Pylori-infected Mice Research Articles

Comparison between targeted and untargeted systemic immunizations with adjuvanted urease to cure Helicobacter pylori infection in mice

Outbred OF1 mice infected in a first step with a mouse-adapted Helicobacter pylori strain were immunized in a second step by systemic or mucosal routes: systemic immunizations were performed subcutaneously with adjuvanted urease either in the infra or supra-diaphragmatic region of the body, while mucosal immunization was done with urease in the presence of E. coli heat Labile toxin. Mucosal and subcutaneous immunizations induced in infected mice a significant reduction in bacterial density whatever the site of injection but complete eradication was preferentially observed in mice immunized subcutaneously in the back. Systemic immunization with appropriate schedules and formulations could constitute a valuable approach to cure Helicobacter pylori infection.

Development of Helicobacter pylori infection model in BALB/c mice with domestic cagA-positive and -negative strains in Taiwan.

We aimed to develop an H. pylori-infected mouse model using clinically stored strains in Taiwan and to test whether development of H. pylori infection in an in vivo animal model is related to the status of the cagA gene. A total of 100 male BALB/c mice, 6-8 weeks old, including 80 in the experimental group and 20 in the control group, were used. Two clinically stored H. pylori isolates, a cagA-positive and a cagA-negative strain, were selected to induce the H. pylori infection in half (N = 40) of the mice in the experimental group. Bacterial isolates of 0.8 x 10(9) CFU/ml were orally inoculated in each mouse of the experimental group for three consecutive days. Ten mice in the control group were sacrificed to confirm the initial absence of H. pylori. Eight weeks after inoculation of the experimental group and no inoculation of the remaining 10 mice of the control group, each mouse was killed. Gastrectomy was then performed for rapid urease test (CLOtest) and histology. In the control group, none of 20 mice had positive results from the CLOtest or histology. In contrast, excluding eight of 80 mice that died before the eighth week, 90.3% (65/72) of the mice challenged with H. pylori showed persistent presence of H. pylori by histology. The severity of gastritis at the eighth week was more evident in H. pylori-infected mice than in control and noninfected mice (P < 0.05). Although gastritis was more severe in mice inoculated with the cagA-positive strain than with the cagA-negative strain, the rates of H. pylori infection in mice were not different between cagA-positive and -negative strains (91.4% vs 89.2%, P > 0.05). In summary, stored strains of H. pylori can be applied to induce an infection model in BALB/c mice. The less virulent cagA-negative strain can induce H. pylori infection in mice as effectively as the cagA-positive strain. The high prevalence of cagA-positive strains in Taiwanese patients may be related to factors other than only the cagA gene of the bacteria.

Mouse model of gastric infection with cytotoxin-expressing strain of Helicobacter pylori in studying of pathogenesis of chronic gastric ulcer.

Helicobacter pylori is a major risk factor for peptic ulcer, but studies on the role of H. pylori infection in gastric pathology are limited due to lack of convenient models resembling H. pylori infection in humans. We studied the effects of inoculation of conventional BALB/c mice with a toxigenic (cytotoxin associated gene A (cagA)+ and vacuolating cytotoxin gene A (vacA+) H. pylori strain on the course of healing of gastric ulcers. Following inoculation of toxigenic H. pylori or vehicle, gastric ulcers were produced in mice, which were then killed either at day 0 or after 2, 4, 7, 14 or 28 days and ulcer area and gastric blood flow (GBF) were determined. Gastric secretions from mice with chronic gastric fistulae were studied before and after inoculation with toxigenic H. pylori or vehicle (saline). The area (7 mm2 ) of ulcers in control mice decreased gradually and disappeared almost completely after 14 or 28 days. The ulcers in H. pylori-infected mice were present at all test days, showing a larger area than in vehicle control animals. The GBF in control mice rose gradually with decreasing ulcer size, being significantly higher at the ulcer margin than the ulcer crater. In contrast, the GBF in H. pylori-infected mice was significantly lower at the ulcer area than that in the vehicle controls but, again, the GBF at the ulcer margin was always higher than at the ulcer crater. Gastric acid output was reduced by more than 50% immediately after H. pylori inoculation and was accompanied by a significant increase in plasma gastrin release and a fall in gastric luminal somatostatin content. These secretory changes persisted at all test days. Oedema/congestion of surface epithelium appeared after 7 days and mucosal inflammatory infiltration appeared after 14 days, to further increase after 28 days, upon the induction of ulcer. Plasma interleukin (IL)-Iß and IL-12 were significantly elevated above the initial values compared with controls. Conventional mice with gastric ulcers can be successfully infected with an H. pylori strain expressing cagA and vacA cytotoxin and this infection markedly delays healing of the ulcers, probably due to the fall in GBF in the ulcer area, mucosal inflammation, cytokine release and impairment of the gastrin-somatostatin link.

