Dolores G. Evans

Epidemiology of Helicobacter pylori in an asymptomatic population in the United States: Effect of age, race, and socioeconomic status

A causative role is now accepted for Helicobacter (formerly Campylobacter) pylori in type B gastritis, and evidence is accumulating that H. pylori infection plays a major contributory role in peptic ulcer disease. Preliminary studies have reported that the prevalence of H. pylori infection increases with age, but detailed information on the prevalence of the bacteria in any defined population and on the factors that may influence the pattern of distribution remains scanty. In the present study, a sensitive enzyme-linked immunosorbent assay and a [13C] urea breath test were used to investigate the prevalence of H. pylori infection among 485 healthy asymptomatic volunteers between the ages of 15 and 80 residing in the Houston metropolitan area. H. pylori infection was present in 52%. The prevalence of H. pylori infection increased rapidly with age at 1%/yr for the overall population. The frequency of H. pylori infection was higher in blacks (70%) than whites (34%) (P less than 0.001); this difference remained after adjustments were made for age, gender, educational level, income, and use of tobacco or alcohol. H. pylori infection was independent of gender but was closely correlated with socioeconomic class. There were significant inverse correlations between age-adjusted frequency of H. pylori infection and income and between educational level and H. pylori infection. There was no association between H. pylori infection and consumption of alcohol or nonsteroidal antiinflammatory drug use or smoking. Having pets was associated with a lower frequency of H. pylori infection, but this was highly associated with higher socioeconomic status. The mode(s) of transmission of H. pylori is unknown, but the social patterns of H. pylori infection are consistent with fecal-oral transmission as one important pathway. Socioeconomic factors seem to determine the age of acquisition.

Seroepidemiology ofHelicobacter pylori infection in India

Helicobacter pylori (previouslyCampylobacter pylori) is now accepted as the major cause of type B gastritis and thus what is known about the epidemiology of type B gastritis can reasonably be transferred toH. pylori. We used a specific ELISA for anti-H. pylori IgG to study the prevalence ofH. pylori infection in a population of lower socioeconomic class from Hyderabad, India. The results from India were compared to studies from other parts of the world. Two hundred thirty-eight individuals ages 3 to 70 participated. The frequency ofH. pylori infection increased with age (P<0.01) and was >80% by age 20.H. pylori infection was present in 79% of the population studied; there was no gender-related difference in prevalence ofH. pylori infection. IgG antibody against hepatitis A (HAV) was rapidly acquired in Hyderabad; in a subset of 58 children between the ages of 3 and 21 tested, the frequency of anti-HAV was 98.2%. The prevalenc ofH. pylori infection increases with age in both developed and developing countries. The high age-specific prevalence ofH. pylori infection in developing countries is probably a reflection of the lower socioeconomic level of those areas.

A sensitive and specific serologic test for detection of Campylobacter pylori infection.

Campylobacter pylori has been associated with gastritis, duodenal ulcer, gastric ulcer, and nonulcer dyspepsia. Evidence that C. pylori may be the causative agent or at least a major contributory factor in peptic ulcer disease has generated intense interest in the development of reliable methods for detecting C. pylori infections. We have developed a specific and sensitive enzyme-linked immunosorbent assay (ELISA) that detects serum immunoglobulin G antibodies directed against high molecular weight cell-associated proteins (HM-CAP) of C. pylori. In a blinded fashion we tested sera from 300 individuals and found that all of 147 HM-CAP ELISA-negative individuals were also negative for C. pylori, as documented by a negative urea breath test; also, 151 of 153 C. pylori-positive (by urea breath test) individuals were HM-CAP ELISA-positive. Campylobacter pylori was cultured from the two ELISA-negative but infected patients and these isolates did possess HM-CAP antigens, showing that these two individuals had failed to seroconvert. Thus, the specificity and positive predictive value of the HM-CAP ELISA were each 100%; the sensitivity of the assay was 98.7%, and the negative predictive value was 98.6%. The HM-CAP ELISA and the urea breath test both proved valuable for detecting C. pylori infection, the urea breath test being a more direct method whereas the ELISA is less expensive and easier to perform. Furthermore, the results of a serologic test such as the HM-CAP ELISA would not be influenced by recent ingestion of bismuth compounds or antimicrobial therapy, which might suppress C. pylori and cause a transient false-negative result in the urea breath test.

