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Experimental Helicobacter pylori infection in an animal model
Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Helicobacter pylori is a microaerophilic Gram-negative bacterium colonizing the human stomach. The prevalence of this infection is between 20-90% depending on geographic location. Comprehensive studies have shown significant relationships between H. pylori infection and chronic gastritis, peptic ulcer and gastric carcinoma. The mechanisms behind these associations are still unclear in many aspects. The objective of this thesis was to elucidate some pathogenetic aspects of H. pylori infection based on an animal model using Mongolian gerbils.

Aims: To determine bacterial profiles in the stomach of gerbils with and without H. pylori infection. To study the long-term morpho-functional development of H. pylori-induced gastritis. To investigate the gastric mucosal barrier function and to explore the effects of dietary antioxidant vitamin supplements on H. pylori-associated chronic gastritis.

Methods and results: Mongolian gerbils were inoculated with H. pylori ATCC 43504 or culture broth (controls). The animals were killed at scheduled time points. The gastric microflora was profiled and identified by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region. TTGE and pyrosequencing revealed the presence of a mixed bacterial flora in the stomach of both H. pylori infected and control animals. In both cases, lactobacilli appeared to prevail. In H. pylori-infected gerbils, serum concentrations of anti-H. pylori IgG and gastrin increased over time. Mucosal epithelial proliferation quantified after immunohistochemical labeling with 5-bromo-2'-deoxy-uridine was increased in the antrum in short-term gastritis, followed by an increase in the corpus in the long-term. Gene expression of pro-inflammatory cytokines was quantitated by real-time RT-PCR. Interleukin-1beta and tumor necrosis factor-alpha expression was increased in H. pylori-infected gerbils. Beta-actin was not a reliable endogenous control for relative quantitative RT-PCR. Histological parameters of gastritis were semiquantitatively assessed and expressed as a "gastritis score". Gastritis scores increased over time and reached a peak 32 weeks after inoculation. With time there was an expansion of gastritis from the antrum to the corpus. Severe inflammation, ulcer development and pseudopyloric metaplasia (glandular atrophy) were characteristic features. Gastric mucosal samples were mounted in Ussing chambers and 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP, protein antigen) were used as indicators of gastric mucosal barrier function. Short-term gastritis showed increased mucosal permeability to 51Cr-EDTA in the antrum. Long-standing gastritis was associated with increased 51Cr-EDTA permeation in both the antrum and corpus and an increased HRP flux in the antrum. In the vitamin supplement study, concentrations of 3-nitrotyrosine (nitrosative protein damage) and thiobarbituric acid reactive substances (TBARS) (oxidative lipid damage) in the gastric mucosa were determined with an immunodot blot and a fluorometric method, respectively. Mucosal concentrations of carbonyl carbons on proteins (oxidative protein damage) and 8-hydroxydeoxyguanosine (oxidative DNA damage) were determined by ELISA. Vitamin supplements had no effect on the colonization with H. pylori. Vitamin C as well as vitamin E supplements reduced mucosal 3-nitrotyrosine concentrations to normal levels in the infected animals. Vitamin E supplement induced decreased mucosal protein carbonyls and TBARS in short-term gastritis. In addition, vitamin C supplement caused attenuated mucosal oxidative DNA damage and milder mucosal inflammation in short-term gastritis.

Conclusions: Lactobacilli, the prevailing indigenous bacterium in the stomach of gerbils, may have a probiotic impact on the colonization of H. pylori. The long-term morpho-functional development in the stomach of H. pylori-infected Mongolian gerbils resembles that of H. pylori-infected humans. H. pylori-induced gastritis in gerbils is associated with a long-standing gastric mucosal barrier dysfunction, which follows the extension of chronic gastritis from the antrum into the corpus over time. This impaired barrier function may contribute to perpetuation of chronic inflammation and may be involved in H. pylori-associated carcinogenesis. Vitamin C as well as vitamin E supplements lead to some short-term protective effects on H. pylori-induced stritis but these effects seem to subside over time when the infection persists.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2004. , 62 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 876
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-24034Local ID: 3590ISBN: 91-7373-850-6 (print)OAI: oai:DiVA.org:liu-24034DiVA: diva2:244350
Public defence
2004-12-10, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-26Bibliographically approved
List of papers
1. Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection
Open this publication in new window or tab >>Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection
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2003 (English)In: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 8, no 2, 149-157 p.Article in journal (Refereed) Published
Abstract [en]

Background. Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model.

Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection.

Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons.

Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant.

Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24966 (URN)10.1046/j.1523-5378.2003.00136.x (DOI)9377 (Local ID)9377 (Archive number)9377 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Long-term morpho-functional development of Helicobacter pylori-induced gastritis in Mongolian gerbils
Open this publication in new window or tab >>Long-term morpho-functional development of Helicobacter pylori-induced gastritis in Mongolian gerbils
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2005 (English)In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 40, no 10, 1157-1167 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE:

Epidemiological studies have shown that Helicobacter pylori infection with associated chronic gastritis is the main risk factor for development of gastric cancer. The aim of this study was to investigate the long-term development of H. pylori-induced gastritis in Mongolian gerbils in terms of morphology, gastrin secretion, epithelial proliferation and gene expression of pro-inflammatory cytokines.

MATERIAL AND METHODS:

A total of 133 gerbils were inoculated with H. pylori and 62 served as controls. The gerbils were killed at different time-points between 6 and 94 weeks after inoculation. Serum concentrations of anti-H. pylori IgG and gastrin were determined by enzyme-linked immunoabsorbent assay (ELISA) and radioimmunoassay (RIA), respectively. Epithelial proliferation was evaluated immunohistochemically after labeling with 5-bromo-2'-deoxy-uridine. Gene expression of beta-actin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Histological parameters of gastritis were assessed semiquantitatively and expressed as a "gastritis score".

RESULTS:

Serum concentrations of anti-H. pylori IgG and gastrin increased over time. Epithelial proliferation in the antrum was increased 6 weeks after inoculation, followed by increased proliferation in the corpus 32 weeks after inoculation. Gene expression of IL-1beta and TNF-alpha were increased in H. pylori-infected gerbils. Beta-actin was not a reliable endogenous control for RT-PCR. With time, gastritis expanded from the antrum to the corpus and the gastritis score increased to reach a peak 32 weeks after inoculation. Pseudopyloric metaplasia (loss of specialized cells) was a characteristic feature in the corpus mucosa. Gastric ulcers, but neither dysplasia nor carcinoma, were observed during 94 weeks of infection.

CONCLUSIONS:

Long-term H. pylori infection in Mongolian gerbils led to progressive gastritis, glandular atrophy, hypergastrinemia, increased epithelial proliferation and elevated gene expression of pro-inflammatory cytokines.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-30811 (URN)10.1080/00365520510023378 (DOI)16441 (Local ID)16441 (Archive number)16441 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
3. Long-standing gastric mucosal barrier dysfunction in Helicobacter pylori-induced gastritis in Mongolian gerbils
Open this publication in new window or tab >>Long-standing gastric mucosal barrier dysfunction in Helicobacter pylori-induced gastritis in Mongolian gerbils
2004 (English)In: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 9, no 3, 217-227 p.Article in journal (Refereed) Published
Abstract [en]

Background and Aims. Helicobacter pylori infection causes chronic gastritis and leads to peptic ulcer and gastric adenocarcinoma. An impaired gastric mucosal barrier could be involved in these processes. Our aim was to investigate gastric barrier function in H. pylori-induced gastritis.

Methods.  Stripped gastric mucosal tissues of H. pylori-infected Mongolian gerbils (4 weeks and 70 weeks after inoculation, respectively) and controls were mounted in Ussing chambers. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP, protein antigen) were used to assess mucosal barrier function. The electrophysiological parameters of the mucosa (transepithelial potential, short circuit current, and transepithelial resistance) were monitored as measurements of barrier integrity and viability. Tissue histology was performed to assess inflammation.

