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I Bölin

Publications and source records attributed to I Bölin.

At least 19 recordsLinked to original sources
PubMed2006

Molecular characterization of Haemophilus ducreyi isolates from different geographical locations.

The technique of random amplified polymorphic DNA (RAPD) was adapted and optimized to study Haemophilus ducreyi isolates. A panel of 43 strains isolated from chancroid patients from different countries in Africa, Europe, North America, and Asia were characterized. The strains were also studied with respect to lipooligosaccharide (LOS) migration and immunoblotting patterns and the presence of cytolethal distending toxin genes. The RAPD method with the OPJ20 primer generated nine banding patterns (1 to 9). The majority of the isolates were clustered into two major profiles, 14 and 13 strains into profiles 1 and 2, respectively, and just a few strains revealed patterns 3 and 4. The isolates from Thailand were exceptional in that they showed greater diversity and were represented by six different RAPD patterns, i.e., patterns 3 and 5 to 9. The LOS migration and immunoblotting analyses revealed two different patterns, which indicated long and short forms of LOS; the former was found in 20/23 tested strains. Two strains that expressed the short form of LOS were grouped into RAPD pattern 4. The absence of cdtABC genes was observed in only 4/23 strains, and three of these isolates were assigned to RAPD pattern 4. Our results showed limited genotypic and phenotypic variations among H. ducreyi strains, as supported by the conserved RAPD and LOS profiles shared by the majority of the studied strains. However, the RAPD method identified differences between strains, including those from different geographic areas, which indicate the potential of RAPD as an epidemiological tool for the typing of H. ducreyi isolates in countries where chancroid is endemic.

Bacterial Toxins↗
PubMed2003

Stimulated murine macrophages as a bioassay for H. pylori-related inhibition of nitric oxide production.

BACKGROUND: Interference with the L-arginine/nitric oxide pathway may be a virulence strategy for the gastric pathogen Helicobacter pylori. This study evaluates a bioassay for such inhibitory actions on nitric oxide synthase. METHODS: Cultured murine macrophages were stimulated by lipopolysaccharide and interferon-gamma. Nitric oxide synthesis and the expression of inducible nitric oxide synthase (iNOS) at increasing concentrations of L-arginine were analysed using chemiluminescence and Western blotting, respectively. RESULTS: The bioassay was evaluated against nitrite accumulation and two established NOS inhibitors. Bacterial extracts or whole cells of one H. pylori strain inhibited nitric oxide production at low L-arginine concentrations (2-20 microM). A higher concentration of L-arginine (200 microM) was not associated with such inhibition. The iNOS expression was not affected by any of the additives compared to stimulated controls. CONCLUSIONS: This bioassay is a reliable and simple method for analysing iNOS inhibition, resolving effects on enzyme activity or enzyme expression. H. pylori water extract and whole cells exert an L-arginine-dependent NOS inhibition, not influencing iNOS expression.

Animals↗
PubMed2002

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.

Adult↗
PubMed2001

HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains.

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.

Adhesins, Bacterial↗
PubMed2001

The 26-kilodalton, AhpC homologue, of Helicobacter pylori is also produced by other Helicobacter species.

BACKGROUND: The 26 kDa protein, which is an alkyl hydroperoxide reductase (AhpC) homologue, has earlier been described as specific for Helicobacter pylori. The aims of this study were to analyse whether this protein, or the corresponding gene, could be identified in other Helicobacter species. MATERIALS AND METHODS: Two different monoclonal antibodies (Mabs), which recognise the 26 kDa protein in H. pylori, were used in immunoblots to determine the presence of the protein in 10 Helicobacter species. PCR was performed in order to analyse whether the gene was detectable and the PCR products were sequenced. Southern and Northern blot analyses were done on chromosomal DNA and total RNA, respectively, isolated from some selected Helicobacter species in order to compare the genes and mRNA transcripts to H. pylori. RESULTS: The 26 kDa protein was identified in H. nemestrinae (primate), H. acinonychis (cheetah), H. bilis (mouse), H. felis (cat) and H. salomonis (dog) but not in H. mustelae (ferret), H. cinaedi (human), H. canis (dog), H. fennelliae (human) or H. pullorum (poultry). By PCR the gene was also recognised in H. mustelae, H. cinaedi and H. pullorum. The PCR products showed high sequence homology (66-98%) compared to H. pylori. The gene was also highly conserved in four H. pylori strains (94-99% homology). Southern blot showed that the H. nemestrinae and H. acinonychis chromosomal DNA contained a single copy of the gene and the Northern blot analyses indicated mono-cistronic transcription of the gene in these two species, as has been found in H. pylori. CONCLUSIONS: A gene similar to ahpC was found in eight out of 10 Helicobacter species analysed.

