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Helicobacter pylori persistently colonizes the human gastrointestinal tract and is associated with chronic gastritis and, in some cases, peptic ulcer disease or gastric neoplasms. One factor in the persistence of this organism may be its inability to elicit a strong inflammatory response. Lipopolysaccharide (LPS) is a proinflammatory substance found in the cell walls of all gram-negative bacteria. H. pylori LPS has been found by several different measures to be less active than LPS from Enterobacteriaceae. This study addresses the role of CD14 and LPS-binding protein in the cellular response to H. pylori LPS. We report that H. pylori LPS activates mammalian cells expressing CD14 at much lower LPS concentrations than those for control cells not expressing CD14. The maximal activation of CD14-70Z/3 cells by H. pylori LPS also requires LPS-binding protein. H. pylori LPS at concentrations as high as 30 microg/ml does not elicit an interleukin-8 (IL-8) response from the epithelial cell line SW620 in the presence of CD14; 10 ng of Escherichia coli LPS per ml elicits a maximal IL-8 response. Furthermore, in contrast to some other types of LPS with little activity, H. pylori LPS does not inhibit the CD14-70Z/3 cell response to E. coli LPS. From these studies, we conclude that H. pylori LPS, though much less active than E. coli LPS, stimulates cells via CD14.

Helicobacter pylori is associated with gastritis and peptic ulcer disease in humans. We have identified a homolog of the chaperonin cpn60 family of heat shock proteins in H. pylori, referred to as Hp54K. Hp54K, purified from water-extractable H. pylori proteins, migrated as a single band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its native molecular mass was 740 kDa; thus, Hp54K apparently comprises a 14-mer. The N-terminal 33 residues of Hp54K exhibited 60.6, 57.6, 54.5, 54.5, 51.5, and 51.5% identity with corresponding sequences in the following cpn60 homologs: HtpB (Legionella pneumophila), P1 (human mitochondria), GroEL (Escherichia coli), BA60K (Brucella abortus), HypB (Chlamydia trachomatis), and the 65-kDa immunodominant protein of Mycobacterium bovis BCG, respectively. Hp54K was the only protein recognized in whole-cell preparations of H. pylori by immunoblotting using monospecific antisera against cpn60 homologs from L. pneumophila, E. coli, C. trachomatis, and M. bovis BCG. Antiserum against Hp54K recognized proteins with molecular masses of 50 to 60 kDa in a large number of gram-negative bacteria, consistent with the known highly conserved nature of cpn60 proteins. Hp54K is a major protein and is immunogenic in humans infected with H. pylori. Thus, Hp54K shares many similarities with known cpn60 homologs. On the basis of the proposed role of other cpn60 proteins in induction of chronic inflammation, immune cross-reactivity between Hp54K and gastric tissue may provide an important link between H. pylori infection and gastritis.

Helicobacter pylori, a Gram-negative pathogen associated with ulcers, chronic gastritis, and gastric cancers, has been a resident of the human stomach since early human history [1]. This association has only recently begun to erode with the advent of antibiotics and modern lifestyles, but even today H. pylori colonizes approximately half the world's population. To have remained a successful colonizer of humans during thousands of years of association, populations of H. pylori must have been able to survive and adapt to countless evolutionary challenges within and between hosts. As a species, H. pylori possesses one of the most fluid genomes within the prokaryotic kingdom [2], a characteristic that has likely aided its continued success. H. pylori exhibits exceptionally high rates of DNA point mutations, intragenomic recombination (facilitated by repetitive elements common in H. pylori genomes), and intergenomic recombination (mediated by natural transformation), all of which contribute to the high genomic variability between isolates. Previous reviews have focused on these processes as agents of evolutionary change within H. pylori [2-8]. The mechanisms of both mutation and natural transformation, and the evolutionary processes that retain genetic variation generated by these mechanisms, dictate the extent to which each contributes to genomic diversity in the context of different bacterial population structures [9-13]. Unlike well-studied evolutionary systems, such as Salmonella and Escherichia coli, H. pylori is notable in its lack of an environmental reservoir outside of human and other primate stomachs, suggesting that between-host survival is a relatively weak determinant of selection pressures [14, 15]. Given that H. pylori exist largely as distinct host-associated populations, it is possible to begin to model the evolutionary mechanisms that affect the long-term persistence of this species. In this chapter, we consider how the attributes of H. pylori's natural history as a long-term resident of the human stomach and the specific mechanisms of mutation and genetic exchange in this organism have shaped the H. pylori genome. We begin with a survey of genome plasticity in H. pylori. We then discuss mechanisms of mutation and natural transformation in H. pylori and examine experimental evidence for the generation of genomic changes within populations. Finally, we consider how different models of H. pylori population structure affect the relative contributions of mutation and recombination to the evolutionary success of this organism. By bridging evolutionary studies with investigations of pathogenesis from a molecular perspective, we hope to shed new light on how H. pylori has and continues to evolve with its human hosts.

