Cure of Helicobacter pylori infection and resolution of gastritis by adoptive transfer of splenocytes in mice

Vaccination suppresses Helicobacter pylori colonization but does not cure infection. Furthermore, postvaccination gastritis, likely induced by enhanced host response to residual colonization, may exacerbate disease. The goal of this study was to determine if adoptive transfer of C57BL/6 splenocytes to C57BL/6scid/scid (severe combined immunodeficient [SCID]) mice cures infection without exacerbating gastritis. H. pylori-infected and uninfected C57BL/6 mice and SCID recipients of normal splenocytes were killed at intervals between 5 and 51 weeks after infection. Colonization and gastritis were quantified, humoral immune responses were determined by enzyme-linked immunosorbent assay, and cellular immune responses were determined by delayed-type hypersensitivity response and by a proliferative response of cultured splenocytes to H. pylori sonicate. In infected C57BL/6 mice, gastritis developed gradually and bacterial colonization diminished but persisted throughout the experiment. In contrast, gastritis in infected recipient SCID mice developed rapidly and bacterial colonization decreased precipitously. Gastritis in those mice peaked 9 weeks after adoptive transfer, however, and began to resolve. By 45 weeks after transfer, gastritis had returned to background levels and bacteria were no longer detectable. Resolution of gastritis and elimination of infection were associated with a cellular but not humoral immune response to H. pylori antigens. These results demonstrate that although the host response fails to clear bacterial colonization in normal mice, enhanced cellular immune responses in recipient SCID mice are capable of clearing H. pylori infection and allowing resolution of gastritis. Thus, immune mechanisms of cure exist, and effective and safe vaccination protocols may be feasible.     

Enhanced disease severity in Helicobacter pylori-infected mice deficient in Fas signaling

Recent evidence suggests that immune-mediated gastric epithelial cell apoptosis through Fas-Fas ligand interactions participates in Helicobacter pylori disease pathogenesis. To define the role of Fas signaling in vivo, H. pylori strain SS1 infection in C57BL/6 mice was compared to that in mice deficient in the Fas ligand (gld). gld mice had a degree of gastritis similar to that of C57BL/6 mice after 6 weeks (gastritis score, 5.2 +/- 0.6 [mean +/- standard error] versus 3.5 +/- 0.8) and 12 weeks (4.0 +/- 0.7 versus 3.4 +/- 0.5) of infection. Bacterial colonization was comparable in each group of mice at 12 weeks of infection (2.1 +/- 0.3 versus 1.6 +/- 0.3 for gld and C57BL/6, respectively; the difference is not significant). Sixty-seven percent of H. pylori-infected gld mice displayed atrophic changes in the gastric mucosa, compared with 37% of infected C57BL/6 mice, at 12 weeks. In addition, atrophic changes were more severe in H. pylori-infected gld mice (P < 0.05). Splenocytes isolated from H. pylori-infected C57BL/6 mice had a twofold increase in production of the Th1 cytokine gamma interferon (IFN-gamma) in response to H. pylori antigens at both 6 and 12 weeks compared to controls (143 +/- 65 versus 69 +/-26 pg/ml and 336 +/- 73 versus 172 +/- 60, respectively). In contrast, there was a lack of detectable IFN-gamma in gld mice infected with the bacterium. H. pylori-infected C57BL/6 mice had increased epithelial cell apoptosis compared with sham-infected C57BL/6 mice (35.0 +/- 8.9 versus 12.3 +/- 6.9; P < 0.05). Epithelial cell apoptosis did not differ between H. pylori-infected and control gld mice (5.2 +/- 1.6 versus 6.5 +/- 2.9 [not significant]). These data demonstrate that mice with mutations in the Fas ligand develop more severe premalignant mucosal changes in response to infection with H. pylori in association with both an impaired gastric epithelial cell apoptotic response and IFN-gamma production. The Fas death pathway modulates disease pathophysiology following murine infection with H. pylori. Deregulation of the Fas pathway could be involved in the transition from gastritis to gastric cancers during H. pylori infection.     

