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Infection and Immunity, March 2007, p. 1214-1222, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01459-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

"Candidatus Helicobacter heilmannii" from a Cynomolgus Monkey Induces Gastric Mucosa-Associated Lymphoid Tissue Lymphomas in C57BL/6 Mice

Masahiko Nakamura,^1* Somay Yamagata Murayama,^2,3 Hiroshi Serizawa,⁴ Yukie Sekiya,³ Masahiro Eguchi,^2,3 Shinichi Takahashi,⁵ Kaori Nishikawa,⁵ Tetsufumi Takahashi,⁶ Tsukasa Matsumoto,^2,3 Haruki Yamada,^2,3 Toshifumi Hibi,⁶ Kanji Tsuchimoto,^1,4 and Hidenori Matsui^2,3

Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences,¹ Kitasato Institute for Life Sciences & Graduate School of Infection Control Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan,² Center for Basic Research,³ Department of Internal Medicine, Kitasato Institute Hospital, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan,⁴ Third Department of Internal Medicine, Kyorin University, Mitaka, Tokyo 181-8611, Japan,⁵ Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan⁶

Received 12 September 2006/ Returned for modification 13 October 2006/ Accepted 14 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both Helicobacter pylori and "Candidatus Helicobacter heilmannii" infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of "Candidatus Helicobacter heilmannii" gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as "Candidatus Helicobacter heilmannii" based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous "Candidatus Helicobacter heilmannii" bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, "Candidatus Helicobacter heilmannii" induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
"Candidatus Helicobacter heilmannii," previously known as Gastrospirillum hominis (14), infects animals and humans, but the incidence is much lower than the incidence of Helicobacter pylori in humans. "Candidatus Helicobacter heilmannii" has not been reliably cultured to date, but it can be maintained in mouse stomachs (2, 23). In humans, "Candidatus Helicobacter heilmannii" infection has been associated with minimal gastritis, occasional gastroduodenal ulcers, and mucosa-associated lymphoid tissue (MALT)-type gastric B-cell lymphomas (MALT lymphomas) (15). Both H. pylori and "Candidatus Helicobacter heilmannii" infections have been reported to be related to these MALT lymphomas, while the relative significance of H. pylori and "Candidatus Helicobacter heilmannii" for MALT lymphoma formation is controversial. Morgner et al. showed that 1.47% of "Candidatus Helicobacter heilmannii"-infected patients and 0.66% of H. pylori-infected patients had primary gastric MALT lymphomas (15). On the other hand, Stolte et al. reported a milder form of gastritis and less common MALT lymphomas in "Candidatus Helicobacter heilmannii" gastritis patients than in H. pylori-infected gastritis and duodenal ulcer patients, who are known to have less gastritis (26). Jhala et al. reported less gastritis and fewer lymphoid aggregates in "Candidatus Helicobacter heilmannii"-infected patients, but their method to prove the presence of "Candidatus Helicobacter heilmannii" depended solely on morphological observation using hematoxylin and eosin or Warthin-Starry silver staining or immunostaining with polyclonal antibodies against H. pylori (11). Suzuki et al. reported that nearly all MALT lymphomas were H. pylori positive, but their diagnosis was dependent solely on the H. pylori serum antibody titer, which could not distinguish between H. pylori and "Candidatus Helicobacter heilmannii" (27).

In animal models, MALT lymphomas have been reported following infection with Helicobacter mustelae in ferrets (7), following infection with Helicobacter felis in normal BALB/c mice (6), following infection with H. pylori in neonatally thymectomized BALB/c mice (9), and following infection with "Candidatus Helicobacter heilmannii" in normal BALB/c mice (19, 21). In this study, we used the "Candidatus Helicobacter heilmannii" previously obtained from a cynomolgus monkey to infect C57BL/6 mice for up to 24 months. We observed the development of low-grade MALT lymphomas in the fundic regions of the stomachs of mice infected with this pathogen. The characteristics of the pathological changes were determined by immunohistochemical, electron microscopic, and in situ hybridization methods.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of bacteria and infection. We identified urease-positive bacteria infecting in the stomach of a cynomolgus monkey in 1994 (10). We then used gastric mucosal and mucus homogenates for inoculation of C3H/HeJ mice by peroral administration, and the infected mice were maintained under standard laboratory conditions for periods ranging from 3 to 24 months. In 20 6-month intervals (total, 120 months), we inoculated uninfected C3H/HeJ mice using gastric mucosal and mucus homogenates from infected mice to maintain the isolate. In the present experiment, 6-week-old C57BL/6 mice were inoculated with gastric mucosal homogenates containing gastric mucus and mucosa from infected C3H/HeJ mice 3, 6, 12, 18, and 24 months prior to the experiment. As a control, noninfected C3H/HeJ mice were used to prepare gastric mucosal and mucus homogenates.