To evaluate the accuracy of IgG and IgA serological tests in establishing a diagnosis of Helicobacter (Campylobacter) pylori gastric infection, 60 children presenting with chronic abdominal pain were prospectively studied. Endoscopic antral biopsies were obtained and analyzed for the presence of H. pylori using three standard methods: culture and identification of bacterial isolates, microscopic examination for morphologically characteristic bacteria, and urease production by the biopsy specimen. Concomitantly obtained serum samples were analyzed for the presence of IgG and IgA antibodies against H. pylori surface antigens using enzyme-linked immunosorbent assay (ELISA). Thirty-four of 60 (56.6%) had histological evidence of chronic active gastritis, eight of whom (13.3%) also had evidence of H. pylori infection by at least one criteria. Six of the eight infected patients had H. pylori demonstrated by all three methods. Of the eight infected patients, seven had IgG antibodies against H. pylori (sensitivity of 87%) and six had IgA antibodies (sensitivity of 75%). Among the six patients who had H. pylori infection confirmed by all three methods, all had IgG antibodies (sensitivity of 100%). In the patients without evidence of H. pylori infection, the IgG ELISA had a specificity of 96% (50/52), and the IgA ELISA had a specificity of 100% (52/52). Our data suggest that serological testing for the presence of antibodies against H. pylori may be a useful diagnostic tool in screening children with chronic abdominal pain for the presence of gastric infection with H. pylori.

To investigate the role of the Helicobacter pylori cytotoxin in the pathogenesis of gastritis, gnotobiotic piglets were colonized with either toxigenic H. pylori or a nontoxigenic isogenic mutant. Only piglets given the toxigenic strain developed toxin-neutralizing antibodies (indicating that toxin is expressed in vivo), but there was no difference in bacterial colonization, epithelial vacuolation, or gastritis between the two groups of piglets.

Gastric colonization by Helicobacter pylori is a risk factor for noncardia gastric cancer. The association between H. pylori and cancer may be attributable to increased epithelial cell turnover, possibly related to antigastric antibodies. Two previous studies reported a disproportionate increase in proliferation relative to apoptosis in patients with H. pylori strains expressing the virulence-related cagA gene. This has led to the hypothesis that an abrogation of apoptosis by cagA-positive strains may promote neoplasia. We, therefore, examined the effect of H. pylori on gastric epithelial proliferation, apoptosis, and the presence of serum antiparietal cell antibodies in a large prospective study. Proliferation and apoptosis were evaluated "blindly" using validated immunohistochemical methods in two antral and two gastric corpus biopsies from 60 patients with nonulcer dyspepsia, and results were correlated with the presence of serum antiparietal cell antibodies. H. pylori colonization was assessed by histology, biopsy urease test, and serology. Proliferation was increased 2-fold in both antrum and corpus in H. pylori-positive patients, was not related to H. pylori cagA status, and was positively correlated with histological gastritis. Apoptosis was increased in the antrum and body only in patients with cagA-positive H. pylori strains. Antiparietal cell antibodies were not more prevalent in H. pylori colonization, and their presence was inversely related to epithelial apoptosis scores we therefore conclude that in patients with nonulcer dyspepsia, H. pylori carriage is associated with increased proliferation. Futhermore the cag pathogenicity island is associated with increased apoptosis. Our results do not support the hypothesis that there is a relative deficiency of gastric epithelial cell apoptosis associated with the carriage of cagA-positive strains. Host factors may be more important than bacterial products in determining the long-term outcome of H. pylori colonization.

The hypothesis that colonization with cagA(+) Helicobacter pylori strains protects against the development of gastroesophageal reflux disease (GERD) and its complications is tested.Patients with reflux esophagitis and Barrett's esophagus were studied. Antral biopsy specimens were obtained for detection of H. pylori. A serum sample was obtained for determination of IgG antibodies to H. pylori and to the CagA protein.736 patients were studied. 118 patients had reflux esophagitis, 36 had Barrett's esophagus, 108 had hiatal hernia without signs of inflammation (the reflux group), and 20 patients had esophageal or stomach cancer. The remaining 454 patients had no signs of GERD. The 262 patients with reflux disease had a significantly lower prevalence of H. pylori (34.9%) than the 454 controls (54.6%; p0. 001). Among 310 H. pylori-positive patients from whom serum was available, colonization with cagA(+) strains was detected in 59% in the control group versus 35% in the reflux group (p0.001).Patients with reflux esophagitis and Barrett's esophagus have a significantly lower prevalence of H. pylori colonization than controls, in particular of the cagA(+) type. These data suggest that colonization with cagA(+) H. pylori strains may be protective against the development of GERD

Helicobacter pylori cag+ strains enhance gastric epithelial cell proliferation and attenuate apoptosis in vivo, which may partially explain the increased risk of gastric cancer associated with these strains. The goals of this study were to identify specific H. pylori genes that regulate epithelial cell cycle events and determine whether these effects were dependent upon p53-mediated pathways. AGS gastric epithelial cells were cultured alone or in the presence of 21 clinical H. pylori isolates, H. pylori reference strain 60190, or its isogenic cagA-, picB-, vacA-, or picB-/vacA- derivatives. Coculture of H. pylori with AGS cells significantly decreased cell viability, an effect most prominent with cag+ strains (P0.001 versus cag-strains). cag+ strains significantly increased progression of AGS cells from G1 into G2-M at 6 h and enhanced apoptosis by 72 h. Compared with the parental 60190 strain, the picB- mutant attenuated cell cycle progression at 6 h (Por = 0.05), and decreased apoptosis with enhanced AGS cell viability at 24 h (Por = 0.04). The vacA- mutant decreased apoptosis and enhanced viability at later (48-72 h) time points (Por = 0.05). Compared with the wild-type strain, the picB-/vacA- double mutant markedly attenuated apoptosis and increased cell viability at all time points (Por = 0.05). Furthermore, cocolonization with H. pylori had no significant effect on expression of p53, p21, and MDM2. The diminished AGS cell viability, progression to G2-M, and apoptosis associated with cag+ H. pylori strains were dependent upon expression of vacA and genes within the cag pathogenicity island. These results may explain heterogeneity in levels of gastric epithelial cell proliferation and apoptosis found within H. pyloricolonized mucosa.

