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STOMACH
Helicobacter pylori infection induces duodenitis and superficial duodenal ulcer in Mongolian gerbils T Ohkusa, I Okayasu, H Miwa, K Ohtaka, S Endo, N Sato ............................................................................................................................. Gut 2003;52:797–803
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Correspondence to: Dr N Sato, Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan;
[email protected] Accepted for publication 20 February 2003
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Background: There is no direct evidence for an animal model of Helicobacter pylori induced duodenal ulcer. Aim: In this study we evaluated the roles of bacterial strain and age of experimental animals in induction of duodenitis and duodenal ulcer in Mongolian gerbils after H pylori infection. Methods: Specific pathogen free Mongolian gerbils were inoculated orally with three bacterial strains (H pylori ATCC 43504, TN2GF4, and K-6, a clinical isolate from a patient with gastric cancer in our clinic). These strains have both the cagA gene and VacA. Five week old gerbils were used to emulate prematurity infection and 14 week old animals were used as mature test subjects. Animals were observed for 12 weeks after inoculation. Interleukin 8 (IL-8) production in gastric epithelial cells (MKN74) after coculture with the H pylori strains was measured by ELISA. Results: Gastritis and gastric ulcers were found in all gerbils infected with the three strains. However, duodenitis and gastric metaplasia were seen more frequently in gerbils infected with TN2GF4 and K-6 strains than in the ATCC 43504 infected or control groups (p0.1) Infection rate (%)
Mongolian gerbils infected at 5 weeks of age
ATCC 43504 (n=3)
TN2GF4 (n=6)
K-6 (n=4)
Control (n=4)
p Value*
5.2 (0.3) 2/3 3/3
5.0 (0.7) 5/6 6/6
5.7 (1.2) 2/4 4/4
N.D. 0/4 0/4
0.174
0.37 (0.12) 3/3 100‡
0.46 (0.32) 0.17 (0.15) 0.05 (0.02) 0.017 6/6 4/4 0/4 100‡ 100‡ 0
0.017
ATCC 43504 (n=5)
TN2GF4 (n=4)
K-6 (n=5)
Control (n=6)
p Value*
5.7 (1.2) 3/5 5/5
4.8 (0.2) 3/4 4/4
4.4 (0.10) 3/5 4/5
ND 0/6 0/6
0.071
0.44 (0.37) 5/5 100‡
0.33 (0.31) 4/4 100‡
0.16 (0.20) 4/5 80‡
0.04 (0.01) 0.025 0/6 0
0.025
*The Kruskal-Wallis test was used for comparisons between the four groups. †Bacterial counts were expressed as log10 colony forming units (CFU)/gastric wall (mean (SD)). ‡p0.2 >0.2 >0.2 >0.2 0.076 0.076
0.074 0.074 0.018 0.018 0.032 >0.2 >0.2
>0.2 >0.2 >0.2 0.049 0.099 >0.2 >0.2
>0.2 >0.2 >0.2 0.074 >0.2 0.074 0.074
>0.2 >0.2 >0.2 >0.2 >0.2 0.074 0.074
0.033 0.033 0.006 0.006 0.020 0.157 0.157
>0.2 >0.2 >0.2 >0.2 0.068 0.165 0.099
0.005 0.006 0.006 0.005 0.010 0.008 0.006
0.032 0.026 >0.2 >0.2 0.169 0.008 0.007
0.171 0.114 >0.2 >0.2 >0.2 0.053 0.046
0.065 0.053 >0.2 >0.2 0.033 0.053 0.046
0.001 0.002 0.002 0.001 0.004 0.002 0.002
0.008 0.008 0.013 0.013 0.019 0.011 0.011
0.074 0.074 >0.2 0.091 0.138 0.023 0.023
>0.2 >0.2 >0.2 0.127 0.147 >0.2 >0.2
0.127 0.127 >0.2 >0.2 >0.2 >0.2 >0.2
0.003 0.003 0.004 0.004 0.010 0.003 0.003
0.176 0.176 0.009 0.005 0.014 >0.2 >0.2
0.040 0.040 0.011 0.008 0.020 0.008 0.008
0.127 0.127 0.011 0.011 0.020 0.008 0.008
>0.2 >0.2 >0.2 >0.2 >0.2 0.024 0.024
>0.2 >0.2 >0.2 >0.2 >0.2 0.024 0.024
0.667 1 0.667 1 2.67 3 2.33 2 473 490 0.333 0 0.333 0
0, 1 0, 1 2, 3 2, 3 420, 510 0, 1 0, 1
1.17 1.33 2.33 2.83 633 2.00 2.33
1 1 2 3 630 2 3
1, 1 1, 2 2, 3 2.5, 3 550, 725 1, 3 1, 3
0.157 0.123 0.371 0.157 0.052 0.033 0.027
1.00 1.00 2.50 2.50 458 1.25 1.25
1 1 2.5 2.5 410 1 1
1, 1 1, 1 2, 3 2, 3 395, 520 1, 2 1, 2
>0.2 >0.2 >0.2 >0.2 >0.2 0.076 0.076
>0.2 >0.2 >0.2 >0.2 0.068 0.165 0.099
0.000 0 0.000 0 0.000 0 0.000 0 178 180 0.000 0 0.000 0
0, 0 0, 0 0, 0 0, 0 150, 200 0, 0 0, 0
0.074 0.074 0.018 0.018 0.032 >0.2 >0.2
0.005 0.006 0.006 0.005 0.010 0.008 0.006
0.008 0.008 0.013 0.013 0.019 0.011 0.011
0.400 0 0.400 0 2.40 2 3.00 3 558 550 0.200 0 0.200 0
0, 1 0, 1 2, 3 3, 3 550, 600 0, 0.5 0, 0.5
>0.2 >0.2 >0.2 0.049 0.099 >0.2 >0.2
0.032 0.026 >0.2 >0.2 0.169 0.008 0.007
0.074 0.074 >0.2 0.091 0.138 0.023 0.023
0.176 0.176 0.009 0.005 0.014 >0.2 >0.2
0, 0, 2, 3,
>0.2 >0.2 >0.2 0.074
0.171 0.114 >0.2 >0.2
>0.2 >0.2 >0.2 0.127
0.040 0.040 0.011 0.008
0.750 0.750 2.25 3.00
1 1 2 3
1 1 2 3
0.157 0.123 0.371 0.157 0.052 0.033 0.027
