Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease.

The gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. pylori pathogenesis and the development of therapeutics and vaccines.

Helicobacter pylori virulence and genetic geography.

Isolated for the first time in 1982 from human gastric biopsy, Helicobacter pylori is responsible for gastritis, peptic ulcer, and gastric cancer. A pathogenicity island acquired by horizontal transfer, coding for a type IV secretion system, is a major determinant of virulence. The infection is now treated with antibiotics, and vaccines are in preparation. The geographic distribution suggests coevolution of man and Helicobacter pylori.

Mutants of pertussis toxin suitable for vaccine development.

Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.

Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.

The bacterium Helicobacter pylori is the causative agent for peptic ulcer disease. Bacterial adherence to the human gastric epithelial lining is mediated by the fucosylated Lewis b (Leb) histo-blood group antigen. The Leb-binding adhesin, BabA, was purified by receptor activity-directed affinity tagging. The bacterial Leb-binding phenotype was associated with the presence of the cag pathogenicity island among clinical isolates of H. pylori. A vaccine strategy based on the BabA adhesin might serve as a means to target the virulent type I strains of H. pylori.

Immunobiology of Helicobacter pylori infection.

Helicobacter pylori is a 'slow' bacterial pathogen. While infection is usually acquired early in life, only decades later does severe pathology appear. During this long period of incubation, the host mounts a vigorous immune response against H. pylori which fails to resolve the infection and may in fact contribute to the severity of the disease. In the past year, evidence has accumulated indicating a role for a polarized T helper 1 cell response in the gastric pathology induced by H. pylori. Furthermore, a pathogenicity island in type I H. pylori strains has been shown to be responsible for H. pylori induced inflammation. Recent advances in animal models have provided the rationale for entering into human clinical trials of an H. pylori vaccine

Helicobacter pylori: molecular evolution of a bacterial quasi-species.

Helicobacter pylori persists chronically within individuals and as they spread the mutating bacteria migrate with them. The continuous selection and microevolution generates a population of closely related but different bacteria that behave like a quasi-species. Within this heterogeneity, H. pylori strains fall into distinct types, into the virulent (type I) and less virulent (type II) strains, based on the presence of a pathogenicity island (cag) that encodes a specialized secretion machinery. We propose that during chronic infection a dynamic equilibrium between bacteria expressing a disparate degree of virulence is established, and that diverse forms prevail at different times.

Cellular responses induced after contact with Helicobacter pylori.

Contact-dependent activation of the cag organelle, a type IV secretion system of Helicobacter pylori, promotes translocation of CagA into the host cell. CagA is an immunodominant antigen of H. pylori, encoded by cag. It is thought to be associated with severe clinical outcomes, but has an unclear role in pathogenesis. Now we know that CagA is injected into the host and is tyrosine-phosphorylated by a membrane-associated eukaryotic tyrosine kinase. After activation, CagA induces morphological changes in the host, as well as actin reorganization, variations in the cell cycle and autocrine effects. Subversion of cell control may ultimately lead to cellular damage and to increased risks for gastric cancer development. cag instability contributes to long-term persistence within the host by attenuating bacterial virulence. We still do not know if additional factors are co-translocated with CagA and we do not know their specific mechanisms of action, but there is a strong experimental evidence that indicates that cag is the major player in the host-pathogen relationship.

Did the inheritance of a pathogenicity island modify the virulence of Helicobacter pylori?

Strains of Helicobacter pylori from patients with peptic ulcer disease and gastric cancer contain a 40-kb fragment of DNA that is not present in isolates from carriers with asymptomatic infections. The discovery of the cag pathogenicity island suggests that virulence has evolved by quantum leaps through the inheritance of one or more DNA insertions.

Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer: potential new therapies and vaccines.

The recognition that peptic ulcer is an infectious disease caused by the bacterium Helicobacter pylori has revolutionized the approach to diagnosis and therapy of this condition. Treatment of the symptoms of peptic ulcer with drugs that block acid secretion is already being replaced by antibiotic eradication of the causative agent. Studies of the molecular events that lead to H. pylori pathogenesis have shown that clinical isolates can be divided into two groups, only one of which produces a cytotoxin and is associated with severe disease. The cloning of the genes coding for molecules specific for disease-associated strains of H. pylori, and the development of animal models that mimic the human pathology, will provide the basis for better strategies to treat and prevent peptic-ulcer disease.