Medical science, as with most organized human activities, is subject to fashion and fad. The major scientific advances in the first half of the twentieth century, encompassed by the development of microbiology and improvements in public health, led to a marked reduction in the morbidity and mortality due to infectious diseases. Following the discovery and widespread use of antibiotics, society had become complacent about the role of infectious diseases as threats to humankind. However, the plagues of the acquired immunodeficiency syndrome (AIDS), and the development of multiply-resistant Mycobacterium tuberculosis, among others, have led to a sea-change in our attitudes about infectious diseases. Now we are at ‘war’ against microbes, and concerned about killer strains (‘flesh-eating bacteria’, for example), and distant epidemics. The threat of infectious diseases, once thought to be under control, clearly is not gone.

The incidence rates of gastric cardia and esophageal adenocarcinomas are increasing, but data suggest that carriage of cagA(+) Helicobacter pylori strains may protect against development of Barrett's esophagus and esophageal adenocarcinoma. Our aims were to examine the relationship between pre-malignant and malignant lesions in the gastric cardia and serum antibodies to H. pylori antigens in patients with and without complications of Barrett's esophagus. The prevalence of carditis was 40% in controls compared with 13% in patients with complicated or uncomplicated Barrett's esophagus and cardia adenocarcinoma (p0.001). Cardia intestinal metaplasia (IM) and atrophy were present and concordant in 28% of controls but less frequent in patients with Barrett's alone or with dysplasia/adenocarcinoma (0% for each, p0.001). Carriage of cagA(+) strains was present in 34% of patients with carditis and significantly associated with increased frequency and severity of cardia inflammation, IM, and atrophy but not with adenocarcinoma. IgA and HspA seropositivity were significantly increased in H. pylori-colonized patients with carditis compared to persons with normal cardia histology (p/= 0.005) but not in persons with esophageal disease or cardia adenocarcinoma. We conclude that carriage of cagA(+) H. pylori strains and induction of particular serological responses are significantly associated with marked histological findings in the gastric cardia but not with adenocarcinoma of either the gastric cardia or esophagus.

Most persons infected with Helicobacter pylori strains that produce vacuolating cytotoxin and possess cytotoxin-associated gene A (cagA) genotype nonetheless remain asymptomatic, suggesting that additional genes are important in virulence. We hypothesized that adherence to gastric epithelium provides stimuli that induce expression of some virulence genes. Our aims were to identify expression of H. pylori genes induced by adherence and to determine if such genes were correlated with peptic ulceration, mucosal interleukin-8 (IL-8) levels, and gastric inflammation. RNA was isolated from an ulcer-derived strain and a gastritis-derived strain that were exposed or not exposed to gastric epithelial cells. These RNAs were used for random arbitrarily primed reverse transcription polymerase chain reaction to identify newly expressed transcripts unique to the ulcer-derived strain following adherence. Clinical isolates of H. pylori were characterized for presence of the newly identified gene, and mucosal IL-8 and inflammation were examined in gastric biopsies from the source patients. A novel H. pylori gene, iceA (induced by contact with epithelium), was identified. DNA sequences revealed two families, iceA1 and iceA2. iceA1 strains were significantly associated with peptic ulceration and increased mucosal concentrations of IL-8. Both iceA1 and iceA2 were expressed in vivo by respective H. pylori strains in gastric biopsies. Adherence to gastric epithelial cells in vitro stimulates the transcription of iceA1, an H. pylori gene that is highly correlated with pathological outcome.

Helicobacter pylori acquisition is the main cause of chronic gastritis in humans. In up to half of the infected subjects, chronic gastritis progresses to atrophic gastritis and intestinal metaplasia. During this course, various mechanisms are triggered that may contribute to the pathogenesis of gastric cancer. Such mechanisms include the inflammation-related cascades of cytokine and free radical reactions, up- and downregulation of growth factors and their receptors, and the atrophy-related impairment of acid output and intraluminal acidity. An array of other factors may also have become significant including overgrowth of bacteria other than H. pylori in the hypochlorhydric or achlorhydric stomach, a high dietary consumption of salt, nitrate, or nitrite, smoking, deficiency of vitamins or micronutrients, influence of sex hormones, or an inherited liability of the dividing epithelial cells to gene errors. These factors may vary in effect between populations and individuals but, if active, may affect the cell genome which may further influence the course and progression of chronic gastritis, and can finally result in overt gastric neoplasia. The molecular biology of gastric cancer has revealed a spectrum of gene errors which vary in type and extent between different histological types of cancer, and between individual cases. There now is evidence that the intestinal metaplasia or the gastric epithelium in atrophic gastritis reveal signs of abnormal expression of various regulatory genes well before the appearance of gastric neoplasia. It is possible that the mechanisms leading to mutation of the genes in epithelial cells are triggered very early in the H. pylori gastritis cascade, and that atrophic gastritis and intestinal metaplasia result from these processes.