K-6
>0.2 >0.2 >0.2 >0.2
>0.2 >0.2 >0.2 >0.2
Control
>0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 0.103 0.103 0.003 0.002 0.006 >0.2 >0.2 0.016 0.016 0.003 0.003
Ohkusa, Okayasu, Miwa, et al
ATCC 43504-14W (n=3) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness Duodenal activity Duodenal inflammation TN2GF4-14W (n=6) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness Duodenal activity Duodenal inflammation K-6-14W (n=4) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness Duodenal activity Duodenal inflammation Control-14W (n=4) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness Duodenal activity Duodenal inflammation ATCC 43504-5W (n=5) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness Duodenal activity Duodenal inflammation TN2GF4-5W (n = 4) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation
Mean
Mongolian gerbils infected at 5 weeks of age
0.000 0.000 0.000 0.000 172 0.000 0.000
*Numbers are 25th and 75th percentile score, respectively. †The Mann-Whitney U test was used for comparison between scores and each other. 14W, 14 weeks; 5W, five weeks.
0.103 0.103 0.003 0.002 0.006 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 0.003 0.003 0.004 0.004 0.010 0.003 0.003 0.001 0.002 0.002 0.001 0.004 0.002 0.002 0.033 0.033 0.006 0.006 0.020 0.157 0.157 0 0 0 0 175 0 0
0.500 0.500 2.25 2.75 473 1.00 1.00
0, 0 0, 0 0, 0 0, 0 140, 200 0, 0 0, 0
>0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 >0.2 0.024 0.024 0.127 0.127 0.011 0.011 0.020 0.008 0.008 0.127 0.127 >0.2 >0.2 >0.2 >0.2 >0.2 0.065 0.053 >0.2 >0.2 0.033 0.053 0.046 >0.2 >0.2 >0.2 >0.2 >0.2 0.074 0.074 0.5 0.5 2 3 515 1 1
568 1.00 1.00
Pyloric mucosal thickness (µM) Duodenal activity Duodenal inflammation K-6-5W (n = 4) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness (µM) Duodenal activity Duodenal inflammation Control-5W (n=6) Fundic activity Fundic inflammation Pyloric activity Pyloric inflammation Pyloric mucosal thickness (µM) Duodenal activity Duodenal inflammation
0, 1 0, 1 2, 2 3, 3 280, 580 1, 1 1, 1
>0.2 0.024 0.024 0.020 0.008 0.008 0.147 >0.2 >0.2 >0.2 0.053 0.046 >0.2 0.074 0.074 505 1 1
Mean
480, 780 1, 1 1, 1
0.016 0.016 0.003 0.003 0.010 0.003 0.003
0.066 0.066 0.003 0.003 0.010 0.003 0.003
0.066 0.066 0.003 0.003 0.010 0.003 0.003
0.010 0.003 0.003 >0.2 >0.2 >0.2
K-6 TN2GF4 ATCC K-6 TN2GF4 ATCC
Mongolian gerbils infected at 14 weeks of age
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Bacteria strain group
Table 3 Continued
Score
Median
Range*
p Value†
Control
Mongolian gerbils infected at 5 weeks of age
Control
H pylori duodenal ulcer in Mongolian gerbils
animals (33%) infected with TN2GF4 at 14 weeks and in none of the ATCC 43504 or K-6 groups. Moreover, no ulcer was found in animals infected at five weeks old, regardless of the strain. These results may explain why duodenal ulcer has not been observed in previous investigations13 14 which used the ATCC 43504 strain to infect gerbils with H pylori at 4–5 weeks old. For unknown reasons, animals in this age group and the ATCC 43504 strain do not appear to be suitable for induction of duodenal ulcers. Histological detection of duodenal ulcer was concurrent with severe duodenitis and gastric metaplasia. Although duodenitis can be found in most TN2GF4 and K-6 inoculation groups (75–100%), it is rarely found in the ATCC 43504 inoculation group (infected at 14 weeks of age, 20%; five weeks, 33%). This result suggests that the strains TN2GF4 and K-6 may have an affinity for the duodenal mucosa. Gastric metaplasia was seen in all animals in the TN2GF4 inoculation group and moreover, duodenal ulcers occurred only in the TN2GF4 inoculation group. The finding of duodenal ulcer with gastric metaplasia in the gerbil model is similar to results in human duodenal ulcer. In human duodenal ulcer, the presence of gastric metaplasia in the duodenum is a major risk factor for duodenal ulcer disease in patients colonised by H pylori.18 19 Gastric metaplasia has been viewed as a protective adaptation in duodenal epithelium exposed to high acidity.20 However, a recent study using a 24 hour gastric pH meter indicated that gastric hyperacidity was not associated with duodenal gastric metaplasia.21 Unfortunately, measurement of gastric acid output in Mongolian gerbils