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.

Helicobacter pylori genetic diversity within the gastric niche of a single human host.

Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.

Analysis of iceA genotypes in South African Helicobacter pylori strains and relationship to clinically significant disease.

BACKGROUND: South African Helicobacter pylori isolates are characterised by the universal presence of cagA but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. However, the candidate virulence marker gene iceA has not been investigated. AIM: To characterise the genetic organisation and heterogeneity of iceA genotypes in different South African clinical isolates. PATIENTS AND METHODS: We studied H pylori strains isolated from 86 dyspeptic patients (30 with peptic ulcer disease (PUD), 19 with distal gastric adenocarcinoma (GC), and 37 with non-erosive gastritis) for the presence of iceA1 or iceA2 genes, and for differences in the genetic organisation of iceA2 by polymerase chain reaction, Southern hybridisation analysis, and sequencing. RESULTS: Genetic analysis of iceA1 demonstrated significant homology (92-95%) with the USA type strain 26695 and probably functions as a transcriptional regulator, while a novel variant (iceA2D') of iceA2 and marked differences in predicted protein secondary structure of the iceA2 protein were defined. iceA1 was detected in 68% and iceA2 in 80% of all clinical isolates. Although approximately 40% of patients had both strains, a higher prevalence (p< 0.01) of GC patients were infected with iceA1 isolates which were invariably vacA s1/iceA1 (p< 0.005 v gastritis). Isolates from PUD patients were distinguished by the structurally altered iceA2D variant (53%; p<0.03 v gastritis) while the iceA2C variant distinguished isolates from patients with gastritis alone (67%; p< 0.005 v PUD). CONCLUSION: In this study, an association between iceA1 and GC was noted while differences in variants of iceA2 differentiated between PUD and gastritis alone. Combination analyses of iceA genotypes and vacA alleles supported these associations.

pathogenesis of Helicobacter pylori-induced gastric inflammation.

Helicobacter pylori causes persistent inflammation in the human stomach, yet only a minority of persons harbouring this organism develop peptic ulcer disease or gastric malignancy. An important question is why such variation exists among colonized individuals. Recent evidence has demonstrated that H. pylori isolates possess substantial phenotypic and genotypic diversity, which may engender differential host inflammatory responses that influence clinical outcome. For example, H. pylori strains that possess the cag pathogenicity island induce more severe gastritis and augment the risk for developing peptic ulcer disease and distal gastric cancer. An alternative, but not exclusive, hypothesis is that enhanced inflammation and injury is a consequence of an inappropriate host immune response to the chronic presence of H. pylori within the gastric niche. Investigations that precisely delineate the mechanisms responsible for induction of gastritis will ultimately help to define which H. pylori-colonized persons bear the highest risk for subsequent development of clinical disease, and thus, enable physicians to focus eradication therapy.

Activation of peroxisome proliferator-activated receptor gamma suppresses nuclear factor kappa B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells.

Helicobacter pylori colonization leads to epithelial cell hyperproliferation within inflamed mucosa, but levels of apoptosis vary, suggesting that imbalances between rates of cell production and loss may contribute to differences in gastric cancer risk among infected populations. Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates inflammatory and growth responses of intestinal epithelial cells. We determined whether activation of PPARgamma modified H. pylori-induced apoptosis in gastric epithelial cells. PPARgamma was expressed and functionally active in gastric epithelial cell lines sensitive to H. pylori-induced apoptosis. PPARgamma ligands 15d-PGJ(2) and BRL-49653 significantly attenuated H. pylomicronri-induced apoptosis, effects that could be reversed by co-treatment with a specific PPARgamma antagonist. Cyclopentanone prostaglandins that do not bind and activate PPARgamma had no effects on H. pylori-induced apoptosis. The ability of H. pylori to activate nuclear factor (NF)-kappaB and increase levels of the NF-kappaB target IL-8 was blocked by co-treatment with PPARgamma agonists, and direct inhibition of NF-kappaB also abolished H. pylori-stimulated apoptosis. These results suggest that activation of the PPARgamma pathway attenuates the ability of H. pylori to induce NF-kappaB-mediated apoptosis in gastric epithelial cells. Because PPARgamma regulates a multitude of host responses, activation of this receptor may contribute to varying levels of cellular turnover as well as the diverse pathologic outcomes associated with chronic H. pylori colonization.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer.

Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.

Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori.

Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strain-specific restriction-modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock-treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.

Characteristics of Helicobacter pylori natural transformation.

For Helicobacter pylori, which exhibits substantial genetic diversity, many strains are naturally competent for transformation by exogenous DNA. To better understand the mechanism of natural transformation and its role in the generation of diversity, we sought to systematically identify factors important for natural transformation in H. pylori. We now show that the highest frequency of H. pylori transformation occurs when DNA is introduced prior to exponential phase growth, and that it is a saturable phenomenon. That transformation can be inhibited by DNA from Helicobacter (H. pylori and Helicobacter bilis) but not Escherichia coli suggests specificity based on DNA source. Finally, the cag island was determined to be unnecessary for high-frequency transformation.

Role of iron in Helicobacter pylori: its influence in outer membrane protein expression and in pathogenicity.

The acquisition of iron is a necessity for bacterial growth in Helicobacterpylori, as it is for other organisms. In addition, iron is a critical factor for the virulence of this organism. Therefore, it is not surprising that H. pylori isolates have the potential to express at least three major iron acquisition mechanisms. The association of H. pylori infection with host iron deficiency might indicate that the iron-scavenging systems play a role in the virulence of H. pylori.

Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host.

Helicobacter pylori isolates show greater genetic diversity than other bacterial species studied, but the basis for this phenomenon is unknown. Whether detectable genomic mutation appears within an H. pylori population during persistent colonization was investigated. Paired H. pylori populations obtained across 7- to 10-year intervals from 13 patients were characterized by use of methods including polymerase chain reaction (PCR) genotyping for cagA, vacA, iceA, recA, and IS605; random arbitrarily primed DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis; and ELISA, to determine Lewis phenotypes. Genotyping, including recA sequence analysis, revealed that initial and follow-up populations represented the same population in 11 patients (85%). Nevertheless, distinct dissimilarities were shown within each of these 11 pairs by both RAPD-PCR and AFLP analyses. During follow-up, Lewis-y levels, but not Lewis-x levels, decreased significantly. The changes detected by RAPD-PCR and AFLP indicate that genetic drift occurs within H. pylori populations over the course of years of colonization of a single host.

HP0333, a member of the dprA family, is involved in natural transformation in Helicobacter pylori.

Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.

Using disease-state management as the key to promoting employer sponsorship of medical nutrition therapy (continuing education credit).

The purpose of this project was to design, implement, and improve a nationwide medical nutrition therapy (MNT) intervention program for nutritionally at-risk employees and their dependents and retirees (hereinafter referred to as clients) with a Fortune 100 company (Texas Instruments, Dallas, Tex) with a dispersed population of 80,000. Preferred Nutrition Therapists (PNT), a network of registered dietitians, with the assistance of the Texas Instruments Health Promotion and Benefits Department, identified International Classification of Diseases, 9th ed (ICD-9), codes for which MNT intervention was appropriate. PNT then negotiated a contract with the Texas Instruments Health Promotion and Benefits Department and implemented clients' self-referral process. The main challenge was to promote utilization of a new service from an outside vendor (PNT) and to measure outcomes in meaningful ways. The goal was to use MNT as a tool to prevent the progression of clients' diseases to states that require more costly treatments. PNT used a continuous quality improvement process to refine the system and improve information gathering and reporting, by providing quarterly reports to the Health Promotion and Benefits Department. These reports summarized the outcomes for all clients seen at least 3 times during the quarter. The cost was less than $0.35 per member per month (less than the employer spent on advertising the program), and 0.5% of the population requested MNT during the first year of implementation (about what was expected for a new carveout benefit).

Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori.

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.

MR of the spine with a fast T1-weighted fluid-attenuated inversion recovery sequence.