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogen Helicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Ley blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea (type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pylori J223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pylori adaptation.

In vivo, gastric infection with Helicobacter pylori leads to substantial production of the inflammatory cytokines IL-1, IL-6, TNF-alpha, and IL-8. H. pylori strains that contain the cag pathogenicity island (cag+) and are associated with ulceration and gastric carcinoma induce greater cytokine production than cag- strains. Expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-kappa B (NF-kappa B) through kappa B-binding elements in the enhancer/promoter regions of their genes. We report that more virulent cag+ H. pylori strains induce increased NF-kappa B-DNA binding activity, which elevates IL-8 expression in AGS gastric epithelial cells. The cag+ H. pylori strains induce significant stimulation of IL-8 promoter-driven reporter activity, while cag- strains do not. Furthermore, mutation of specific genes within the cag island (picA1 and picB) ablates enhanced NF-kappa B activation and IL-8 transcription. Increased IL-8 expression is inhibited by mutation in either the NF-kappa B or NF-IL-6 binding element. The cag+ strains, compared with the cag- strains, induce enhanced nuclear localization of a RelA-containing NF-kappa B binding complex, but no increase in NF-IL-6 binding activity. These studies demonstrate that the ability of different types of H. pylori strains to activate NF-kappa B correlates with their ability to induce IL-8 transcription and indicate a mechanism for the heightened inflammatory response seen in subjects infected with cag+ H. pylori strains.

Gastric colonization with Helicobacter pylori, especially cagA+ strains, is a risk factor for noncardia gastric adenocarcinoma, but its relationship with gastric cardia adenocarcinoma is unclear. Although incidence rates for noncardia gastric adenocarcinoma have declined steadily, paralleling a decline in H. pylori prevalence, rates for adenocarcinomas of esophagus and gastric cardia have sharply increased in industrialized countries in recent decades. To clarify the role of H. pylori infection in these tumors with divergent incidence trends, we analyzed serum IgG antibodies to H. pylori and to a recombinant fragment of CagA by antigen-specific ELISA among 129 patients newly diagnosed with esophageal/gastric cardia adenocarcinoma, 67 patients with noncardia gastric adenocarcinoma, and 224 population controls. Cancer risks were estimated by odds ratios (OR) and 95% confidence intervals (CI) using logistic regression models. Infection with cagA+ strains was not significantly related to risk for noncardia gastric cancers (OR, 1.4; CI, 0.7-2.8) but was significantly associated with a reduced risk for esophageal/cardia cancers (OR, 0.4; CI, 0.2-0.8). However, there was little association with cagA- strains of H. pylori for either cancer site (OR, 1.0 and 1.1, respectively). These findings suggest that the effects of H. pylori strains on tumor development vary by anatomical site. Further studies are needed to confirm these results and to assess whether the decreasing prevalence of H. pylori, especially cagA+ strains, may be associated with the rising incidence of esophageal/gastric cardia adenocarcinomas in industrialized countries.

The discovery of a Helicobacter pylori antigenic protein1, now called the cagA product2,3, that is not conserved in all H. pylori strains, has led to an understanding of a fundamental distinction among H. pylori strains: the presence of the cag island4–6. From the earliest observations1,7, it has become increasingly clear that persons who carry cag + H pylori strains have important differences in outcome compared with persons carrying cag - strains (reviewed in refs 8 and 9). In this chapter I will review some of the important pathophysiological differences between cag + and cag - H. pylori strains, and relate them to a general hypothesis of the relationship between H. pylori strain type and risk of disease. The specific hypothesis to be addressed is that cag + strains are more interactive with their human hosts than are cag - strains.

Whether or not a microbe is virulent is at its root a biological question. Clinical and pathological consequences of the host-microbial interaction may be important determinants of the relationship, but ultimately the question turns on the role of the microbe in enhancing or diminishing the evolutionary fitness of its host. It is from this context that we must consider the relationship of Helicobacter pylori to humans.

Helicobacter pylori strains may be either cagA+ or cagA-, and in logitudinal studies, infection with a cagA+ strain has been associated with increased risk for the development of atrophic gastritis and cancer of the distal stomach. We sought to determine the relative proportion of strains producing CagA in different geographic locales, and the extent to which CagA seroprevalence varied in countries with different gastric and esophageal cancer rates. Using an enzyme-linked immunosorbent assay (ELISA) to detect serum IgG to CagA, we examined sera from 468 asymptomatic H. pylori-infected adults from Canada, Peru, China, Thailand, The Netherlands and 3 different ethnic groups in New Zealand. The CagA seroprevalence in Peru and Thailand (82.2% and 78.8%, respectively) were each substantially higher than for the Chinese (37.9%), Canadian (41.9%), Dutch (39.0%) and New Zealand (28.2%) subjects, but within each population, rates were relatively constant across gender and age groups. Reported gastric but not esophageal cancer rates for the 8 studied populations were significantly associated with H. pylori seroprevalence. Variation in CagA positivity rates was not significantly associated with variation in either gastric or esophageal cancer rates. Our data suggest that CagA seroprevalence is not the major factor influencing gastric cancer rates.