DNA extraction. The entire bacterial and host cell DNA mixture was extracted from the gastric homogenates with phosphate-buffered saline containing 0.001% (wt/vol) gelatin (pH 7.4) by using an UltraClean microbial DNA isolation kit (MO Bio, Solana Beach, CA) according to the manufacturer's instructions. The DNA was stored at –20°C until it was required.

PCR amplification and sequencing. PCR was used to amplify the 16S rRNA and urease genes from gastric samples. The reaction mixtures each contained universal eubacterial primers TM16S-27F and TM16S-1492R (12) for the 16S rRNA gene and either primers U430F and U1735R or primers U430F and U2235R (20) for the urease genes. The cycling conditions for the 16S rRNA gene were initial denaturation at 94°C for 1 min and then 35 cycles of 94°C for 45 s, 55°C for 30 s, and 72°C for 2 min, followed by an extension step of 72°C for 5 min. The cycling conditions for the urease genes were initial denaturation at 94°C for 3 min and then 35 cycles of 94°C for 10 s, 52°C for 30 s, and 72°C for 1.5 min, followed by a final extension step of 72°C for 5 min. Alternatively, for the urease gene, the conditions were the same as those described above but with the following modifications: annealing at 42°C for 30 s and extension at 72°C for 2 min. Both strands were sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) with an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). Sequences were analyzed by using the NCBI BLAST programs (1).

Quantitative real-time PCR. Real-time PCR was performed using a Bio-Rad iCycler iQ detection system (Bio-Rad Laboratories, Hercules, CA) with the SYBR green fluorophore. Reactions were performed in 20-µl (total volume) mixtures which included 10 µl of 2x SYBR green PCR master mix (Bio-Rad Laboratories), 5 µl of each primer at a concentration of 5 µM, and 1 µl of the template DNA. A 112-bp fragment of the 16S rRNA gene was amplified with primers HeilF (5' AAG TCG AAC GAT GAA GCC TA 3') and HeilR (5' ATT TGG TAT TAA TCA CCA TTT C 3') (3). Data analyses were performed with an iCycler iQ real-time detection system (Bio-Rad Laboratories). The following protocol was used: 50 cycles consisting of 95°C for 15 s, 55°C for 15 s, and 72°C for 30 s. A melting curve analysis was performed following every run to ensure that there was a single amplified product for every reaction. At the extension time, the PCR fluorophore was acquired. All reactions were carried out at least three times for each sample. The same reference standard dilution series was used on every experimental plate, and semiquantification of 16S rRNA gene normalization was based on the averaged results. We used the PCR products amplified with primers TM16S-27F and TM16S-1492R as the standards. The copy number of the 16S rRNA gene of "Candidatus Helicobacter heilmannii" has not been identified yet, but it has been reported that H. pylori has two separate sets of rRNA genes on its chromosome (28). Based on this information, we calculated the number of 16S rRNA genes in "Candidatus Helicobacter heilmannii," which is thought to have two sets of 16S rRNA genes on its chromosome (13). Duplicate negative controls (no template DNA) were also included on every experimental plate in order to assess specificity and to detect potential contamination.

Macroscopic, immunohistochemical, and electron microscopic observations. The percentage of surface area for the protrusive lesions in the entire fundic mucosa was estimated by obtaining macroscopic images with a stereomicroscope (Keyence VHX-500; Keyence, Osaka, Japan) and by measuring the lesions and fundic mucosa in three mice in each group using the NIH Image public domain image processing and analysis program for Macintosh.