Dietary factors influence the recovery rates of Helicobacter pylori in a BALB/cA mouse model

The aim of this study was to assess the ability of different mouse diets to sustain an H. pylori infection in BALB/cA mice. Four commercially available mouse diets were compared. Experiment 1: Mice were fed the four diets for seven days before infection, infected three times at two-day intervals with 0.1 ml of 10(9) colony-forming units/ml H. pylori cells. H. pylori strains (n = 4) were cultured on GAB-Camp agar for 2 days, harvested and suspended in PBS. All animals were sacrificed at 2 and 4 weeks post inoculation. Experiment 2: Mice infected for 8 weeks were fed RM2, changed to the different diets for 10 days and sacrificed. Stomachs were collected, cultured on GAB-Camp agar to estimate H. pylori growth and stomach biopsies were analyzed by PCR. There were significant differences between diets in their ability to sustain growth of H. pylori. The range was from a few hundred colonies to no growth at all on the GAB-Camp agar. PCR signals showed good correlation with the culture results. All H. pylori-infected mice gave a significantly higher inflammation score compared to non-infected mice. The diet RM2, having the highest number of culturable H. pylori in the mouse stomach, also showed the highest inflammation. These results suggest that the dietary factors affect the amounts of H. pylori in an infection of BALB/cA mice.

Delayed healing of chronic gastric ulcer after Helicobacter pylori infection in mice.

It has been suggested that there is a close relationship between Helicobacter pylori and the onset or recurrence of gastroduodenal disease. The aim of this study was to examine the effect of H. pylori on the healing of chronic gastric ulcers induced in mice. H. pylori administered to nude mice delayed the healing of experimental acetic acid-induced gastric ulcers. Histological examination showed the occurrence of high densities of H. pylori on the surface of epithelial cells and in the ulcerated area. Repeated administration of amoxicillin (10 mgkg(-1) daily for 5 days) eradicated H. pylori and increased the rate of healing of gastric ulcers in H. pylori-infected mice, but metronidazole, which also eradicated the organisms, did not significantly affect the rate of healing. In conclusion, H. pylori-infection delayed the healing of gastric ulcers induced by the serosal application of acetic acid in mice, possibly by aggravation or prolongation of the mucosal inflammation. Amoxicillin eradicated H. pylori and promoted gastric ulcer healing in mice infected with H. pylori.

Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection.

A model permitting the establishment of persistent Helicobacter pylori infection in mice was recently described. To evaluate murine immune responses to H. pylori infection, specific-pathogen-free Swiss mice (n = 50) were intragastrically inoculated with 1.2 x 10(7) CFU of a mouse-adapted H. pylori isolate (strain SS1). Control animals (n = 10) received sterile broth medium alone. Animals were sacrificed at various times, from 3 days to 16 weeks postinoculation (p.i.). Quantitative culture of gastric tissue samples from inoculated mice demonstrated bacterial loads of 4.0 x 10(4) to 8 x 10(6) CFU per g of tissue in the animals. Infected mice had H. pylori-specific immunoglobulin M (IgM) and IgG antibodies in serum (at day 3 p.i.) and IgG and IgA antibodies in their gastric contents (weeks 4 and 16 p.i.) and saliva (week 16 p.i.). Mucosal IgM antibodies were not detected. Histological examination of the gastric mucosae from control and infected mice revealed mild chronic gastritis, characterized by the presence of polymorphoneutrophil cell infiltrates and submucosal lymphoid aggregates, in infected animals at 16 weeks p.i. Differences in the quantities of IgG1 and IgG2a subclass antibodies detected in the sera of mouse strains (Swiss, BALB/c, and C57BL/6) infected by H. pylori suggested that host factors influence the immune responses induced against this bacterium in the host. In conclusion, immune responses to H. pylori infection in mice, like those in chronically infected humans, appear to be ineffective in resolving the infection.

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: 1. protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and 2. this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection.

Chronic infection of the gastroduodenal mucosae by the gram-negative spiral bacterium Helicobacter pylori is responsible for chronic active gastritis, peptic ulcers, and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. The success of eradication by antibiotic therapy is being rapidly hampered by the increasing occurrence of antibiotic-resistant strains. An attractive alternative approach to combat this infection is represented by the therapeutic use of vaccines. In the present work, we have exploited the mouse model of persistent infection by mouse-adapted H. pylori strains that we have developed to assess the feasibility of the therapeutic use of vaccines against infection. We report that an otherwise chronic H. pylori infection in mice can be successfully eradicated by intragastric vaccination with H. pylori antigens such as recombinant VacA and CagA, which were administered together with a genetically detoxified mutant of the heat-labile enterotoxin of Escherichia coli (referred to as LTK63), in which the serine in position 63 was replaced by a lysine. Moreover, we show that therapeutic vaccination confers efficacious protection against reinfection. These results represent strong evidence of the feasibility of therapeutic use of VacA- or CagA-based vaccine formulations against H. pylori infection in an animal model and give substantial preclinical support to the application of this kind of approach in human clinical trials.