An ELISA based on a pool of United States strains of Helicobacter pylori was compared with a newly developed ELISA based on a pool of Chinese strains. Both assays were tested using sera from 132 Chinese study subjects with biopsy-proven H. pylori infection. Using cutpoints designed to yield equal specificities of 94.9% in an uninfected control population, the sensitivity of the Chinese assay was 100.0%, compared to 97.7% for the United States assay (P = 0.25 by McNemar test). These results suggest that a H. pylori assay based on pooled antigens from United States strains will perform as well in the rural Chinese population as one based on antigens from Chinese strains.

Several epidemiologic investigations have established a link between Helicobacter pylori infection and gastric malignancies. Because the stomach is in continuity with the oral cavity and the bacterium has been isolated from dental plaque and saliva, we hypothesized that H. pylori infection of the upper aerodigestive tract might result in mucosal disruption, allowing for subsequent transformation by known carcinogens such as tobacco and alcohol.To test this hypothesis, we assayed for the presence of IgG antibodies to H. pylori in the serum of 21 patients with squamous cell carcinoma of the head and neck (SCCHN) and 21 matched controls without a history of head and neck cancer.The incidence of seropositivity in the SCCHN patients was 57% and in the controls, 62% (p0.05).These data do not support an etiologic role for H. pylori infection in head and neck cancer.

Although many physicians view Helicobacter pylori strains as a homogenous group of organisms, it has become increasingly clear that populations in humans are highly diverse. This heterogeneity can be analyzed at two different levels: genotypic variation among strains and variations in H. pylori populations within an individual host. Genotypic variation includes point mutations in conserved genes (e.g. ureC), variation in the gene order on physical maps, mosaicism in conserved genes (e.g. vacAs1a), non-conserved genes (e.g. cagA) and extragenetic elements (e.g. IS605). Population differences include the observations that humans can be simultaneously infected with two or more H. pylori strains and that a single strain may represent a cluster of closely related organisms (a 'quasispecies'). The presence of multiple organisms within a host may occur as a result of recombination events leading to genetic shift, whereas ongoing mutation within a strain can lead to the formation of quasispecies by genetic drift. Over recent years it has become increasingly clear that observations on the fundamental biology of H. pylori have considerable clinical relevance. Several genotypic markers (e.g. cagA, vacA, sIa and iceA1) are associated with an increased risk of disease. Also, the multiplicity of infection and quasispecies indicates that analysis of a single H. pylori isolate is inaccurate for defining the genotype of H. pylori strains present in a patient. Global assays, such as serology, are more suitable. The aim of this paper is to review the general phenomenon of diversity in H. pylori and to describe particular heterogeneities that are related to clinical outcome.

To determine the role of preexisting Helicobacter pylori infection in the development of acute upper gastrointestinal (GI) hemorrhage in intensive care unit (ICU) patients in relation to other potential predisposing risk factors.Prospective, multicenter, cohort study.Medical and surgical ICUs in six tertiary care Department of Veterans Affairs Medical Centers.Eight-hundred seventy-four patients without previous GI bleeding or peptic ulcer disease who were enrolled in a multicenter, randomized, controlled trial of prophylactic intravenous immunoglobulin to prevent ICU-associated infections.This substudy of the larger intravenous immunoglobulin study only involved data analysis and had no intervention. All patients were enrolled in the larger study where they received intravenous immunoglobulin or placebo as intervention.Patients were prospectively evaluated for the development of acute upper GI hemorrhage while in an ICU. Anti-H. pylori immunoglobulin G and immnoglobulin A concentrations were determined by enzyme immunoassay on preintervention serum samples. Seventy-six (9%) patients had over upper GI bleeding and a mortality rate of 49%, as compared with a 15% mortality rate in patients who did not bleed (p.001). By logistic regression analysis, the following factors were associated with an increased risk of bleeding: acute hepatic failure, prolonged duration of nasogastric tube placement, alcoholism, and an increased serum concentration of anti-H. pylori immunoglobulin A.Increased anti-H. pylori immunoglobulin A concentrations, prolonged nasogastric intubation, alcoholism, and acute hepatic failure were found to be independently correlated with the development of acute GI bleeding in an ICU setting. These observations should be prospectively confirmed in an independent population before being used for treatment guidelines.

We investigated whether Helicobacter pylori cytotoxin induces vacuolation in primary epithelial cells from normal human mucosa. Epithelial cells purified by enzyme digestion and elutriation were evaluated for vacuolation in a blinded protocol by light and electron microscopy before and after incubation with culture supernatant (CS) from H. pylori 60190, which has vacuolating activity for HeLa cells (Tox+), and isogenic H. pylori mutant 60190-v1, which lacks this activity (Tox-). Primary epithelial cells (>98% pure) exposed to CS from Tox+ H. pylori exhibited marked vacuolation (52% +/- 5% of cells) compared with epithelial cells exposed to either CS from Tox- H. pylori (23% +/- 3.2%) or uninoculated control broth (23% +/- 3.7%) (P < 0.05) by light microscopy, which was confirmed by electron microscopy and antibody inhibition studies. These are the first data to show that H. pylori cytotoxin causes vacuolation of primary human mucosal epithelial cells.