Coinfection with Enterohepatic Helicobacter species can ameliorate or promote Helicobacter pylori-induced gastric pathology in C57BL/6 mice

To investigate how different enterohepatic Helicobacter species (EHS) influence Helicobacter pylori gastric pathology, C57BL/6 mice were infected with Helicobacter hepaticus or Helicobacter muridarum, followed by H. pylori infection 2 weeks later. Compared to H. pylori-infected mice, mice infected with H. muridarum and H. pylori (HmHp mice) developed significantly lower histopathologic activity index (HAI) scores (P < 0.0001) at 6 and 11 months postinoculation (MPI). However, mice infected with H. hepaticus and H. pylori (HhHp mice) developed more severe gastric pathology at 6 MPI (P = 0.01), with a HAI at 11 MPI (P = 0.8) similar to that of H. pylori-infected mice. H. muridarum-mediated attenuation of gastritis in coinfected mice was associated with significant downregulation of proinflammatory Th1 (interlukin-1beta [Il-1β], gamma interferon [Ifn-γ], and tumor necrosis factor-alpha [Tnf-α]) cytokines at both time points and Th17 (Il-17A) cytokine mRNA levels at 6 MPI in murine stomachs compared to those of H. pylori-infected mice (P < 0.01). Coinfection with H. hepaticus also suppressed H. pylori-induced elevation of gastric Th1 cytokines Ifn-γ and Tnf-α (P < 0.0001) but increased Th17 cytokine mRNA levels (P = 0.028) at 6 MPI. Furthermore, mRNA levels of Il-17A were positively correlated with the severity of helicobacter-induced gastric pathology (HhHp>H. pylori>HmHp) (at 6 MPI, r2 = 0.92, P < 0.0001; at 11 MPI, r2 = 0.82, P < 0.002). Despite disparate effects on gastritis, colonization levels of gastric H. pylori were increased in HhHp mice (at 6 MPI) and HmHp mice (at both time points) compared to those in mono-H. pylori-infected mice. These data suggest that despite consistent downregulation of Th1 responses, EHS coinfection either attenuated or promoted the severity of H. pylori-induced gastric pathology in C57BL/6 mice. This modulation was related to the variable effects of EHS on gastric interleukin 17 (IL-17) responses to H. pylori infection.     

Effect of bacterial flora on postimmunization gastritis following oral vaccination of mice with Helicobacter pylori heat shock protein 60

In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increased in all animals at 10 weeks p.i. Anti-H. pylori HSP60 immunoglobulin G was detected in serum at 3 weeks p.i. in mice vaccinated with either H. pylori HSP60 or GroEL. Significant increases in the gastritis scores were observed only in SPF mice immunized with H. pylori HSP60. These results indicate that oral vaccination with H. pylori HSP60 has partial protective effects on subsequent H. pylori infection but also induces postimmunization gastritis. However, GF mice immunized with H. pylori HSP60 did not suffer from severe gastritis. Therefore, the presence of bacterial flora appears to contribute to the induction of postimmunization gastritis.     

Macrophages are mediators of gastritis in acute Helicobacter pylori infection in C57BL/6 mice

Helicobacter pylori is the etiological agent of human chronic gastritis, a condition seen as a precursor to the development of gastrointestinal ulcers or gastric cancer. This study utilized the murine model of chronic H. pylori infection to characterize the role of macrophages in the induction of specific immune responses and gastritis and in the control of the bacterial burden following H. pylori infection and vaccination. Drug-loaded liposomes were injected intravenously to deplete macrophages from C57BL/6 mice, and effective removal of CD11b+ cells from the spleens and stomachs of mice was confirmed by immunofluorescence microscopy. Transient elimination of macrophages from C57BL/6 mice during the early period of infection reduced the gastric pathology induced by H. pylori SS1 but did not affect the bacterial load in the stomach. These data suggest that macrophages are important to the severity of gastric inflammation during H. pylori infection.     

Wild-type and interleukin-10-deficient regulatory T cells reduce effector T-cell-mediated gastroduodenitis in Rag2-/- mice, but only wild-type regulatory T cells suppress Helicobacter pylori gastritis

