Helicobacter pylori-induced activation of human endothelial cells.

Helicobacter pylori infection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa. To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterized H. pylori strains as well as purified proteins. Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation with H. pylori bacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-alpha) and the cytokine IL-6. However, there were large variations in the ability of the different H. pylori strains to stimulate endothelial cells. These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens. In addition, one or several unidentified proteins which act via NF-kappaB activation seem to induce endothelial cell activation. In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certain H. pylori strains. These effects probably contribute to the continuous recruitment of neutrophils to H. pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.

Differences in surface-exposed antigen expression between Helicobacter pylori strains isolated from duodenal ulcer patients and from asymptomatic subjects.

We have analyzed possible qualitative and quantitative differences in antigen expression between Helicobacter pylori strains isolated from the antrum and different locations in the duodenum of 21 duodenal ulcer (DU) patients and 20 asymptomatic subjects (AS) by enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA. Almost all antral and duodenal strains grown in vitro expressed the N-acetyl-neuroaminyllactose-binding hemagglutinin, flagellins (subunits FlaA and FlaB), urease, a 26-kDa protein, and a neutrophil-activating protein. In 75% of both the DU patients and the AS, antral H. pylori strains expressed either the blood group antigen Lewis y (Le(y)) alone or together with the Le(x) antigen. However, duodenal H. pylori strains of DU patients expressed Le(y) antigen more frequently than corresponding strains of AS (P < 0.05). Presence of Le(y) on H. pylori was related to the degree of active duodenitis (P < 0.05). Duodenal H. pylori strains isolated from AS were significantly more often Lewis nontypeable than duodenal strains of DU patients (P < 0.01). Presence of H. pylori blood group antigen-binding adhesin (BabA) was significantly higher on both antral and duodenal strains isolated from DU patients than on corresponding strains isolated from AS (P < 0.05). BabA-positive duodenal H. pylori strains isolated from DU patients were associated with active duodenitis more frequently than corresponding strains isolated from AS (P < 0.01). Infection with H. pylori strains positive for Le(y) and BabA in the duodenum is associated with development of duodenal ulcer formation.

Different Helicobacter pylori strains colonize the antral and duodenal mucosa of duodenal ulcer patients.

BACKGROUND: We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O-side chains of LPS. MATERIALS AND METHODS: Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies. RESULTS: Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient. CONCLUSION: The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient.

[When is H pylori a cause of duoenal ulcer? Hypersecretion of gastric acid, active duodenitis and reduced bicarbonate secretion are links in the chain]. / När ger H pylori ulcus duodeni? Hypersekretion av syra, aktiv duodenit och nedsatt bikarbonatsekretion är länkar i kedjan.

Helicobacter pylori infection engaging mainly the gastric antrum causes hypersecretion of gastric acid. The increased duodenal acid load gives rise to islands of gastric mucosa in the proximal duodenum. As these bacteria thrive only on gastric mucosa it presents an opportunity for Helicobacter pylori to colonize the duodenum. A much higher density of virulent Helicobacter pylori has been found in the duodenum of duodenal ulcer patients in comparison to infected subjects without duodenal ulcer. The high density of virulent Helicobacter pylori in the proximal duodenum results in a strong inflammatory reaction with active duodenitis and impaired bicarbonate secretion. These characteristics of duodenal ulcer patients, together with the acid hypersecretion, seem to be the key factors in evoking a duodenal ulcer.

Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results.

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.

Duodenal Helicobacter pylori infection differs in cagA genotype between asymptomatic subjects and patients with duodenal ulcers.

BACKGROUND & AIMS: It is unclear why only a minority of subjects infected by Helicobacter pylori develop duodenal ulcers (DU). The aim of this study was to investigate whether the number and type of H. pylori strains in the duodenum of patients with DU may play a critical role. METHODS: Twenty-one patients with DU and 20 asymptomatic subjects with antral H. pylori infection were studied. Paired biopsy specimens were taken from the antrum and from each quadrant of the duodenal bulb. Analyses included extent of duodenal gastric metaplasia, severity of duodenitis, bacterial density, presence of the cagA gene, and vacuolating cytotoxin activity. RESULTS: H. pylori was cultured from duodenal biopsy specimens in 95% of patients with DU and 80% of asymptomatic subjects. Both groups had a similar bacterial density and proportion of cagA-positive strains in the antrum (86% vs. 75%), but patients with DU had a 20-fold higher density of H. pylori and a higher proportion of cagA-positive strains in the duodenal bulb (81% vs. 30%). Active duodenitis was present only in patients with DU infected by cagA positive strains in the duodenum. CONCLUSIONS: The results suggest that a high density of cagA-positive strains in the duodenum with severe duodenitis are important determinants of DU disease.

An analysis of seven different methods to diagnose Helicobacter pylori infections.

BACKGROUND: It has been debated which diagnostic test should be preferred for the diagnosis of Helicobacter pylori in patients with gastroduodenal diseases. METHODS: The H. pylori infection was diagnosed prospectively in 97 untreated patients. H. pylori was diagnosed by means of tests based on five different principles: 1) culture, 2) microscopy (HLO), 3) urease activity (urea breath test (UBT) and urease test on biopsy specimens (CLO test)), 4) DNA detection (polymerase chain reaction (PCR)), and 5) IgG antibody detection (enzyme-linked immunosorbent assay (EIA) and Western blotting (WB)). RESULTS: This study showed that two positive tests out of five tests, based on different principles, were most reliable for predicting the H. pylori infection. Most tests had specificities and predictive values for a negative result greater than 90%. The most important difference between the tests was the sensitivity and the predictive value for a positive result (PPV). WB, HLO, UBT, and PCR had sensitivities and PPV greater than 75%. CONCLUSIONS: The non-invasive tests UBT and WB are reliable, both alone and in combination, and they are recommended for the pre-endoscopic diagnosis of H. pylori. WB is recommended as a confirmative test for antibody detection by EIA. When patients have an upper endoscopy, we recommend taking biopsy specimens for culture and histology because of the additional information obtained about susceptibility, virulence determinants, and morphology, including the degree of inflammation.

Development of a PCR-based technique for detection of Helicobacter pylori.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.