Helicobacter pylori can establish chronic infection in the human gastric mucosa, and it is a major cause of peptic ulcer disease and a principal risk factor for gastric cancer. This creates a need for H. pylori infection models that mimic the human condition. To test the suitability of rhesus monkeys as infection models, H. pylori-free animals were inoculated intragastrically with mixtures of H. pylori strains, bacteria recovered from colonized animals were typed by arbitrarily primed PCR, and host inflammatory and immunologic responses were monitored. Among five H. pylori-free animals inoculated with a mixture of two human strains plus one monkey strain, one became persistently infected and one became only transiently infected. The recovered bacteria matched the monkey input strain in DNA fingerprint. A subsequent trial using two new human isolates and three animals that had resisted colonization by the monkey strain resulted in persistent infection in one animal and transient infection in two others. Antral gastritis, anti-H. pylori serum immunoglobulin G, and atrophy all increased, but with patterns that differed among animals. We conclude that (i) rhesus monkeys can be infected experimentally with H. pylori, (ii) individuals differ in susceptibility to particular bacterial strains, (iii) infections may be transient, and (iv) the fitness of a particular strain for a given host helps determine the consequences of exposure to that strain.

Helicobacter pylori infection is a major cause of gastritis and may be a key risk factor for stomach cancer, but its role in the process of gastric carcinogenesis is not well understood. Herein, we examine H. pylori prevalence in relation to demographic and lifestyle factors and to severity of precancerous lesions in an area of China with one of the highest rates of stomach cancer in the world. H. pylori serum IgG antibody positivity was assayed among 2646 adults, ages 35-64, participating in a population-based gastroscopic screening survey in the high-risk area. The prevalence of positivity was evaluated according to gastric histology, environmental and lifestyle variables determined by interviews during the screening, and level of serum pepsinogens. The odds of advanced precancerous lesions (intestinal metaplasia and dysplasia) of the stomach among those with antibody positivity were estimated by logistic regression. Seventy-two % of the population was H. pylori antibody-positive, with nonsignificant variation by sex, age, income, education, family size, and cigarette smoking habits. H. pylori positivity was higher among those who ate sour pancakes, a fermented indigenous staple that is a risk factor for gastric dysplasia and stomach cancer in this population. The prevalence of H. pylori varied most notably, however, with gastric pathology. The percent of H. pylori positivity increased from 55 to 60 to 87% among those with superficial (nonatrophic) gastritis, mild chronic atrophic gastritis, and severe chronic atrophic gastritis, respectively, before falling to 78% among those with intestinal metaplasia or dysplasia. H. pylori antibody positivity also was strongly correlated with serum pepsinogen concentrations, particularly pepsinogen II, but knowledge of H. pylori status did not markedly improve serological identification of advanced precancerous lesions above that provided by pepsinogen ratios alone. The findings suggest that H. pylori infection contributes to the process of gastric carcinogenesis, particularly during the early stages, in this high-risk area.

Diversity, a characteristic of all living things, has long been recognized among microorganisms and has been well appreciated among bacteria1. For pathogenic microbes, those whose lifestyle results in injury to their larger hosts, a central notion is that these often represent clonal populations. In this context clonality represents a common ancestry, and a very close if not identical genetic relationship among the members of a group.

Helicobacter pylori strains that possess the cytotoxin-associated gene (cagA) are highly associated with peptic ulcer disease, but the role of cagA in pathogenesis is unknown.To test the hypothesis that cagA+ stains elicit a greater proinflammatory cytokine response in the gastric mucosa than cagA- strains, gastric biopsies were obtained from 52 patients and studied by histology, culture, enzyme-linked immunosorbent assay, and reverse transcription polymerase chain reaction.Of 52 patients, 32 (62%) were infected with H. pylori based upon both serology and histology or culture, 16 (31%) were negative by serology, histology, and culture, and four (7%) were positive by serology only. Of 15 H. pylori-infected patients with peptic ulceration, 14 (92%) were infected with cagA+ strains compared with 8 (50%) of 16 patients with gastritis alone, and those infected with cagA+ strains had significantly higher grades of inflammation in the gastric mucosa. Antral inflammation score was significantly associated with IL-8 production. Antral biopsies from infected patients, compared with uninfected patients, significantly more often demonstrated IL-1 beta, IL-2, and IL-8 expression, and those infected with cagA+ compared with cagA- strains significantly more often expressed IL-1 alpha and IL-1 beta and showed elevated antral IL-8 protein levels. Similarly, patients with ulcer disease significantly more often expressed antral IL-1 alpha and IL-8 than those without ulceration.These results indicate that infection with cagA+ H. pylori strains is associated with higher grades of gastric inflammation, correlating with enhanced mucosal levels of IL-8, and increased risk of peptic ulceration.

Approximately 50% of Helicobacter pylori strains produce a cytotoxin, encoded by vacA, that induces vacuolation of eukaryotic cells. Analysis of a clinically isolated tox- strain (Tx30a) indicated secretion of a 93-kDa product from a 3933-base pair vacA open reading frame. Characterization of 59 different H. pylori isolates indicated the existence of three different families of vacA signal sequences (s1a, s1b, and s2) and two different families of middle-region alleles (m1 and m2). All possible combinations of these vacA regions were identified, with the exception of s2/m1 (p0.001); this mosaic organization implies that recombination has occurred in vivo between vacA alleles. Type s1/m1 strains produced a higher level of cytotoxin activity in vitro than type s1/m2 strains; none of 19 type s2/m2 strains produced detectable cytotoxin activity. The presence of cagA (cytotoxin-associated gene A) was closely associated with the presence of vacA signal sequence type s1 (p0.001). Among patients with past or present peptic ulceration, 21 (91%) of 23 harbored type s1 strains compared with 16 (48%) of 33 patients without peptic ulcers; only 2 (10%) of 19 subjects harboring type s2 strains had past or present peptic ulcers (p0.005). Thus, specific vacA genotypes of H. pylori strains are associated with the level of in vitro cytotoxin activity as well as clinical consequences.