Animals↗
PubMed2001

Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phases.

Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.

Adhesins, Bacterial↗
PubMed2000

A 26 kDa protein of helicobacter pylori shows alkyl hydroperoxide reductase (AhpC) activity and the mono-cistronic transcription of the gene is affected by pH.

The 26 kDa protein of Helicobacter pylori, with 67% amino acid identity to alkyl hydroperoxide reductase (AhpC) of Campylobacter jejuni, was studied. We wanted to evaluate it the protein has AhpC activity. Therefore, an Escherichia coli mutant defective for alkyl hydroperoxide reductase and a plasmid expressing the 26 kDa protein from H. pylori were used in complementation studies. The complemented E. coli mutant showed a decreased sensitivity to cumene hydroperoxide indicating that the 26 kDa protein of H. pylori has AhpC activity and could be of importance in the defence against oxidative stress. Furthermore, Northern blot analysis detected one mRNA transcript of approximately 700 bp which is in agreement with the gene being transcribed as a single gene with its own promoter. This promoter region was further characterized by primer extension experiments. Additional studies on how environmental factors, such as long term growth and pH, can affect the transcription of the gene were performed on two H. pylori strains. We found that low pH and long term growth repressed transcription of the gene. Attempts to construct a mutant deficient for the gene in H. pylori were unsuccessful.

Base Sequence↗
PubMed2000

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.

DNA Fingerprinting↗
PubMed1998

Specific antibodies in sera and gastric aspirates of symptomatic and asymptomatic Helicobacter pylori-infected subjects.

In this study we have determined systemic and local antibody responses against different Helicobacter pylori antigens in H. pylori-infected and noninfected subjects. In addition, we studied whether differences in antibody responses between patients with duodenal ulcers and asymptomatic H. pylori carriers might explain the different outcomes of infection. Sera and in most instances gastric aspirates were collected from 19 duodenal ulcer patients, 15 asymptomatic H. pylori carriers, and 20 noninfected subjects and assayed for specific antibodies against different H. pylori antigens, i.e., whole membrane proteins (MP), lipopolysaccharides, flagellin, urease, the neuraminyllactose binding hemagglutinin HpaA, and a 26-kDa protein, by enzyme-linked immunosorbent assay. The H. pylori-infected subjects had significantly higher antibody titers against MP, flagellin, and urease in both sera and gastric aspirates compared with the noninfected subjects. Furthermore, the antibody titers against HpaA were significantly elevated in sera but not in gastric aspirates from the infected subjects. However, no differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pylori carriers, either in sera or in gastric aspirates.

Adult↗
PubMed1997

Local and systemic immune responses in humans against Helicobacter pylori antigens from homologous and heterologous strains.

The capacity of Helicobacter pylori to induce strain specific immune responses was studied in adult Swedish volunteers. Sera and gastric aspirates from 11 H. pylori-infected subjects were tested for specific antibody levels against, respectively, lipopolysaccharides (LPS) and total membrane preparations (MPs) prepared from the study subjects' own strains, as well as with corresponding antigens from two reference H. pylori strains or heterologous strains collected from other subjects within the study. It was found that sera from five of the 11 subjects had significantly higher IgA antibody titres against LPS from the homologous strain than against LPS from either of the reference strains and in five cases sera reacted with higher IgG titres against the homologous LPS than with LPS preparations from the reference or heterologous patient strains. Analyses of specific titres against MPs revealed that six sera had higher IgA titres and four sera had higher IgG titres against MPs prepared from the subjects' own strains than against MPs from either of the two reference strains. Determination of specific antibodies in gastric aspirates revealed significantly higher IgA titres against LPS from the homologous H. pylori isolate than against LPS from the two reference strains in five cases, and six aspirates reacted in higher IgA titre with the homologous H. pylori MPs. Results from immunoblotting analyses of sera support induction of strain specific immune responses against H. pylori LPS. By means of specific monoclonal antibodies against H. pylori LPS, antigenic heterogeneity between the different LPS preparations tested was confirmed.