CD4(+) CD45RB(hi) CD25(-) effector T cells (T(E)) promote Helicobacter pylori gastritis in mice, and CD4(+) CD45RB(lo) CD25(+) regulatory T cells (T(R)) are anti-inflammatory. Using adoptive transfer into H. pylori-infected Rag2(-/-) mice, we evaluated effects of wild-type (wt) C57BL/6 or congenic interleukin-10-deficient (IL-10(-/-)) T(R) cells on gastritis, gastric cytokines, and H. pylori colonization. Infected Rag2(-/-) mice colonized in the corpus and antrum with 10(5) to 10(6) H. pylori CFU/gram without associated gastritis. T(E) cell transfer caused morbidity and an H. pylori-independent pangastritis and duodenitis (gastroduodenitis) associated with increased expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha. T(E) cell transfer to H. pylori-infected mice led to additive corpus gastritis associated with inflammatory cytokine expression and reduced colonization. wt T(R) cells reduced morbidity, H. pylori corpus gastritis, gastroduodenitis, and inflammatory cytokine expression and reversed the decline in H. pylori colonization attributable to T(E) cells. Although less effective than wt T(R) cells, IL-10(-/-) T(R) cells also reduced morbidity and gastroduodenitis but did not reduce H. pylori corpus gastritis or impact T(E) cell inhibition of colonization. Gastric tissues from mice receiving wt T(R) cells expressed higher levels of Foxp3 compared to recipients of IL-10(-/-) T(R) cells, consistent with lower regulatory activity of IL-10(-/-) T(R) cells. These results demonstrate that wt T(R) cells suppressed T(E)-cell-mediated H. pylori-independent gastroduodenitis and H. pylori-dependent corpus gastritis more effectively than IL-10(-/-) T(R) cells. Compartmental differences in T(E)-cell- and H. pylori-mediated inflammation and in regulatory effects between wt T(R) and IL-10(-/-) T(R) cells suggest that IL-10 expression by wt T(R) cells is important to regulatory suppression of gastric inflammation.     

Tumor necrosis factor-alpha is required for gastritis induced by Helicobacter felis infection in mice

Helicobacter pylori colonizes the gastric mucosa of human and causes chronic gastritis. The previous studies have demonstrated that gamma interferon (IFN-gamma) but not tumor necrosis factor-alpha (TNF-alpha) plays a critical role in pathogenesis of gastritis induced by H. pylori infection. In this study we investigated the induction of gastritis induced by H. felis infection in TNF-alpha-deficient mice, comparing with IFN-gamma-deficient mice. The scores of gastritis and epithelial changes of TNF-alpha-deficient mice and IFN-gamma-deficient mice were significantly lower than that of C57BL/6 mice. Moreover, the degrees of gastritis and epithelial changes of TNF-alpha-deficient mice were rather low compared with that of IFN-gamma-deficient mice. In spleen cell cultures stimulated with heat-killed H. felis, IFN-gamma production by TNF-alpha-deficient mice and TNF-alpha production by IFN-gamma-deficient mice were significantly reduced compared with those in C57BL/6 mice. These results suggested that TNF-alpha is involved in pathogenesis of gastritis induced by H. felis infection as IFN-gamma and that an interaction between TNF-alpha and IFN-gamma might be required in pathogenesis of gastritis induced by Helicobacter infection.     

Absence of CD4+CD25+ regulatory T cells is associated with a loss of regulation leading to increased pathology in Helicobacter pylori-infected mice

Helicobacter pylori induces symptomatic chronic gastritis in a subpopulation of infected individuals. The mechanism(s) determining the development and severity of pathology leading to symptoms are not fully understood. In a mouse model of H. pylori infection we analysed the influence of immunoregulatory CD4+CD25+ T cells on H. pylori colonization and gastritis. Athymic C57BL/6 nu/nu mice were reconstituted with (a) lymph node (LN) cells (b) LN cells depleted of CD25+ T cells (CD25(-) LN) or (c) not reconstituted at all. Mice were then infected orally with 3 x 10(8)H. pylori SS1 bacteria. At 2 and 6 weeks after the inoculation there was a significant (P < 0.001) reduction in H. pylori colonization in athymic mice transferred with CD25(-) LN cells compared to mice transferred with LN cells. Colonization was still reduced at 12 weeks after inoculation. Mice transferred with CD25(-) LN cells showed an earlier onset and increased severity of gastritis as compared to mice receiving LN cells. Splenic cells isolated from mice receiving CD25(-) LN cells produced the highest level of IFN-gamma on stimulation with H. pylori antigens in vitro, had a higher H. pylori-specific DTH response and increased infiltration of CD4+ T cells and macrophages in the gastric mucosa. Athymic mice not transferred with T cells had persistent high H. pylori colonization and displayed a normal gastric epithelium without inflammatory cells. In conclusion, CD4+CD25+ cells reduce immunopathology in H. pylori infection, possibly by reducing the activation of IFN-gamma producing CD4+ T cells, even at the expense of a higher H. pylori load in the gastric mucosa.     