Seroprevalence of cytotoxin-associated gene A positive Helicobacter pylori strains in Changle, an area with very high prevalence of gastric cancer in south China.

BACKGROUND: Helicobacter pylori, especially the CagA-positive strains, are closely associated with peptic ulcers and gastric cancers. We performed a large scale gastric cancer screening project and examined the prevalence of H. pylori and CagA-positive strains in Changle, China, an area with one of the World's highest gastric cancer mortality. We also compared the prevalence with that in Hong Kong which has one-tenth of the gastric cancer mortality of that in Changle. METHODS: A total of 2424 subjects in Changle and 523 subjects in Hong Kong had endoscopic examination and venesection. Sera were tested for anti-H. pylori antibody and anti-CagA antibody and correlated with endoscopic findings. RESULTS: In Changle, 80. 9% of the subjects were H. pylori carriers. Out of 551 carriers, 408 (74%) were positive for anti-CagA antibody. A total of 76% and 87% of the asymptomatic and gastric cancer patients were positive for anti-CagA antibody, respectively (P > 0.05). Compared to Hong Kong, there was a significantly (P < 0.0001) higher prevalence of CagA-positive strains in asymptomatic subjects in Changle (76%) than in Hong Kong (28%), but not in peptic ulcers or gastric cancers. CONCLUSIONS: Subjects in Changle had a high prevalence of H. pylori infection and a high prevalence of the CagA-positive strains. The contrast in the prevalence of CagA-positive strains, in asymptomatic subjects in two areas with differing gastric cancer mortality, supports the pathogenic role of CagA-positive strains in gastric carcinogenesis.

Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.

Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype.

AIMS: To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS: Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS: Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS: These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration.

Antibodies to CagA protein are associated with gastric atrophy in Helicobacter pylori infection.

Strains of Helicobacter pylori which express the product of the cytotoxin associated gene A(CagA) are associated with duodenal ulceration. Also there is evidence that the presence of serum IgG antibodies to CagA is associated with an increased risk of gastric cancer of the intestinal type. Gastric atrophy is a precursor of intestinal-type gastric cancer so we have investigated whether antibodies to CagA are associated with gastric atrophy. In H. pylori infected patients, IgG antibodies to CagA were present in 24/38 (63%) of non-ulcer patients with atrophy compared with 13/40 (33%) of patient-controls with neither atrophy nor ulcer (P < 0.02). CagA antibodies were also more prevalent in patients with duodenal ulcers; 15/20 (75%) or gastric ulcers 5/5 (100%) than in the patient-controls (P < 0.005 and < 0.02 respectively). These results show that circulating IgG antibodies to CagA are associated with gastric atrophy, as well as peptic ulcer disease. Atrophy is a precursor of gastric cancer so support the hypothesis that certain strains of H. pylori are more likely to cause gastric cancer.

CagA protein seropositivity in a random sample of adult population and gastric cancer patients in Estonia.

OBJECTIVE: The prevalence of antibodies to CagA protein, associated with the risk of developing gastric cancer (GC), was studied in an Estonian adult population with a high prevalence of Helicobacter pylori (HP) infection and in a group of GC patients. DESIGN: In a representative sample of a random adult population from the South Estonian town of Karksi-Nuia, containing 199 subjects (86 M, 113 F, mean age 42.4) and in 45 (22 M, 23 F, mean age 64.5) consecutive patients with gastric adenocarcinoma, recruited during the periods 1986-87 and 1995-96 in the Hospital of Oncology, University of Tartu, anti-CagA IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA) using a recombinant fragment of CagA protein. The occurrence of anti-CagA IgG in ELISA was compared with immunoblot results for 141 subjects. RESULTS: Seropositivity to acid glycine extracted cell surface proteins of HP was 85% in the population and 91% in GC patients (p = 0.39). Anti-CagA IgG antibodies were present in 63% of the population and in 87% of GC patients (p = 0.004). The highest prevalence of anti-CagA IgG in the population sample occurred in the age group 20-29 (76%). A comparison of anti-CagA positivity evaluated by using ELISA and immunoblot showed an agreement of results in 80% of cases. CONCLUSION: HP seropositivity was similarly high in the Estonian random adult population sample and in GC patients, however, the prevalence of anti-CagA IgG was significantly higher in GC patients. Moreover, persons aged 20-29 years in the population possess the highest prevalence of anti-CagA IgG and should be given further attention with respect to the development of GC later in life.