After the macroscopic observations had been carried out, some of the tissues were fixed with Zamboni's fixative (26), and immunohistochemical studies were performed using monoclonal antibodies against CD3 (R&D Systems, Minneapolis, MN), CD45R (clone RA3-6B2; Chemicon International, Temecula, CA), and Bcl-2 (R&D Systems) and VEGF-A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Polyclonal anti-H. pylori antisera were prepared by immunization of two male New Zealand White rabbits with a formalin-killed bacterial lysate of H. pylori Sydney strain SS-1. Subsequently, polyclonal anti-H. pylori antibody was used in order to observe the localization of "Candidatus Helicobacter heilmannii" using cross-reactivity. After treatment with Zamboni's fixative (25), 10-µm cryostat sections were obtained, and indirect immunohistochemical procedures using a diaminobenzidine reaction were carried out; the sections were then postfixed with a 1% osmium tetroxide solution and embedded in Epon. Ultrathin sections were then cut, counterstained with uranyl acetate, and observed with a JEOL 1200 EX-II electron microscope at an accelerating voltage of 80 kV.

Some of the tissues were embedded in paraffin, and 4-µm sections were cut; in situ hybridization using a microprobe method (30) was then carried out with previously described specific primers (Hhe1, Hhe2, Hhe3, Hhe4, and Hhe5) (29). Sense probes were used in the control experiment.

Statistics. All data were expressed as means ± standard deviations. Comparisons between groups were made by using a two-tailed Student's t test. P values of <0.05 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analysis of 16S rRNA and urease genes. The DNAs prepared from gastric biopsies of a cynomolgus monkey were amplified by PCR with all of the 16S rRNA gene primers used in this study. Previous attempts to amplify a section of the urease gene using previously described methods failed (17, 24), as noted by O'Rourke and colleagues (20). Using newly designed primers U430F, U1735R, and U2235R (17, 18), amplification of the urease genes was successfully carried out. The amplified 1,661-bp urease genes (ureA and ureB; DDBJ accession number AB252065) and the 1,473-bp 16S rRNA gene (DDBJ accession number AB252066; "Candidatus Helicobacter heilmannii" strain TKY) were sequenced and analyzed using NCBI BLAST programs. The urease gene of strain TKY was 98% identical (1,549/1,575 bases) to that of "Candidatus Helicobacter heilmannii" strain CM2 (DDBJ accession number AF508007), and the 16S rRNA gene of TKY exhibited 99% identity (1,449/1,451 bases) with that of "Candidatus Helicobacter heilmannii" strain MM2 (DDBJ accession number AF506783). The results demonstrated that there were high levels of homology with cluster 1 (16S rRNA) and cluster A (urease) genes (20). No contamination of H. pylori 16S rRNA genes was detected.

Macroscopic observation. Macroscopic observation of the stomach revealed several small, round, protrusive lesions located primarily in the fundic area in one-half of the infected mice at 3 months after infection; moreover, the number of lesions increased gradually in almost all of the infected mice that were infected for more than 6 months (Fig. 1). Quantitative analysis of the protrusive lesion area revealed significant increases in lesion density at 3 months after infection, and significant, gradual increases were observed thereafter (Fig. 2). In the control mouse, no protrusive lesions were observed.

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FIG. 1. Macroscopic observation of control and "Candidatus Helicobacter heilmannii"-infected mouse gastric mucosal surfaces. (a) Uninfected surface. (b) Three months after infection, several round protrusive lesions were observed in the fundic area. (c) Six months after infection, the number of protrusive lesions increased. (d) Twelve months after infection, almost all fundic mucosa samples contained round, protrusive lesions. (e) Eighteen months after infection, the lesions united to form larger lesions.

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FIG. 2. Time course of occupancy of the protrusive lesions in the entire fundic mucosa. The surface area occupied by round, protrusive lesions gradually increased. *, P < 0.05 for a comparison with the control group; #, P < 0.05 for a comparison with the 3-month group; &, P < 0.05 for a comparison with the 12-month group; @, P < 0.05 for a comparison with the 18-month group.