Role of Helicobacter pylori cag region genes in colonization and gastritis in two animal models

The Helicobacter pylori chromosomal region known as the cytotoxin-gene associated pathogenicity island (cag PAI) is associated with severe disease and encodes proteins that are believed to induce interleukin (IL-8) secretion by cultured epithelial cells. The objective of this study was to evaluate the relationship between the cag PAI, induction of IL-8, and induction of neutrophilic gastric inflammation. Germ-free neonatal piglets and conventional C57BL/6 mice were given wild-type or cag deficient mutant derivatives of H. pylori strain 26695 or SS1. Bacterial colonization was determined by plate count, gastritis and neutrophilic inflammation were quantified, and IL-8 induction in AGS cells was determined by enzyme-linked immunosorbent assay. Deletion of the entire cag region or interruption of the virB10 or virB11 homolog had no effect on bacterial colonization, gastritis, or neutrophilic inflammation. In contrast, these mutations had variable effects on IL-8 induction, depending on the H. pylori strain. In the piglet-adapated strain 26695, which induced IL-8 secretion by AGS cells, deletion of the cag PAI decreased induction. In the mouse-adapted strain SS1, which did not induce IL-8 secretion, deletion of the cagII region or interruption of any of three cag region genes increased IL-8 induction. These results indicate that in mice and piglets (i) neither the cag PAI nor the ability to induce IL-8 in vitro is essential for colonization or neutrophilic inflammation and (ii) there is no direct relationship between the presence of the cag PAI, IL-8 induction, and neutrophilic gastritis.     

Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 microgram, or 2 microgram of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 x 10(7) CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 microgram/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice were considered protected. Lower levels of H. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.     

Two-year follow-up of Helicobacter pylori infection in C57BL/6 and Balb/cA mice

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. We previously found high-grade lymphoma after 13 months' H. pylori infection in C57BL/6 mice. In this study we followed H. pylori infection by three different isolates in C57BL/6 and Balb/cA mice for 23 months. Six-week-old C57BL/6 and Balb/cA mice were infected with H. pylori strains 119p (CagA+, VacA+), SS1 (CagA+, VacA+) and G50 (CagA-, VacA-). Mice were followed at 2 weeks, 10 weeks and 23 months post-inoculation (p.i.) by culture, histopathology and serology. Strain G50 was only reisolated from mice 2 weeks p.i. There was no difference in colonization between strain 119p and SS1 at 10 weeks p.i., whereas SS1 gave 100% colonization versus 119p gave 50% 23 months p.i. Interestingly, the inflammation score was higher in mice infected with strain 119p than with SS1 10-week p.i., and there were lymphoepithelial lesions in mice infected with strain 119p and G50 but not with SS1 at 23 months post-infection. Eight mice infected with strains 119p and G50 developed gastric lymphoma (grade 5 and 4). One C57BL/6 mouse infected with strain 119p developed hepatocellular carcinoma after 23 months. Immunoblot showed specific bands of 26-33 kDa against H. pylori in infected mice, and two mice infected with strain SSI reacted with antibodies to the 120 kDa CagA toxin. Conclusion: A reproducible animal model for H. pylori-induced lymphoma and possibly hepatocellular carcinoma is described. Strain diversity may lead to different outcomes of H. pylori infection.     

Clearance of Helicobacter pylori Infection and Resolution of Postimmunization Gastritis in a Kinetic Study of Prophylactically Immunized Mice