Seropositivity to Helicobacter pylori and CagA protein in schoolchildren of different ages living in urban and rural areas in southern Estonia.

OBJECTIVE: To evaluate Helicobacter pylori and CagA seropositivity in a non-selected group of schoolchildren in southern Estonia, with reference to previous studies where high seroprevalence to H. pylori (87%) and anti-CagA positivity (63%) in an adult population from the same region were found. STUDY POPULATION: A total of 421 schoolchildren selected haphazardly from a random population (n = 1018, ages 9, 12 or 15 years) and living in urban or rural areas. METHODS: H. pylori status was determined by evaluation of IgG antibodies against cell surface proteins of H. pylori, strain CCUG 17874, using standard ELISA. Anti-CagA IgGs were determined by ELISA using a recombinant fragment of CagA (CCUG 17874) as solid-phase antigen. Absorbance values > 0.3 (405 nm) were taken as a CagA-positive result based on a study of 25 sera from H. pylori-negative children. RESULTS: Of the 421 subjects, 235 (56%) were H. pylori-ELISA positive, and 109 out of the 235 (46%) were anti-CagA positive. Neither H. pylori nor CagA positivity were significantly different in girls and boys, or in children aged 9, 12 or 15 years. The H. pylori prevalence rate (118/181, 65%) as well as CagA positivity (64/181, 35%) in rural areas were higher compared with those in towns (117/240, 49% and 54/240, 22%, respectively; P = 0.001 and P = 0.005). CONCLUSION: Of schoolchildren living in southern Estonia, 56% were seropositive to H. pylori. Half of them had anti-CagA antibodies. Schoolchildren living in rural areas were infected significantly more often with CagA-seropositive strains compared with those living in towns.

"Cervia Working Group Report": guidelines on the diagnosis and treatment of Helicobacter pylori infection.

Different national attitudes exist between countries in Europe concerning eradication of Helicobacter pylori infection due to the wide differences in Helicobacter pylori prevalence, gastric cancer risk, bacterial resistance to antibiotics, health care systems and financial resources. The Cervia Working Group Report has been established in order to fill the gap in the absence of National Guidelines in Italy concerning the diagnosis and treatment of Helicobacter pylori infection. The recommendations made are, by and large, similar to the European Guidelines but differ slightly with regard to the "test-and-treat" approach to young dyspeptics without sinister symptoms. In the absence of a national validation of this strategy a case-by-case assessment of dyspepsia has been promoted, both at primary care and specialist level. Another area of partial disagreement concerns the eradication of Helicobacter pylori in patients undergoing long-term proton pump inhibitor treatment which has not been generally recommended as scientific evidence in support of this policy is at present rather weak.

A recombinant fragment of Helicobacter pylori CagA affects proliferation of human cells.

The outcome of H. pylori infectins depends on proliferation of various host cells, including lymphocytes, monocytes and epithelial cells. In this study we showed that a recombinant fusion protein carrying an immunodominant region of H. pylori CagA antigen affected the proliferation of human cells. The rCagA inhibited PHA-driven T cell proliferation but enhanced the growth of epithelial HeLa cells, especially in the presence of granulocyte macrophage colony stimulating factor (GM-CSF). When THP-1 monocytes and Kato-3 epithelial cells from metastasis of gastric carcinoma were stimulated with GM-CSF, they were also susceptible to the inhibitory effect of rCagA. These results confirmed our earlier suggestion on the inhibition of T cell function by H. pylori CagA protein. However, antiproliferative activity of CagA antigen appears to be not restricted to T lymphocytes but modulatory effect of this protein seems to depend on the cell type.

Pathogenicity island mediates Helicobacter pylori interaction with the host.

In Helicobacter pylori, a pathogenicity island (PAI) of approximately 40 kb, named cag, is present in a subset of strains. The strains containing the PAI are more virulent than those that do not contain it, and are associated with peptic ulcer and gastric cancer. A putative secretory mechanism is encoded by this PAI. This secretory system is thought to be involved in the induction of the proiflammatory lymphokine IL-8 and tyrosine phosphorylation of proteins in the gastric cells. We are currently investigating the potential toxic factors exported by this region.