Colonization of "Candidatus Helicobacter heilmannii" after oral inoculation. At 3, 6, 12, and 18 months after oral inoculation with 1 x 10⁴ copies of 16S rRNA genes per C57BL/6 mouse, the levels of "Candidatus Helicobacter heilmannii" in the gastric mucosal homogenate were about 10⁵ copies of the 16S rRNA genes per stomach (Fig. 3). Thereafter, the level of bacteria decreased until it reached approximately 5 x 10³ copies of the 16S rRNA genes per stomach at month 24.

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FIG. 3. Time course showing the copy numbers of "Candidatus Helicobacter heilmannii," as determined by real-time PCR. At 3, 6, 12, and 18 months after infection, the number of "Candidatus Helicobacter heilmannii" obtained from the gastric homogenate was about 105 CFU per stomach, and this number decreased until it reached approximately 5 x 103 CFU at 24 months after infection. The asterisk indicates that the P value is <0.01 for a comparison with the data for 18 months.

Pathological analysis. Light and electron microscopic observations revealed that the lesions consisted of an accumulation of small centrocyte-like cells in the mucosa concomitant with the destruction of the glandular tissues; this destruction included the appearance of lymphoepithelial lesions that coincided with low-grade MALT lymphomas (Fig. 4 and 5). Small, erosive lesions were also detected at the top of the protrusive lesions. In situ hybridization also showed that there was a positive reaction in the glandular lumen, especially near the intracellular canaliculi of parietal cells (Fig. 6). Electron microscopic immunocytochemical observations using polyclonal antibody against H. pylori, which cross-reacts with "Candidatus Helicobacter heilmannii," revealed numerous "Candidatus Helicobacter heilmannii" bacilli not only in the mucus layer but also in the lumen of the body and base of the fundic glands and the intracellular canaliculi of the parietal cells. In addition, a large number of parietal cells were damaged in areas adjacent to the lymphocytic infiltration, and some bacilli were detected in the lamina propria mucosae near these cells.

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FIG. 4. Light microscopic observation of hematoxylin- and eosin-stained, "Candidatus Helicobacter heilmannii"-infected fundic mucosa. (a) Lymphocytic accumulation is recognized in the lamina propria mucosa (arrow) just above the muscularis mucosa 3 months after infection. Magnification, x60. (b) Lymphoid follicle in the gastric body mucosa, with some accumulation of lymphocytes in the adjacent submucosal layer of mice infected for 6 months. Magnification, x400. (c) Mouse infected for 12 months with a low-grade MALT lymphoma involving the submucosal layer. Multiple follicles are spanned by profuse bridging aggregates of the hypochromic marginal zone. Magnification, x80. (d) At higher magnification, clear cell variety is predominant, with larger CCL cells with predominant nucleoli. Magnification, x1,000.

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FIG. 5. Light and electron microscopic observations of lymphoid follicles and lymphoepithelial lesions. (a) Lymphoid follicle (L) observed in the fundic mucosa of a mouse infected for 6 months. Toluidine blue-stained 1-µm Epon section. Magnification, x100. (b) Lymphoepithelial lesion in the stomach of a mouse infected for 6 months. Toluidine blue-stained 1-µm Epon section. Magnification, x600. (c) Invasion of a lymphocyte between fundic glandular cells in a mouse infected for 6 months. Magnification, x3,000. (d) Accumulation of centrocyte-like cells in a lymphoid follicle. Magnification, x2,000.

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FIG. 6. Localization of "Candidatus Helicobacter heilmannii" by in situ hybridization, immunohistochemistry, and electron microscopic cytochemistry. (a and b) Many reactive bacilli were recognized by in situ hybridization at the luminal side of the body of the fundic gland (a). No reaction was detected with a sense probe (b). Magnification, x800. (c and d) Indirect fluorescent immunohistochemistry using anti-H. pylori polyclonal antibody revealed immunoreactive bacilli at the luminal side of the body of the fundic gland (c), and Alexa-phalloidin fluorescence (d) revealed that the localization of bacilli coincided approximately with the localization of f-actin-rich parietal cells. Magnification, x800. (e, f, and g) Electron microscopy revealed the presence of extremely tortuous bacilli (arrows) in the fundic glandular lumen near the parietal cells (e and f) and in the intracellular canaliculi (e and g). (e) Magnification, x1,500; (f and g) Magnification, x6,000. (h and i) Some bacilli (white arrow) were detected in the lamina propria mucosae. An adjacent parietal cell (P) was destroyed. (h) Magnification, x2,000; (i) Magnification, x6,000. Just adjacent to the disrupted nucleus of the cell (arrowheads), a substance (arrow) having the same electron density as the bacilli was recognized.