was not available and therefore it is difficult to determine whether gastric metaplasia found in this study was due to high acid output. Nevertheless, development of duodenal ulcer seems to be strongly associated with duodenitis and antral gastritis as severe duodenitis with gastric metaplasia and thickened antral mucosa due to antral gastritis were found in gerbils infected with strain TN2GF4 at 14 weeks of age. It has been postulated that H pylori strains which possess the cagA gene and functional VacA are associated with an increased prevalence of duodenal ulcer.10 11 The three strains of H pylori used in the study had both the cagA gene and VacA, so there may be other strain determinants for inducing duodenal ulcers. In the present study, we assessed the functionality of the cag island in these three strains by analysing IL-8 induction in vitro. The TN2GF4 strain stimulated significantly higher levels of IL-8 than strains ATCC 43504 and K6. Similar findings were reported by Israel and colleagues22 who infected gerbils with two different H pylori isolates that were cagA+ and toxigenic, and found one of their strains induced much more severe inflammation than the other. Microarray analysis of the two strains showed that the less inflammatory strain had a mid region deletion in the cag island but it still had the cagA gene. This indicates that the presence of cagA per se does not always signify the presence of a complete and functional cag island. Glandular atrophy and intestinal metaplasia were found in Mongolian gerbils, 26 weeks after H pylori inoculation.23 In our study however, we observed the infected animals for 12 weeks after inoculation and neither atrophy nor intestinal metaplasia in the stomach was found. Hansson et al reported that duodenal ulcer disease has a protective effect against the development of gastric cancer.24 As all duodenal ulcers in the present study were concomitant with gastric ulcers, this model is not useful for verifying this protective effect. Some patients with gastric ulcers also have or have had duodenal ulcers, and the incidence of coexistent gastric and duodenal ulcers ranges from 7% to 64%.25 Recently, a case study reported that H pylori infection carried the strongest association with gastroduodenal ulcer compared with gastric ulcer in the gastric body and prepyloric region.26 In most instances it was thought that duodenal ulcer was the primary disease, with gastric ulcer occurring secondarily, but one study
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Ohkusa, Okayasu, Miwa, et al
Figure 2 (A) Duodenitis characterised by dense neutrophil and mononuclear cell infiltration is seen in a gerbil infected with bacterial strain Helicobacter pylori K-6 (haematoxylin-eosin, original magnification ×100). (B) The duodenal mucosa in a gerbil infected with bacterial strain H pylori TN2GF4 at 14 weeks was positive for periodic acid- Schiff staining, indicating gastric metaplasia (original magnification ×200). (C) The mucus of gastric metaplasia in the duodenum in an infected gerbil from the TN2GF4 group, inoculated at 14 weeks, was colonised by H pylori (arrows) (Giemsa, original magnification ×400). (D) Superficial duodenal ulcer (arrows) in the gastric metaplasia with severe duodenitis was evident in a gerbil from the TN2GF4 group, inoculated at 14 weeks. Brunner’s glands can be seen beneath the gastric metaplasia (haematoxylin-eosin, original magnification ×100). (E) No duodenitis is seen in a non-infected gerbil of the control group (haematoxylin-eosin, original magnification ×200).
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inoculation in the Mongolian gerbil. In addition, both duodenitis and duodenal ulcer were induced by H pylori infection in an age and strain dependent manner. This fulfills the requirements of Koch’s postulates for establishing a role for H pylori as a causative agent of duodenal ulcer and validates a model system for studying this disease. However, the occurrence rate of duodenal ulcer was not significant with respect to controls; larger numbers of animals are needed to determine its significance.
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Figure 3 Interleukin 8 (IL-8) production in MKN74 cells after coculture with the three bacterial strains of Helicobacter pylori. **p