We determined the seroepizootiology of Helicobacter pylori infection in rhesus monkeys. Plasma was obtained from 196 animals (age range, 1 to 22 years) that were housed in social environments, either in indoor gang cages, in outdoor corrals, or in free-ranging forested conditions. Plasma immunoglobulin G levels were determined with a specific enzyme-linked immunosorbent assay, and the cutoff immunoglobulin G value for H. pylori seropositivity was determined from a study of 25 monkeys whose infection status was assessed by light microscopy and culture. One-year-old animals of both genders in all housing conditions had the lowest rate of positivity (60% in monkeys 1 year old versus 81% in monkeys 2 to 10 years old, P = 0.026). In addition, females tended to have higher rates of positivity than males. Seroconversion during a 1-year observation period occurred in 7 (28%) of 25 seronegative animals. Seroreversion occurred in 3 (4%) of the 78 positive animals; all 3 of these animals had received antimicrobial agents during the year. These observations demonstrate that the epizootiology of H. pylori infection in rhesus monkeys may serve as a model for human infection.

To determine whether infection with a Helicobacter pylori strain possessing cagA is associated with an increased risk of development of adenocarcinoma of the stomach, we used a nested case-control study based on a cohort of 5443 Japanese-American men in Oahu, Hawaii, who had a physical examination and a phlebotomy during 1967 to 1970. We matched 103 H. pylori-infected men who developed gastric cancer during a 21-year surveillence period with 103 H. pylori-infected men who did not develop gastric cancer and tested stored serum specimens from patients and controls for the presence of serum IgG to the cagA product of H. pylori using an ELISA. The serum IgG assay using a recombinant CagA fragment had a sensitivity of 94.4% and a specificity of 92.5% when used in a clinically defined population; serological results were stable for more than 7 years. For men with antibodies to CagA, the odds ratio of developing gastric cancer was 1.9 (95% confidence interval, 0.9-4.0); for intestinal type cancer of the distal stomach, the odds ratio was 2.3 (95% confidence interval, 1.0-5.2). Age72 years and advanced tumor stage at diagnosis were significantly associated with CagA seropositivity. We conclude that infection with a cagA-positive H. pylori strain in comparison with a cagA-negative strain somewhat increases the risk for development of gastric cancer, especially intestinal type affecting the distal stomach.

Approximately 50% of Helicobacter pylori isolates produce a cytotoxin in vitro that induces vacuolation of eukaryotic cells. Screening a lambda ZapII library of H. pylori 60190 chromosomal fragments permitted the identification of a 3864-base pair (bp) open reading frame (vacA) that encoded the vacuolating cytotoxin, and aor = 567-bp upstream gene that was homologous to Escherichia coli cysteinyl-tRNA synthetase. The sequence data suggest that a 33-amino-acid leader sequence and a C-terminal peptide are cleaved from a 139-kDa protoxin to yield the mature 87-kDa cytotoxin. The vacA gene product contains a C-terminal motif that is present in several other bacterial proteins that undergo C-terminal cleavage, including IgA proteases of Haemophilus influenzae and Neisseria gonorrhoeae. Isogenic H. pylori mutants with insertional mutation of the vacA gene lacked vacuolating cytotoxin activity and failed to produce the 87-kDa protein. Southern analysis of naturally occurring tox-H. pylori strains with vacA probes indicated the presence of hybridizing bands, but both Southern analysis and polymerase chain reaction studies suggested that the vacA sequences of tox- strains differed from those of tox+ strains. Sequence analysis of a 1541-bp region of polymerase chain reaction-amplified vacA from tox- strain 87-203 indicated 64.8% amino acid identity with the corresponding region from tox+ strain 60190. Thus, sequence divergence in vacA genes may explain the lack of functionally active cytotoxin production by some H. pylori isolates.

Persistent infection with Helicobacter pylori occurs in a large percentage of the population, particularly in countries with low socioeconomic status. Such infection nearly always produces chronic gastric inflammation, although in most individuals it is clinically silent and only a minority of infected persons develop H. pylori-induced peptic ulcers. In this review, the hypothesis that diversity among H. pylori strains is at least partly responsible for the observed variability in the outcome of infection is explored. To date, four phenotypes that vary among H. pylori strains have been identified: variations in lipopolysaccharide structure; expression of the cagA-encoded product; production of a vacuolating cytotoxin, and enhanced activation of neutrophils. These phenotypes are associated with one another, with enhanced tissue inflammation, and with peptic ulceration, suggesting that H. pylori strain characteristics have an important influence on the clinical outcome of H. pylori infection.

Current regimens to eradicate Helicobacter pylori usually consist of metronidazole plus a bismuth compound, as well as a third agent such as tetracycline. Such regimens are not ideal because organisms may be metronidazole-resistant, side-effects occur, and compliance is often poor. This randomized, double-blind study was designed to assess the ability of clarithromycin, a new macrolide antimicrobial, as monotherapy to eradicate H. pylori. Thirty-seven healthy volunteers who were H. pylori positive by 13C-urea breath test plus histology and/or culture completed 14 days of oral therapy with clarithromycin in one of three dosages. Eradication, defined as all three tests negative at 4-6 wk after the end of therapy, was achieved in 2/13 (15%) with clarithromycin 500 mg bid, 4/11 (36%) with 1000 mg bid, and 7/13 (54%) with 500 mg qid. Isolates of H. pylori were resistant to clarithromycin prior to therapy in 12% of subjects, and became resistant during therapy in 21% of subjects. Taste perversion, the most common side effect, resulted in one subject terminating therapy.Whereas clarithromycin is a promising antimicrobial in the eradication of H. pylori, it is not sufficient to be used as monotherapy.

