Helicobacter pylori populations in Peruvian patients.

Helicobacter pylori is an extremely diverse species. The characterization of strains isolated from individual patients should give insights into colonization and disease mechanisms and bacterial evolution. We studied H. pylori isolates from patients in the Japanese-Peruvian Polyclinic in Lima, Peru, by determining metronidazole susceptibility or resistance and by random amplified polymorphic DNA (RAPD) fingerprinting (a measure of overall genotype). Strains isolated from several biopsy specimens from each of 24 patients were studied. Both metronidazole-susceptible and -resistant strains were isolated from 13 patients, whereas strains of more than one RAPD type were isolated from only seven patients. We propose that the homogeneity in RAPD fingerprints for strains isolated from most persons reflects selection for particular H. pylori genotypes during chronic infection in individual hosts and the human diversity in traits that are important to this pathogen. Carriage of related metronidazole-resistant and -susceptible strains could reflect frequent metronidazole use in Peru and alternating selection for resistant and susceptible phenotypes during and after metronidazole therapy.

H. pylori DNA Fingerprinting Using the Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD) Method.

An ability to distinguish individual strains of Helicobacter pylori with sensitivity and efficiency is valuable for studies of the epidemiology, population genetic structure, and evolution of this gastric pathogen. The arbitrarily primed polymerase chain reaction (AP-PCR), or random amplified polymorphic DNA (RAPD) method (1-4), provides one of the most sensitive and efficient means for distinguishing individual strains, and has been particularly useful for H. pylori (5-7). In overview, the method entails PCR amplification with an oligonucleotide primer of arbitrarily chosen sequence and no known match to sequences in the target genome. This allows initiation of DNA synthesis from genomic sites to which the primer is fortuitously, and usually only partially, matched (Fig. 1). The method detects DNA sequence diversity throughout the genome, rather than just at individual loci; less DNA is needed than in most other DNA fingerprinting methods; the DNA need not be very large nor be double-stranded; and no DNA labeling or hybridization, nor information about target DNA sequences, is needed. There are two principal variants of the AP-PCR protocol, one using oligonucleotide primers of about 10 nucleotides (nt) (3,4), and a second using longer primers, which often may have been constructed for other purposes, such as conventional PCR or DNA sequencing (1,2). Fig. 1. Strategy for DNA fingerprinting by the arbitrarily primed (AP) PCR or random amplified polymorphic DNA (RAPD) method. In the top left are diagrammed the genomes of related but genetically distinct strains of H. pylori that may have diverged from a common ancestor by mutation and/or gene transfer from other strains. Pairs of thick half-arrows indicate primer annealing to pairs of sites that result in AP-PCR products; thin half-arrows indicate the same primer annealing to individual sites that are not near enough to other potential primer binding sites in opposite orientation to yield AP-PCR products. The annealing of primers to pairs of incompletely matched sites, which is postulated to be responsible for many AP-PCR bands from prokaryotic genomes, is diagrammed in the DETAIL section (lower left), and the array of products that would be generated from the two strains compared here is diagrammed at the right.

The stimulation and inhibition of T cell proliferation by Helicobacter pylori components.

The purified T cells from peripheral blood of healthy human volunteers, seronegative for anti-Helicobacter pylori antibody were stimulated in cultures with live or heat-killed H. pylori rods or with bacterial sialic acid-specific surface haemagglutinin (sHA), a crude surface (SF) or cytoplasmic (CF) fractions. It is demonstrated that H. pylori bacteria contain both stimulatory and inhibitory components for T cells of healthy individuals. The sHA as well as SF (5-20 micrograms) induced the proliferative response of T lymphocytes. By contrast, CF inhibited in dose dependent manner, the proliferation of T cells in the cultures stimulated with H. pylori bacteria or PHA. The result suggest that in vivo, a dominance of activation or immunosuppression could depend on the concentration of the bacteria and their products in infective foci.

Helicobacter pylori in endoscopy patients in Zimbabwe: value of enzyme-linked immunosorbent assay and a rapid urease test.