In situ hybridization using primer Hhe1 also revealed a positive reaction in the glandular lumen, especially near the intracellular canaliculi of the parietal cells just adjacent to the lymphoma (Fig. 7). Some hybridization reaction products were also found within the lymphoma tissues. The immunohistochemical observations indicated that most of the aggregated lymphocytes were B220-positive and CD3-negative cells. Bcl-2 immunoreactivity was observed in some of the accumulated lymphocytes in the mice that had been infected for more than 6 months. In addition, most of the cells in the MALT lymphomas were immunoreactive with VEGF-A.

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FIG. 7. Immunohistochemistry and in situ hybridization of MALT lymphomas. (a and b) In situ hybridization revealed a positive reaction in a lymphoid follicle that reacted with primer Hhe1. (a) Magnification, x400; (b) Magnification, x1,000. (c) Positive B220 immunoreactivity was recognized in the lymphoid tissue (L). Magnification, x400. (d) Only a few CD3-immunoreactive cells were detected in the lymphoid follicle (L). Magnification, x400. (e) Some of the lymphocytes in and near the lymphoid follicle (L) were immunoreactive with Bcl-2. Magnification, x400. (f) Most of the lymphocytes in the lymphoid follicle (L) were immunoreactive with VEGF-A. Magnification, x400.

Top Abstract Introduction Materials and Methods Results Discussion References

 
There have been at least four reports of animal studies demonstrating the formation of gastric MALT lymphomas induced by Helicobacter species. Erdman and colleagues described a case study of H. mustelae-associated gastric MALT lymphoma in four ferrets, and they found two low-grade lymphomas and two high-grade lymphomas in the antrum (7). Enno et al. reported observing H. felis-associated gastric MALT lymphomas in normal BALB/c mice, and they found lymphoepithelial lesions in 88% of the animals and lymph follicles in 38% of the animals 22 months after infection (6). Fukui and colleagues reported observing H. pylori-associated gastric MALT lymphomas in neonatally thymectomized BALB/c mice, and they found lymph follicles and lymphoepithelial lesions in 60% of the mice in the absence of Bcl-X immunoreactivity (9). In addition, Bcl-X-immunoreactive cells were found in these mice 6 and 12 months later. O'Rourke and colleagues reported finding "Candidatus Helicobacter heilmannii"-associated gastric MALT lymphomas in normal BALB/c mice; furthermore, 15 months after infection with "Candidatus Helicobacter heilmannii"-like isolates from bobcats and other animals, they reported finding lymphoepithelial lesions in up to 80% of the mice, and at 18 months after infection they found lymphomas in up to 25% of the mice (19). The present study clearly demonstrated that "Candidatus Helicobacter heilmannii" infection with cynomolgus monkey mucosal and mucous homogenates resulted in MALT lymphoma formation in 50% of the C57BL/6 mice 3 months after infection and in 100% of the animals 6 months after infection; therefore, this could be a useful animal model of gastric MALT lymphomas, provided that these bacilli can be successfully cultured. In addition, the "Candidatus Helicobacter heilmannii" used in the present experiments was passaged in mice for up to 12 years, and it could possibly be adapted to mice to increase virulence.

H. pylori infection has been thought to be a prerequisite for the formation of MALT lymphomas, and chronic inflammation is probably a factor leading to malignant transformation via chronic stimulation of the lymphoid tissue (5, 11, 26, 27). However, the diagnosis of H. pylori infection in these previous reports was dependent on pathological data showing the presence of bacteria that were longer and more spiral than H. pylori obtained by hematoxylin and eosin or Warthin-Starry silver staining and immunostaining with polyclonal antibodies against H. pylori. Now that the 16S rRNA gene sequence of "Candidatus Helicobacter heilmannii" is available, as it has been since 2003, the previous conclusions should be reevaluated by performing PCR analyses.