The presence of Helicobacter pylori in the human stomach has been highly associated with gastritis and peptic ulcer disease [1–4]. Adherence of H. pylori to gastric cells of infected patients has been directly observed and may be important in the maintenance of infection [5–8]. The attachment of H. pylori to gastric cells may involve bacterial components able to bind to specific cellular receptors. The cellular receptors may be restricted to the gastric cells explaining the exclusive association of H. pylori with those cells, but the specificity of H. pylori for the gastric mucosa may also result from the particular micro-environment of the stomach; in that case, the cellular receptors binding H. pylori could be present on other human cells. It is thus reasonable to study the colonization factors of H. pylori using epithelial cell lines as has been previously described [8–10]. Using microtiter assays, we previously reported [11] a bacterial material easily extracted from the surface of H. pylori which included components that specifically adhered to HeLa cell membranes. This superficial adhering material (SAM) binds to a cellular receptor unrelated to N-acety1-neuraminic acid and is thus different from the N-acetyl-neuraminyl-lactose-binding hemagglutinin previously described [12]. By gel exclusion chromatography, the adhering components of SAM copurify with urease activity in fractions which included antigens of 60, 52, 30, and 15 kDa. In this work, we further identified the adhering components of SAM and we examined the fate of viable epithelial cells coated with these adhering bacterial components.

A vacuolating toxin was purified to homogeneity from broth culture supernatant of the human gastric bacterium, Helicobacter pylori. The procedure for isolating the toxin included ammonium sulfate precipitation, hydrophobic interactive chromatography, size exclusion chromatography, and anion exchange chromatography, which together resulted in a greater than 5000-fold purification of toxin activity. The molecular mass of the purified, denatured toxin was 87,000 +/- 320 daltons, and the native toxin was an aggregate with a molecular mass greater than or equal to 972,000 daltons. The amino-terminal sequence of the purified toxin was partially homologous with internal sequences of numerous transport or ion channel proteins. Antiserum raised against the M(r) = 87,000 protein neutralized toxin activity, whereas preimmune serum did not. When reacted with specific antiserum to the M(r) = 87,000 protein in an enzyme-linked immunosorbent (ELISA) assay, culture supernatants from eight tox+ H. pylori strains produced significantly higher optical density readings than eight tox- supernatants (0.614 +/- 0.11 versus 0.046 +/- 0.01, p less than 0.0001). Sera from H. pylori-infected humans recognized the purified M(r) = 87,000 protein significantly better by ELISA than sera from uninfected persons (0.424 +/- 0.06 versus 0.182 +/- 0.02, p = 0.0009). Finally, ELISA recognition of the purified M(r) = 87,000 protein by human sera was significantly associated with toxin-neutralizing activity (p = 0.019, r = 0.518).

Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.

Enzyme-linked immunosorbent assays (ELISAs) have been developed to diagnose Helicobacter pylori infection. However, the methods are not standardized. We therefore prospectively evaluated the sensitivities and specificities of ELISAs developed in the United States and the United Kingdom in a study population comprising 41 consecutive symptomatic outpatients and 35 volunteers. At endoscopy, multiple biopsies were obtained for histology and culture and stained sections were graded for chronic gastritis, active chronic gastritis, and density of H. pylori. Serum samples were analyzed for H. pylori by ELISA. The first set of assays for immunoglobulin G (IgG) and IgA used a pool of sonicated isolates of H. pylori from five patients in the United States (antigen A). The second set of assays, developed in the United Kingdom, used three different antigens: antigen 1, an acid-extractable surface antigen; antigen 2, an acid-extractable antigen from an aflagellate variant; and antigen 3, a urease-containing fraction. Cutoff scores for positive results were determined a priori on the basis of previous serological studies. There was close agreement between histology and culture. In the study population, 36% of the individuals were H. pylori positive. The diagnostic value of the different ELISAs were highly comparable, and the crude antigens performed as well as the more purified antigens. The antigen A IgG had a sensitivity and specificity of 96 and 94%, respectively; the values for antigen 1 were 93 and 96%, respectively. The antigen A IgA and antigen 3 assays were the least sensitive tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Helicobacter pylori (formerly known as Campylobacter pylori) has been strongly associated with type B gastritis and with peptic ulcer disease. Although this organism was first discovered only 7 years ago, an increasing body of evidence now indicates that it contributes to the pathogenesis of these common and important disorders of the upper gastrointestinal tract. In this report, I will briefly outline how the development of an appropriate diagnostic test has facilitated our understanding of the epidemiology of this infection, and then turn to the specific epidemiologic characteristics that have been defined.

The presence of Helicobacter pylori on gastric mucosa has been associated with type B gastritis and peptic ulcer disease. Despite its uncertain role in pathogenesis, gastric mucosa colonization by H. pylori is at least a good indicator for gastroduodenal inflammatory diseases [1,14,19].

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.