Biopsy and serum specimens were obtained from 95 patients undergoing endoscopy at the University of Zimbabwe Medical School. Common presenting features were epigastric pain, bleeding and dyspepsia. Ulcers were detected in 16 patients (17%), and were more common in men (24%) than in women (7%). Histological examination of biopsies showed that all 95 patients had spiral-shaped organisms that were indistinguishable microscopically from Helicobacter pylori, though the numbers of organisms varied considerably. There was evidence that the degree of inflammation in the mucosa was related to the numbers of H. pylori-like organisms (HPLO) present. Fifty-one biopsy specimens (55%) gave a positive rapid urease test (RUT), with colour change occurring within 4 h. In all but one case, the gastric mucosa from these patients contained moderate to numerous HPLO. We defined the 'gold standard' of H. pylori-associated gastritis as the presence of both moderate to numerous HPLO and moderate to severe inflammation in the gastric mucosa. Using these criteria, RUT had a sensitivity of 67% and a specificity of 68%. Sera from 92 patients were tested for immunoglobulin G antibodies reactive with a glycine-extract antigen of H. pylori, using an enzyme-linked immunosorbent assay (ELISA). Sera giving an indeterminate reaction in the ELISA were also tested by Western blotting. In all, 36 sera (39%) gave a positive ELISA or Western blot reaction. There was poor correlation between serology and RUT results, with only 57% of biopsy specimens from seropositive patients giving a positive RUT, compared with 45% from seronegative patients. Positive serology was found in only 35 patients (61%) with histological evidence of H. pylori-associated gastritis, and the specificity of the test was only 54%. When used in combination with the RUT result, however, 79% of patients with a positive RUT and positive serology had histological evidence of H. pylori-associated gastritis. There was a general trend for increased seroprevalence in patients with mild to moderate atypia. These findings indicate that serology, using an antigen derived from the type strain of H. pylori, is unreliable in detecting H. pylori infection in Zimbabwe. Current studies are aimed at characterizing antigens from organisms isolated from Zimbabwean patients.

Immunological properties of the cell surface haemagglutinins (sHAs) of Helicobacter pylori strain NCTC 11637.

Rabbits were immunised with stage 1 and stage 2 soluble haemagglutinins (sHA) of Helicobacter pylori strain NCTC 11637 and with rabbit erythrocytes coated with stage 1 sHA. After adsorption of stage 1 sHA on erythrocytes, SDS-PAGE analysis showed that 4 major protein bands were removed from the preparation. The anti-sHA coated erythrocyte serum had the highest HA inhibition titre of 16. Crossed immunoelectrophoresis of the stage 1 sHA, against stage 1 and 2 antisera showed multiple precipitin arcs; however, the anti-sHA coated erythrocyte serum produced only two arcs. One arc produced by the anti-stage 2 serum was absent with the anti-stage 1 serum. This arc could have been produced against a 20 kDa polypeptide which was absent in the stage 1 sHA. The other arc was stronger when compared with that produced by anti-stage 1 serum. These two arcs corresponded to the two arcs produced by the anti-sHA coated erythrocyte serum, which had the highest inhibition titre. The two arcs were markedly reduced in crossed immunoelectrophoresis with an adsorbed stage 1 sHA preparation, which indicates that these arcs were produced against the sHAs.

Interaction of cells of Helicobacter pylori with human polymorphonuclear leucocytes: possible role of haemagglutinins.