The difference between the length of the period before MALT lymphoma occurrence in the study of O'Rourke et al. (19) and the length of the period in our study may have been due to differences between the experimental animals and the bacteria used. When the animal species used in the studies are considered, the differences may have resulted from the Th1/2 status of the Th1-predominant C57BL/6 mice and the Th2-predominant BALB/c mice. In this context, the report of Fukui and colleagues (9) is of interest, because these workers demonstrated the formation of MALT lymphomas induced by H. pylori in neonatally thymectomized BALB/c mice, which suggested that Th1 predominance was important in the formation of MALT lymphomas; however, in that study, Fukui et al. did not provide an explanation for why MALT lymphomas did not form in normal C57BL/6 mice, which are neonatally Th1 predominant.

Another common feature of "Candidatus Helicobacter heilmannii"-infected C57BL/6 mice and thymectomized BALB/c mice is the hypoacidity induced by parietal cell damage. In neonatally thymectomized BALB/c mice, autoimmune gastritis has been found (18). Administration of H. pylori to these mice decreased parietal cell damage, but antiparietal cell antibodies were still detectable. In the present study, direct invasion of parietal cell intracellular canaliculi and the cytoplasm by "Candidatus Helicobacter heilmannii" was observed. Several previous reports demonstrated this characteristic of "Candidatus Helicobacter heilmannii" infection in rhesus monkeys and C3H mice, although identification of the bacteria was based on morphology (4), which could have a tremendous effect on parietal cell function; it is well known that parietal cells change their cytoskeletal structure according to acid secretion status (8). When differences between bacillus species are considered, more prompt and effective formation of MALT lymphomas by "Candidatus Helicobacter heilmannii" is expected. O'Rourke and colleagues used "Candidatus Helicobacter heilmannii"-like isolates whose DNA sequences have yet to be identified (20), whereas we used genetically well-defined "Candidatus Helicobacter heilmannii," which was classified as having cluster 1 16S rRNA and cluster A urease genes, although it should be noted that in the present study, the bacilli were obtained from a cynomolgus monkey stomach.

We previously observed a sustained increase in myofibroblasts (i.e., activated fibroblasts) in "Candidatus Helicobacter heilmannii"-infected C3H mice (16); this finding is in good agreement with the bacterial copy number observed in the present study. The continuous influence of "Candidatus Helicobacter heilmannii" on the gastric mucosa may also be related to the formation of MALT lymphomas.

With regard to the mechanism of formation of gastric MALT lymphomas, soluble FAS antigen and apoptosis have been reported to be associated with the malignancy grade of MALT lymphomas. Thus, the relationship between parietal cell damage and apoptosis still needs to be clarified. In situ hybridization revealed the presence of some bacilli in the lymphoma tissues, which was suggestive of a direct influence of this bacterium or its DNA on the formation of lymphomas. Relevant to this point, Serna et al. (22) reported the presence of Helicobacter-like organisms within the lymphoid follicles in cats, suggesting that the bacteria could serve as a stimulus for the development of lymph follicles.

The immunological cross-reactivity of H. pylori with "Candidatus Helicobacter heilmannii" (11) and "Candidatus Helicobacter suis" is well known and has been considered one of the criteria for identification of these helicobacters (20). The present study also clearly revealed the cross-reactivity of "Candidatus Helicobacter heilmannii" with the anti-H. pylori antibody.

 
We express our sincere thanks to David Y. Graham for expert advice and suggestions.

This study was supported by the Research Fund of Mitsukoshi Health and Welfare Foundation 2005 and in part by a grant from the 21st Century COE Program, Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

 
* Corresponding author. Mailing address: School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Phone and fax: 81-3-3446-9036. E-mail: nakamuram{at}pharm.kitasato-u.ac.jp.

Published ahead of print on 28 December 2006.