Appearance of a metronidazole-resistant Helicobacter pylori strain in an infected-ICR-mouse model and difference in eradication of metronidazole-resistant and -sensitive strains

We tested whether antibiotic-resistant strains appeared in vivo after the failure of treatment using the Helicobacter pylori-infected euthymic mouse model. The numbers of colonies isolated from 56 ICR mice 2 weeks after 4 days of treatment with metronidazole (3.2, 10, or 32 mg/kg of body weight) or amoxicillin (1, 3.2 or 10 mg/kg), with treatment started 4 days after H. pylori CPY2052 inoculation, were counted, and the isolated strains were tested for their sensitivities to two antibiotics to rule out the presence of antibiotic-resistant strains. One metronidazole-resistant strain was detected in a mouse treated with 10 mg of metronidazole per kg, and the MIC of metronidazole for this strain was 25 microg/ml, compared to a MIC of 1.56 microg/ml for the original strain. However, no resistant strain was detected in the amoxicillin treatment group. After the examination described above, mice challenged with a metronidazole-resistant or -sensitive strain isolated from the stomach of a mouse were treated with metronidazole or amoxicillin. The metronidazole-resistant strain was more difficult to eradicate in vivo than the sensitive strain after treatment with metronidazole but not after treatment with amoxicillin. Thus, a metronidazole-resistant H. pylori strain was selected by insufficient treatment, but no resistant strain was selected with amoxicillin. Eradication of a metronidazole-resistant H. pylori strain in vivo required a higher dosage than eradication of a metronidazole-sensitive H. pylori strain. These results may explain one of the reasons for H. pylori treatment failure.

13C-UREA BREATH TEST FOR HELICOBACTER PYLORI MOUSE MODEL

A non-invasive method to evaluate Helicobacter pylori (H. pylori) infection status in a mouse model would be of great value. A rapid and inexpensive breath testing protocol was developed, using a modified disposable 50 ml syringe as a breath collection chamber. The 13C-urea breath test was adapted to the Sydney Strain 1 H. pylori mouse model. Effects of oral intake on breath test results were evaluated in vitro and in vivo. Both diet and coprophagia were found to contribute significantly to the urease activity detected. We found the minimum time required to restrict feed and coprophagia was 13 hours. Optimal dose of 13C-urea was 50 micrograms, which we dissolved in 0.1 ml of water, and administered by oro-gastric gavage after a baseline breath sample was collected. The optimal time to collect the other breath sample was found to be 8 minutes. The modified disposable 50 ml syringe provided a simple, reliable and economical breath collection chamber. Mean breath test results for a group of H. pylori infected mice were higher than mean results for control mice. For use in individual mice we found the breath test to have a sensitivity of 89% and a specificity of 52%. An inverse relationship between breath test values in control mice and time interval between tests was shown which led to difficulties in our attempts to follow establishment of H. pylori infection with frequent testing. This is the first report of adaptation of the 13C-urea breath test to a mouse model of H. pylori infection. Both feed type and coprophagia are critical determinants of gastric urease activity in vivo and must be controlled for 13C urea breath testing in this mouse model to be valid.

Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis.

The role of host factors has been neglected in studies of the pathogenesis of Helicobacter associated disease. The aim of this study was to assess the response of different mouse strains to infection with a single strain of Helicobacter felis. Six strains of inbred mice were infected with the identical H felis culture and were killed at one month, two months, and six months after infection to assess histopathological changes. In addition, two strains of mice were infected with a mouse adapted strain of H pylori and examined at six months after infection. In SJL, C3H/He, DBA/2, and C57BL/6 infected mice, severe to moderate chronic active gastritis was observed only in the body of the stomach, which increased in severity over time with specialised cells in the body glands being replaced. As the severity of this damage in the body increased and atrophic changes were seen, the level of bacterial colonisation of the antrum decreased. In contrast, in BALB/c and CBA mice, there was only mild gastritis in the antrum, no remarkable changes were detected in their body mucosa, and no atrophy was seen over time. In both these strains of mice, heavy bacterial colonisation was seen, which tended to increase over the period of the experiment. Of particular importance in this experiment was that bacterial colonisation was mainly restricted to the antrum yet the atrophy, when present, was only observed in the body of the stomach. H pylori infected C3H/He mice showed moderate colonisation of the antrum, which persisted up to six months with little development of atrophy. In contrast, H pylori in C57BL/6 mice showed excellent colonisation of the antrum at two months but six months after infection there was moderate to severe body atrophy, which was associated with a loss of bacteria from the antrum. These findings challenge current concepts of the development of Helicobacter induced atrophy in that active chronic gastritis of antrum or the body mucosa, or both, is not a prerequisite. They also suggest an autoimmune basis for the pathology although no autoantibody or antibody to the H+/K+ ATPase was detected. Loss of infecting helicobacters from the stomach together with development of an atrophic gastritis in the body of the stomach is similar to the pattern found in certain H pylori infected human subjects.