Mechanisms involved in Helicobacter pylori—Induced inflammation

BACKGROUND Helicobacter pylori infection is associated with mucosal inflammation. The aims of the present study were to assess whether a water extract of H. pylori promotes neutrophil (polymorphonuclear leukocyte [PMN]) adherence to endothelial cells and define the molecular basis of this adhesive interaction. METHODS Intravital microscopy was used to study leukocyte adhesive interactions in rat mesenteric venules in situ. PMN-endothelial cell adhesive interactions were studied in vitro using human PMNs and monolayers of human umbilical vein endothelial cells (HUVEC). RESULTS In vivo, superfusion of rat mesentery with the H. pylori extract increased leukocyte adhesion and emigration in venules. In vitro, adhesion of human PMNs to HUVEC was increased by the H. pylori extract in a concentration-dependent manner. Pretreatment of HUVEC alone with H. pylori extract had no effect on PMN adherence, whereas pretreatment of PMN alone significantly increased their adherence to HUVEC. The extract-induced adhesion was significantly diminished by monoclonal antibodies (MAb) directed against either CD11a, CD11b, or CD18 on neutrophils, and by MAbs against intercellular adhesion molecule-1 (ICAM-1), but not E- or P-selectin, on endothelial cells. CONCLUSIONS These studies suggest that products of H. pylori elicit gastrointestinal inflammation by promoting PMN adhesion to endothelial cells via CD11a/CD18- and CD11b/CD18-dependent interactions with ICAM-1.

Transmission of Helicobacter pylori Infection Studies in Families of Healthy Individuals

Helicobacter pylori is accepted as the commonest cause of type-B gastritis. Detailed information about the mode of transmission remains scanty. We investigated the frequency of H. pylori infection within families, defined as consisting of a husband and wife with at least one biologic child, all living in the same household. Inclusion criteria required that both the parents and the children had been born in the United States, had used no antibiotic or bismuth for the previous 2 months, had no recent major illness or surgical operation, and had no symptoms referable to the upper gastrointestinal tract. H. pylori infection was identified with a 13C-urea breath test and an enzyme-linked immunosorbent assay for anti-H. pylori IgG. Forty-one families (151 healthy individuals) were enrolled. Before the results of the H. pylori tests were known, one parent was selected as the index subject. H. pylori infection clustered; that is, 68% of spouses of H. pylori-infected index subjects were also H. pylori-infected, compared with 9% of spouses of H. pylori-negative index subjects (p less than 0.0001). The children of infected index parents were also more likely to be infected than children of uninfected index parents--40% versus 3%, respectively (p less than 0.0001)--and the results in the children were independent of whether the father or the mother was the index subject. Clustering of H. pylori infection within families suggests person-to-person transmission or common source exposure. The high frequency of H. pylori infection in spouses suggests that genetic factors are less important than living conditions for transmission of H. pylori infection.

Carbohydrate Binding Specificity of the Neutrophil-activating Protein of Helicobacter pylori

The possible interaction of the neutrophil-activating protein of Helicobacter pylori with target cell glycoconjugates was investigated by the binding of125I-labeled recombinant protein to glycosphingolipids from human neutrophils in solid phase assays. Thereby, a distinct binding of the neutrophil-activating protein to four bands in the acid glycosphingolipid fraction from human neutrophils was detected, whereas no binding to the non-acid glycosphingolipids or polyglycosyl ceramides from these cells was obtained. When using glycosphingolipids not present in the cell membrane of human neutrophils, it was found that the neutrophil-activating protein also bound to sulfated glycosphingolipids as sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that in human granulocytes, the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminally unsubstituted NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ sequence.

Characterization of the Helicobacter pylori urease and purification of its subunits.

Helicobacter pylori (formerly Campylobacter pylori) is the causative agent of gastritis in man. Helicobacter pylori cells contain a large amount of an extremely active urease (E.C.3.5.1.5). This enzyme is suspected to be a virulence factor since the ammonium ion produced from urea may be responsible for tissue injury and/or survival of H. pylori in the gastric environment. Helicobacter pylori urease, native relative molecular mass approximately 600,000, was purified by agarose gel filtration and ion exchange chromatography. DEAE-purified urease is highly active and has a Km of 0.48 mM for urea. The enzyme has a pI of 5.93 and is active from pH 4.0 to 10.0, with an optimum at pH 8.0. The purified urease contains nickel and is composed of two protein subunits, with relative molecular masses of 66,000 and 31,000. The subunits were separated and purified and the first 30 N-terminal amino acid residues were determined. A remarkably close relationship was found between both H. pylori urease subunits and jack bean (Canavalia ensiformis) urease, the subunit of which is a single 840 amino acid polypeptide. This subunit is also largely identical to the high molecular mass subunits of the ureases of Klebsiella aerogenes and Proteus mirabilis, evidence that these four ureases are derived from a common ancestral protein.