Editor: A. D. O'Brien

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
2
2. Andersen, L. P., A. Norgaard, S. Holck, J. Blom, and T. L. Elsborg. 1996. Isolation of a "Helicobacter heilmanii"-like organism from the human stomach. Eur. J. Clin. Microbiol. Infect. Dis. 15:95-96.[CrossRef][Medline]
3
3. Chisholm, S. A., and R. J. Owen. 2003. Development and application of a novel screening PCR assay for direct detection of ‘Helicobacter heilmannii’-like organisms in human gastric biopsies in southeast England. Diagn. Microbiol. Infect. Dis. 46:1-7.[CrossRef][Medline]
4
4. Dubois, A., L. M. Heman-Ackah, E. S. Drazek, A. Tarnawski, W. N. Fishbein, G. I. Perez-Perez, and M. J. Blazer. 1994. Natural gastric infection with Helicobacter pylori in monkeys: a model for spiral bacterial infection in humans. Gastroenterology 160:1405-1417.
5
5. Eidt, S., M. Stolte, and R. Fischer. 1994. Helicobacter pylori gastritis and primary gastric non-Hodgkin's lymphomas. J. Clin. Pathol. 47:436-439.[Abstract/Free Full Text]
6
6. Enno, A., J. L. O'Rourke, C. R. Howlett, A. Jack, M. F. Dixon, and A. Lee. 1995. MALToma-like lesions in the murine gastric mucosa after long-term infection with Helicobacter felis. A mouse model of Helicobacter pylori-induced gastric lymphoma. Am. J. Pathol. 147:217-222.[Abstract]
7
7. Erdman, S. E., P. Correa, L. A. Coleman, M. D. Schrenzel, X. Li, and J. G. Fox. 1997. Helicobacter mustelae-associated gastric MALT lymphoma in ferrets. Am. J. Pathol. 151:273-280.[Abstract]
8
8. Forte, J. G., T. M. Forte, J. A. Black, C. Okamoto, and J. M. Wolosin. 1983. Correlation of parietal cell structure and function. J. Clin. Gastroenterol. 5(Suppl. 1):17-27.[Medline]
9
9. Fukui, T., K. Okazaki, H. Tamaki, K. Kawasaki, M. Matsuura, M. Asada, T. Nishi, K. Uchida, M. Iwano, M. Ohana, H. Hiai, and T. Chiba. 2004. Immunogenetic analysis of gastric MALT lymphoma-like lesions induced by Helicobacter pylori infection in neonatally thymectomized mice. Lab. Investig. 84:485-492.[Medline]
10
10. Itoh, T., Y. Yanagawa, M. Singaki, N. Masubuchi, S. Takahashi, and S. Saito. 1994. Isolation of Helicobacter heilmannii like organism from the stomachs of cynomolgus monkeys and colonization of them in mice. Gastroenterology 106:A99.
11
11. Jhala, D., N. Jhala, J. Lechago, and M. Haber. 1999. Helicobacter heilmannii gastritis, association with acid peptic diseases and comparison with Helicobacter pylori gastritis. Mod. Pathol. 12:534-538.[Medline]
12
12. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E.Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley and Sons, Chichester, West Sussex, England.
13
13. Linton, D., M. Moreno, R. J. Owen, and J. Stanley. 1992. 16S rrn gene copy number in Helicobacter pylori and its application to molecular typing. J. Appl. Bacteriol. 73:501-506.[Medline]
14
14. McNulty, C. A., J. C. Dent, A. Curry, J. S. Uff, G. A. Ford, M. W. Gear, and S. P. Wilkinson. 1989. New spiral bacterium in gastric mucosa. J. Clin. Pathol. 42:585-591.[Abstract/Free Full Text]
15
15. Morgner, A., N. Lehn, L. P. Andersen, C. Thiede, M. Bennedsen, K. Trebesius, B. Neubauer, A. Neubauer, M. Stolte, and E. Bayerdorffer. 2000. Helicobacter heilmannii-associated primary gastric low-grade MALT lymphoma: complete remission after curing the infection. Gastroenterology 118:821-828.[CrossRef][Medline]
16
16. Nakamura, M., S. Takahashi, H. Matsui, K. Nishikawa, Y. Akiba, and H. Ishii. 2002. Persistent increase in myofibroblasts in Helicobacter heilmannii-infected mice but not in Helicobacter pylori-infected Mongolian gerbils: colocalization of COX-2 and bFGF immunoreactivity. Aliment. Pharmacol. Ther. 16(Suppl. 2):174-179.