Adult↗
PubMed1997

Water extract of Helicobacter pylori inhibits duodenal mucosal alkaline secretion in anesthetized rats.

BACKGROUND & AIMS: The pathophysiology behind Helicobacter pylori-induced gastroduodenal dysfunction is incompletely understood. The aim of this study was to investigate if a water extract of H. pylori distorts acid-induced duodenal mucosal alkaline secretion. METHODS: Chloralose-anesthetized rats were prepared for duodenal luminal perfusion and in situ pH-stat titration of mucosal alkaline secretion. RESULTS: Mucosal bicarbonate secretion increased approximately 55%-60% after a 5-minute exposure to 10 mmol/L HCl. This response was absent when water extracts of three strains of H. pylori (protein content, 0.2-20 microg/mL) had been added to the perfusate. Presence of 3 mmol/L L-arginine, but not the stereoisomer D-arginine, in the luminal perfusate reversed the H. pylori extract blockade of acid-induced mucosal alkaline secretion. High-performance liquid chromatography-based analyses showed that the endogenous nitric oxide synthase inhibitor asymmetric dimethyl arginine (ADMA) increased fourfold in duodenal perfusate and fivefold in duodenal tissue after H. pylori extract exposure. In vitro proteolysis of H. pylori extract also resulted in a substantial accumulation of ADMA. Exogenously administered ADMA, giving similar tissue concentrations, inhibited the mucosal alkaline response to acid exposure. CONCLUSIONS: Water extracts of H. pylori inhibit acid-induced mucosal alkaline secretion via interference with mucosal NO synthase.

Anesthesia↗
PubMed1995

Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein.

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.

Antibodies, Monoclonal↗
PubMed1991

Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV.

Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca2(+)-regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order lcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the lcrV and lcrH gene products were found to be regulatory proteins having important roles in the Ca2(+)-controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.

Amino Acid Sequence↗
PubMed1988

The plasmid-encoded Yop2b protein of Yersinia pseudotuberculosis is a virulence determinant regulated by calcium and temperature at the level of transcription.

The basic Yop2b protein, encoded by the virulence plasmid pIBI of Yersinia pseudotuberculosis, is produced under Ca2+-deficient conditions. A mutant deleted for the entire yopH gene, which encodes the Yop2b protein, was found to be avirulent. Virulence could be restored by trans-complementation. The DNA-sequence of yopH predicted a 50 737 D polypeptide lacking a typical signal peptide. Transcription of yopH is regulated by both temperature and Ca2+-concentration. Mutations within the region of the virulence plasmid known to be involved in regulating gene expression in response to Ca2+ abolished transcription of yopH. Other temperature-sensitive mutations in the Ca2+-regulatory locus showed a high level of transcription regardless of Ca2+-concentration. These responses were similar to those of the yopE gene. The promoter region of the yopE and yopH genes were compared and four conserved motifs identified.

Amino Acid Sequence↗
PubMed1988

Identification and mapping of the temperature-inducible, plasmid-encoded proteins of Yersinia spp.

The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. Bölin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.

Antigens, Bacterial↗
PubMed1988

Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein.

Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2+-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a cytotoxic effect.

Animals↗
PubMed1987

Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis.

A number of plasmid-associated proteins (YOPs) of Y. pseudotuberculosis are induced and expressed at high levels when the pathogen is grown at 37 degrees C in absence of Ca2+ ions. These proteins were recovered both from the outer membrane fraction and the culture supernatant. Two hours after a temperature-shift the YOPs were only found in the culture supernatant, amounting to about 5% of the total cell protein. After 4 h of incubation they were also detected in the outer membrane fraction. Separation by 2-D gel electrophoresis revealed that the YOPs could be separated into 6 different polypeptides; YOP2a (45 kDa), YOP2b (45 kDa), YOP3 (41-42 kDa), YOP4a (34 kDa), YOP4b (34 kDa) and YOP5 (26 kDa). The structural genes of all of these YOPs, except the YOP2a gene, were cloned to pBR322 and their respective genetic localization was established. It was found that the genes were not part of a common operon but scattered around plasmid plB1. Only the YOP4b protein was found to map within the Ca2+ region. The hybrid plasmid plB572 coded for a number of plasmid plB1 specific proteins, one of which showed a molecular weight of 38 kDa. This polypeptide could be precipitated by monospecific V-antiserum, showing that this protein is the V-antigen.

Antigens, Bacterial↗