The interaction of fluorescein isothiocyanate (FITC)-labelled cells of Helicobacter pylori with human polymorphonuclear leucocytes (PMNs) was studied. Two strains with surface haemagglutinins expressing different receptor specificity were used in order to decide if cell surface haemagglutinins of H. pylori may play a role in lectin-mediated binding to/uptake by phagocytes: (1) strain 17874 (NCTC 11637) which expresses sialic acid-specific haemagglutinin; and (2) strain 17875 (NCTC 11638) which expresses a sialic acid-independent haemagglutinin. Cells of strain 17874 were poorly attached to/ingested by PMNs compared to cells of strain 17875. Pre-treatment of bacteria with fetuin or rabbit antibodies against partly purified sialic acid-specific haemagglutinin enhanced interaction of cells of strain 17874 with PMNs. The enhancement did not occur in the case of strain 17875. Phagocytosis of H. pylori 17874 bacteria was slightly increased by fresh human sera positive for anti-H. pylori antibodies. The results suggest that the sialic acid-specific haemagglutinin complex of 17874 bacteria might disturb their uptake by human PMNs.

Intravenous Helicobacter pylori induces low levels of TNF-alpha and IL-1 alpha in a murine model.

A murine model for testing cytokine production stimulated by Helicobacter pylori is described. H. pylori induced significantly lower levels of TNF-alpha and IL-1 alpha compared to Escherichia coli or Pseudomonas aeruginosa when injected intravenously. The mean TNF-alpha concentration in serum during 6 h after stimulation with H. pylori was 0.2 ng/ml, whereas E. coli induced 4.7 ng/ml and P. aeruginosa 6.0 ng/ml. This was not explained by rapid elimination of H. pylori as bacteria were present for at least 3 h in the blood. The difference in cytokine induction may be a reflection of the bacteria's different biological qualities. E. coli and P. aeruginosa are both capable of causing systemic disease, whereas H. pylori causes only a local, often low grade, inflammation in the gastric mucosa.

Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637.

A deionized water extract of Helicobacter pylori NCTC 11637 contained haemagglutinin activity that was (i) soluble (i.e., not associated with particulate material sedimented by centrifugation at 100,000 x g for 1 h), (ii) stable to lyophilization, (iii) heat-labile, (iv) chymotrypsin-sensitive, (v) inhibited by fetuin, orosomucoid, and NANLac, but not by asialofetuin and (vi) inactive against guinea pig erythrocytes incubated with Clostridium perfringens neuraminidase, but active against untreated guinea pig erythrocytes. The data support the idea that the haemagglutinin is a protein which recognizes the alpha-(2-3) structure of sialylated glycoconjugates. Fractionation of the extract by isoelectric focusing and by gel filtration with Sephacryl S-400 indicated that the haemagglutinin has a pI of 3.7 and consist of high molecular-weight-protein aggregates. SDS-PAGE analysis of the preparation purified by gel filtration showed 3 protein bands at ca. 64 kD, 56 kD and 20 kD. Electron microscopy of H. pylori incubated with gold-labelled fetuin indicated that the haemagglutinin was associated with loosely adherent material on the bacterial surface, and that the purified haemagglutinin did not reveal a fimbrial structure. The ability to bind to sialoglycoconjugates on the erythrocyte membrane suggests that the haemagglutinin may be an important colonization factor enabling H. pylori to bind to similar saccharide structures on epithelial cells.

Rapid detection and characterization of sialic acid-specific lectins of Helicobacter pylori.

A particle agglutination assay (PAA) using fetuin (Ft) covalently coupled to carboxylate-modified latex (CML) particles was evaluated for rapid detection of sialic acid-specific haemagglutinins/lectins (SALs) of Helicobacter pylori isolates which bind sialoglycoconjugates. Sixty-three percent (20/32) of the isolates examined gave a positive PAA test. Cell-bound SALs were extracted by washing the bacteria with deionized water or isotonic saline, and their expression was influenced by pH and culture conditions. The Ft-CML reactivity of the PAA-positive isolates was inhibited by bovine submaxillary mucin, transferrin, fetuin, orosomucoid, vitronectin and lactoferrin in a manner which suggested that the isolates contain a lectin recognizing the alpha(2-6) linkage of terminal sialic acid. Western blots of strain NCTC 11637 SALs probed with horseradish peroxidase (HRP)-labelled Ft identified three bands (MW 64 kD, 62 kD, 56 kD) which also reacted with HRP-labelled mucin, transferrin, lactoferrin, orosomucoid, vitronectin and laminin. Sera from patients with a H. pylori infection and one polyclonal rabbit antiserum (strain NCTC 11637) also reacted with the SALs. Immunogold labelling of a polyclonal rabbit antiserum raised against the 64 kD protein of strain NCTC 11637 that reacted strongly with Ft-CML showed that abundant SALs were loosely cell-associated with the cell surface of both spiral and coccoidal forms of H. pylori. SALs were also present in low amounts on the surface of strain NCTC 11638 and 66, a clinical isolate that did not react with Ft-CML.