Adherence and internalization of Helicobacter pylori by HEp-2 cells.

Helicobacter pylori colonizes the mucous layer of the stomach and the surface of gastric mucous cells. Although H. pylori is not generally thought of as invasive, it has been observed in the lamina propria and within vacuoles in the cytoplasm of epithelial cells. The authors report that isolates of H. pylori can enter into the cytoplasm of tissue culture epithelial cell lines such as HEp-2 cells. Intracellular uptake of H. pylori by HEp-2 cells is rapid and appears to require both the N-acetylneuraminyllactose-binding adhesin and another factor present only in living bacteria. Uptake of H. pylori was inhibited by ammonium chloride and chloroquine at concentrations that did not effect either adherence or bacterial viability. Dansylcadaverine, an inhibitor of receptor clustering and internalization, also inhibited uptake but not adherence of H. pylori. Uptake was completely inhibited when H. pylori and HEp-2 cells were incubated at 4 degrees C under conditions that did not effect bacterial adherence. Cytochalasin B, an inhibitor of phagocytosis, did not inhibit uptake. It is concluded that H. pylori is internalized either by receptor-mediated endocytosis or by a closely related pathway.

Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences

The nucleotide (nt) sequence of the Helicobacter pylori (Hp) napA gene, encoding neutrophil-activating protein A (HPNAP) was determined. Alignment of this sequence with those of known bacterioferritins (Bfr) revealed sequence homology and conservation of a 7-amino-acid (aa) motif constituting the ferroxidase (Frx) center of Bfr in the HPNAP. The N-terminal aa sequence deduced from the iron-regulated mrgC gene of Bacillus subtilis [Chen et al., J. Bacteriol. 175 (1993) 5428-5437] is highly similar to that of HPNAP and contains five Frx center aa residues. The deduced aa sequences for proteins of unknown function in Treponema pallidum [Walfield et al., Infect. Immun. 57 (1989) 633-635] and in the cyanobacterium Anabaena variabilis [Sato, GenBank accession No. JU0384 (1991)] identify these two proteins as Bfr. Although the DNA-binding protein from starved cells of Escherichia coli [Almiron et al., Genes Dev. 6 (1992) 2646-2654] is clearly a HPNAP/Bfr homologue, a significant part of its Frx center is missing. It is unlikely that the intracellular function of HPNAP is related to its ability to activate neutrophils.

Administration of purified colonization factor antigens (CFA/I, CFA/II) of enterotoxigenic Escherichia coli to volunteers. Response to challenge with virulent enterotoxigenic Escherichia coli.

Colonization factor antigens (CFAs) were administered orally to volunteers, and the mucosal immune response was assessed by measuring secretory immunoglobulin A (IgA) in saliva and intestinal secretions and by challenge with virulent enterotoxigenic Escherichia coli (ETEC). A combination of CFA/I and CFA/II (8 mg each) administered orally in four doses in milk failed to induce a mucosal IgA response and also failed to protect against challenge with CFA-positive ETEC. When CFA was administered orally (1.5 mg divided into three doses with bicarbonate) to volunteers without preexisting serum anti-CFA levels, it failed to elicit a serum IgG or intestinal secretory IgA response or to protect against challenge with virulent ETEC. The CFA is destroyed by acid gastric contents but it induced significant antibody titer rises in 8 of 11 (73%) volunteers with preexisting serum anti-CFA IgG levels after oral administration of 1 mg of CFA/I with sodium bicarbonate. Subcutaneous priming (50 micrograms CFA/I) did not induce a serum IgG response but, when followed by oral boosting (1 mg CFA/I in two divided doses with sodium bicarbonate), induced intestinal anti-CFA secretory IgA in 4 of 8 volunteers and protected against challenge with CFA/I-positive ETEC. These results, although preliminary, are encouraging and demonstrate that it may be possible to develop an effective oral vaccine based on soluble nonreplicating antigens such as purified CFAs.