PURPOSE: To optimize a T1-weighted fast fluid-attenuated inversion recovery (FLAIR) sequence using computer-simulated data and to study its clinical utility for imaging the spine. METHODS: Relative signal intensities and contrast of relevant normal and pathologic tissues in the spine were computed using an inversion recovery equation modified to account for a hybrid RARE (rapid acquisition with relaxation enhancement) readout. A range of inversion time (TI) and repetition time (TR) pairs that null the signal from CSF was generated. A contrast-optimized heavily T1-weighted fast FLAIR sequence, based on the generated data, was qualitatively compared with conventional T1-weighted spin-echo sequences for imaging various spinal abnormalities. RESULTS: A T1/TR pair of approximately 862/2000 was extracted from the computer-generated data to produce effective nulling of CSF signal, to achieve heavy T1 weighting, and to optimize contrast between abnormal tissues and cord/bone marrow. Clinical implementation of the optimized T1-weighted fast FLAIR sequence revealed superior contrast at the CSF-cord interface, better conspicuity of lesions of the spinal cord and bone marrow, and reduced hardware-related artifacts as compared with conventional T1-weighted spin-echo sequences. CONCLUSION: The optimized T1-weighted fast FLAIR technique has definite advantages over spin-echo sequences for imaging the spine. Comparable acquisition times render the FLAIR sequence the method of choice for T1-weighted imaging of the spine.

Time delays in propagation of cardiac action potential.

Recent work has suggested that discrete time delays could occur between cardiac cells under physiological conditions. To investigate the existence of such time delays, monolayers of 10-day chick-embryo ventricular myocytes were grown in cell culture on arrays of extracellular microelectrodes. The most closely spaced recording electrodes were on 20-microns centers. Some pairs of electrodes recorded from different points on the same cell, whereas other pairs straddled intercellular junctions. The preparation was electrically paced to obtain a repeatable propagation pathway. Arrival times of activation at each microelectrode were measured. After control recordings, the preparation was exposed to medium containing 20 mM NH4Cl for 15 min and then returned to normal medium. This intervention produces transient intracellular acidification that decreases intercellular coupling. After acidification, the average (multicellular) conduction velocity decreased to about two-thirds of the control value. Propagation velocity measured between most electrode pairs decreased proportionately. However, disproportionately long propagation delays of up to 410 microseconds were observed between some pairs of electrodes in various experiments. The delays recovered as pH returned to normal. The localized long delays were thought to be due to decreased intercellular coupling at gap junctions.

Role of cell membrane rupture in the pathogenesis of electrical trauma.

Heating due to current flow may not always be the primary cause of tissue damage in electrical injury. We have demonstrated that electric field strengths relevant to the clinical electrical injury problem are capable of permanently disrupting isolated skeletal muscle cells and altering the electrical properties of intact skeletal muscle in the absence of Joule heating effects. The field strengths used in these experiments are theoretically representative of fields at sites distant from the surface entry and exit points in many cases of electrical injury. These results provide further evidence that cell membrane disruption by large induced transmembrane potentials may explain the changes in the electrical properties of muscle observed in experimental electrical trauma (M. Chilbert, et al., J. Trauma 25: 209, 1985) and thus may contribute significantly to the extensive tissue destruction associated with electrical trauma.

An array of microelectrodes to stimulate and record from cardiac cells in culture.

An array of extracellular microelectrodes containing 25 recording and 6 stimulating electrodes was fabricated using microelectronics technology. Ventricular myocardial cells from 8- to 10-day chick embryos were cultured on the surface of the microelectrode array. Extracellular potentials were recorded simultaneously from multiple sites. Simultaneous recordings of extracellular and transmembrane potentials were made from single sites. Extracellular potentials were also recorded simultaneously with cell motion from single sites. Cells were paced by means of electrical stimuli applied via the stimulating electrodes. Conduction velocity in a strip of cells varied linearly as a function of temperature from 0.21 m/s at 26 degrees C to 0.38 m/s at 36.5 degrees C.