AND BRONCHOSPASM IN CHILDREN WITH ASTHMA. ▴ 2330