Haemagglutination patterns of Helicobacter pylori. Frequency of sialic acid-specific and non-sialic acid-specific haemagglutinins.

Thirty-two Helicobacter pylori strains were screened for haemagglutination (HA) activity with erythrocytes of 11 different animal species. Twenty-three strains (72%) that agglutinated human erythrocytes exhibited a broad-spectrum HA profile. Human, guinea pig and bovine erythrocytes high in sialoglycoconjugates were strongly agglutinated by most strains. Except for two, seven strains (22%) that did not agglutinate human erythrocytes exhibited a narrow-spectrum HA profile, commonly not inhibitable by sialoglycoconjugates or N-acetylneuraminlactose (NANLac). Strains were classified into three major HA classes. HA of 10 strains (31%) in class I was inhibited by different combinations of NANLac, orosomucoid or fetuin, but not by asialofetuin, suggesting the presence of sialic acid-specific HAs probably recognizing NeuAc alpha-(2-3)- Gal isomer. Twelve strains (38%) in class II exhibited a different receptor specificity binding to different combinations of NANLac, orosomucoid and fetuin, as well as asialofetuin. No inhibition was observed with 10 strains (31%) in class III; thus, this receptor seems different from both the other classes. Of 21 strains (66%) in classes I and II, HA of 11 strains (34%) was inhibited with NANLac, 14 strains (44%) with orosomucoid and 15 strains (47%) with fetuin. The great heterogeneity observed in HA patterns indicates that the HAs of different strains may recognize a heterogeneous class of sialoglycoconjugates on the erythrocyte membrane.

Cell surface proteins of Helicobacter pylori as antigens in an ELISA and a comparison with three commercial ELISA.

Cell surface proteins of Helicobacter pylori were solubilized by extraction with acidic glycine buffer, N-octyl-glucoside, lithium chloride, and distilled water, and by sonication. The preparations were evaluated as antigens in ELISA to detect serum IgG responses in patients and healthy subjects. SDS-PAGE analyses of the preparations from a type strain (NCTC 11637) and of acidic glycine extracts of 4 clinical isolates showed multiple protein bands. The sera were classified as HP+ve and HP-ve by culture of biopsy and immunoblotting. Sera were considered positive for H. pylori if they detected the specific 120kD antigen or 4-5 other bands. 49 sera were HP+ve; the 51 HP-ve sera did not react in immunoblotting. 35/44 sera (80%) that reacted with the 120kD antigen demonstrated high titers in ELISA with all antigen preparations, and the remaining 9(20%) sera gave discordant results. 4/5 HP+ve sera that did not react with the 120kD antigen, demonstrated high ELISA titers with all 5 antigen preparations. Glycine extracts of 3 isolates did not exhibit the 120kD protein, but were equally sensitive in ELISA. The role of 120kD antigen in our ELISA was not clear. Immunoblotting demonstrated that the 5 antigen preparations share similar antigenic components. All preparations were similarly high in sensitivity and specificity, indicating that surface antigens could be satisfactorily used in our ELISA. Our ELISA using the glycine extract was compared with commercial H. pylori ELISAs developed by Bio-Rad Laboratories, USA (GAP ELISA), Roche, Switzerland (EIA 2G), and Whittaker Bioproducts, USA (Pyloristat).(ABSTRACT TRUNCATED AT 250 WORDS)