17
17. Neiger, R., C. Dieterich, A. Burnens, A. Waldvogel, I. Corthesy-Theulaz, F. Halter, B. Lauterburg, and A. Schmassmann. 1998. Detection and prevalence of Helicobacter infection in pet cats. J. Clin. Microbiol. 36:634-637.[Abstract/Free Full Text]
18
18. Okazaki, K., M. Ohana, C. Oshima, K. Uchida, T. Nishi, M. Iwano, T. Fukui, K. Kawasaki, M. Matsuura, M. Asada, H. Tamaki, H. Hiai, and T. Chiba. 2003. Interaction of Helicobacter pylori-induced follicular gastritis and autoimmune gastritis in BALB/c mice with post-thymectomy autoimmune gastritis. J. Gastroenterol. 38:1131-1137.[CrossRef][Medline]
19
19. O'Rourke, J. L., M. F. Dixon, A. Jack, A. Enno, and A. Lee. 2004. Gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma in an animal model of ‘Helicobacter heilmannii’ infection. J. Pathol. 203:896-903.[CrossRef][Medline]
20
20. O'Rourke, J. L., J. V. Solnick, B. A. Neilan, K. Seidel, R. Hayter, L. M. Hansen, and A. Lee. 2004. Description of ‘Candidatus Helicobacter heilmannii’ based on DNA sequence analysis of 16S rRNA and urease genes. Int. J. Syst. Evol. Microbiol. 54:2203-2211.[Abstract/Free Full Text]
21
21. Peterson, R. A., S. J. Danon, and K. A. Eaton. 2001. Comparison of gastritis and gastric epithelial proliferation in Helicobacter heilmannii-infected nude and BALB/c mice. Vet. Pathol. 38:173-183.[Abstract/Free Full Text]
22
22. Serna, J. H., R. M. Genta, L. M. Lichtenberger, D. Y. Graham, and F. A. el-Zaatari. 1997. Invasive Helicobacter-like organisms in feline gastric mucosa. Helicobacter 2:40-43.[Medline]
23
23. Solnick, J. V., J. O'Rourke, A. Lee, B. J. Paster, F. E. Dewhirst, and L. S. Tompkins. 1993. An uncultured gastric spiral organism is a newly identified Helicobacter in humans. J. Infect. Dis. 168:379-385.[Medline]
24
24. Solnick, J. V., J. O'Rourke, A. Lee, and L. S. Tompkins. 1994. Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter. Infect. Immun. 62:1631-1638.[Abstract/Free Full Text]
25
25. Stefanini, M., C. De Martino, and L. Zamboni. 1967. Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173-174.[CrossRef][Medline]
26
26. Stolte, M., G. Kroher, A. Meining, A. Morgner, E. Bayerdorffer, and B. Bethke. 1997. A comparison of Helicobacter pylori and H. heilmannii gastritis. A matched control study involving 404 patients. Scand. J. Gastroenterol. 32:28-33.[CrossRef][Medline]
27
27. Suzuki, T., K. Matsuo, H. Ito, K. Hirose, K. Wakai, T. Saito, S. Sato, Y. Morishima, S. Nakamura, R. Ueda, and K. Tajima. 2006. A past history of gastric ulcers and Helicobacter pylori infection increases the risk of gastric malignant lymphoma. Carcinogenesis 27:1391-1397.[Abstract/Free Full Text]
28
28. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G., Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547.[CrossRef][Medline]
29
29. Trebesius, K., K. Adler, M. Vieth, M. Stolte, and R. Haas. 2001. Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial DNA sequencing. J. Clin. Microbiol. 39:1510-1516.[Abstract/Free Full Text]
30
30. Wang, J. Y., and K. T. Montone. 1994. A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotin-labeled oligonucleotide probe: a comparison with immunohistochemical methods for HSV detection. J. Clin. Lab. Anal. 8:105-115.[Medline]

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Infection and Immunity, March 2007, p. 1214-1222, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01459-06
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