Results.  In the antrum, both short-term gastritis [4.68 (3.88–5.74) × 10−6 vs. control 2.86 (2.34–3.77) × 10−6 cm/s, p < .001] and gastritis of long-standing [5.72 (3.88–10.94) × 10−6 cm/s, p < .001 vs. control] showed increased permeability to 51Cr-EDTA. In long-standing antral gastritis there was also an increased HRP flux [9.01 (2.98–45.02) vs. control 0.52 (0.06–1.20) pmol/h/cm2, p < .001]. In the corpus, permeability to 51Cr-EDTA was increased only in long-standing gastritis [4.63 (3.64–7.45) × 10−6 vs. control 2.86 (2.12–3.98) × 10−6 cm/s, p < .01]. Gastric mucosal permeability to 51Cr-EDTA was correlated to histological inflammation and inflammatory activity. The levels of serum anti-H. pylori immunoglobulin G were positively correlated to HRP flux and 51Cr-EDTA permeation.

Conclusions. Helicobacter pylori-induced gastritis in Mongolian gerbils was associated with a long-standing gastric mucosal barrier dysfunction. The barrier defect extended from the antrum into the corpus over time. This impaired barrier function may contribute to perpetuation of chronic inflammation and may be involved in H. pylori-associated carcinogenesis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22344 (URN)10.1111/j.1083-4389.2004.00227.x (DOI)1545 (Local ID)1545 (Archive number)1545 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Effects of antioxidant vitamin supplements on Helicobacter pylori-induced gastritis in Mongolian gerbils
Open this publication in new window or tab >>Effects of antioxidant vitamin supplements on Helicobacter pylori-induced gastritis in Mongolian gerbils
Show others...
2005 (English)In: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 10, no 1, 33-42 p.Article in journal (Refereed) Published
Abstract [en]

Background.  Epidemiological studies show that high intake of food-bound vitamin C and E reduces the risk of gastric cancer. Whether dietary supplementation with antioxidant micronutrients interferes with Helicobacter pylori infection and associated diseases is unclear. The aim of this study was to investigate if dietary vitamin C or E supplementation influences the progression of gastritis, gastric mucosal nitrosative and oxidative protein damage, gastric mucosal lipid peroxidation, or gastric mucosal oxidative DNA damage in H. pylori-infected Mongolian gerbils.

Materials and methods.  Gerbils were divided into four groups: H. pylori-infected animals fed with vitamin C- or vitamin E-supplemented food, and infected and uninfected animals given standard rodent food. Subgroups of animals were killed at different time-points until 52 weeks postinfection. Concentrations of 3-nitrotyrosine and thiobarbituric acid-reactive substances (TBARS) in the gastric mucosa were determined with an immunodot blot and a fluorometric method, respectively. Mucosal concentrations of carbonyl carbons on proteins and 8-hydroxydeoxyguanosine were determined by enzyme-linked immunosorbent assay. Gastritis was scored semiquantitatively.

Results.  Vitamin supplements had no effect on the colonization with H. pylori. Vitamin C as well as vitamin E supplements reduced mucosal 3-nitrotyrosine concentrations to normal levels in infected animals. Vitamin E supplements decreased mucosal protein carbonyls and TBARS in short-term gastritis. In addition, vitamin C supplements caused attenuated mucosal oxidative DNA damage and milder mucosal inflammation in short-term gastritis.

Conclusion.  Vitamin C or vitamin E supplementation leads to some short-term protective effects on H. pylori-induced gastritis in Mongolian gerbils. These effects seem to subside over time when the infection persists.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-29786 (URN)10.1111/j.1523-5378.2005.00289.x (DOI)15197 (Local ID)15197 (Archive number)15197 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved

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