Patients infected with Helicobacter pylori mount an immune response which fails to clear the infection and may contribute to disease. Mice can be protected by immunization. To further characterize the H. pylori-mouse model, stomachs of unimmunized or intranasally immunized C57BL/6 mice were quantitatively cultured 3 days and 1, 2, 4, 8, 16, 32, and 52 weeks after challenge with H. pylori. At 3 days and 1 week after challenge, colonization was the same in the immunized and unimmunized mice. By 2 weeks after challenge, the immunized mice had a >2-log decrease in bacterial load, and at all later time points, they either were culture negative or had at least a 2-log decrease in bacterial load. Gastritis in the immunized mice peaked at 1 to 2 weeks after challenge and was characterized by a mixed inflammatory infiltrate and epithelial proliferation centered at the transition between corpus and antrum. By 52 weeks postchallenge, the gastric histology in the immunized mice was not different from that in control unchallenged mice. The unimmunized group began to show a reduction in bacterial load as early as 16 weeks after challenge, and by 52 weeks seven of eight unimmunized mice had developed gastritis and reduced bacterial loads. These results indicate that prophylactic immunization does not prevent colonization by H. pylori but enables mice to clear the infection or significantly reduce the number of colonizing bacteria. The reduction in bacterial load is associated with gastric inflammation that subsides over time.     

Roles of Innate and Adaptive Immunity in Respiratory Mycoplasmosis

Current evidence suggests that host defense in respiratory mycoplasmosis is dependent on both innate and humoral immunity. To further delineate the roles of innate and adaptive immunity in antimycoplasmal defenses, we intranasally infected C3H/HeSnJ-scid/scid (C3H-SCID), C3H/HeSnJ (C3H), C57BL/6J-scid/scid (C57-SCID), and C57BL/6N (C57BL) mice with Mycoplasma pulmonis and at 14 and 21 days postinfection performed quantitative cultures of lungs and spleens, quantification of lung lesions, and histopathologic assessments of all other major organs. We found that numbers of mycoplasmas in lungs were associated with genetic background (C3H susceptible, C57BL resistant) rather than functional state of adaptive immunity, indicating that innate immunity is the main contributor to antimycoplasmal defense of the lungs. Extrapulmonary dissemination of mycoplasmas with colonization of spleens and histologic lesions in multiple organs was a common occurrence in all mice. The absence of adaptive immune responses in severe combined immunodeficient (SCID) mice resulted in increased mycoplasmal colonization of spleens and lesions in extrapulmonary sites, particularly spleens, hearts, and joints, and also reduced lung lesion severity. The transfer of anti-M. pulmonis serum to infected C3H-SCID mice prevented extrapulmonary infection and disease, while the severity of lung lesions was restored by transfer of naive spleen cells to infected C3H-SCID mice. Collectively, our results strongly support the conclusions that innate immunity provides antimycoplasmal defense of the lungs and humoral immunity has the major role in defense against systemic dissemination of mycoplasmal infection, but cellular immune responses may be important in exacerbation of mycoplasmal lung disease.     

Accelerated progression of gastritis to dysplasia in the pyloric antrum of TFF2 -/- C57BL6 x Sv129 Helicobacter pylori-infected mice

Trefoil factor family 2 (TFF2) is up-regulated in Helicobacter spp.-infected gastric tissues of both humans and mice. To ascertain the biological effects of TFF2 in vivo, TFF2(-/-) C57BL/6 x Sv129 and wild-type (WT) C57BL/6 x Sv129 mice were orally infected with Helicobacter pylori SS1. Mice were evaluated for gastric H. pylori colonization, pathology, and cytokine profiles at 6 and 19 months post inoculation (pi). At 6 months pi, there was a significant difference (P < 0.05) for epithelial criteria (mucosal defects, atrophy, hyperplasia, pseudopyloric metaplasia, and dysplasia) in the corpus of TFF2(-/-) versus WT mice. At 19 months pi, a similar statistical difference in epithelial parameters was noted in the antrum of TFF2(-/-) versus WT mice (P < 0.01). All of the TFF2(-/-) H. pylori-infected mice had high-grade antral dysplasia, including gastric intraepithelial neoplasia, which was statistically significant (P < 0.05) compared with the infected WT mice. Levels of interferon-gamma were markedly elevated in the gastric mucosa of infected TFF2(-/-) mice at both 6 and 19 months pi. TFF2 provided a cytoprotective and/or anti-inflammatory effect against the progression of premalignant lesions of the gastric corpus at 6 months pi and in the pyloric antrum in H. pylori-infected mice at 19 months pi. These data support a protective role for TFF2 in part by modulating levels of gastric interferon-gamma in the development of H. pylori-associated premalignancy of the distal stomach.     

Development of an interleukin-12-deficient mouse model that is permissive for colonization by a motile KE26695 strain of Helicobacter pylori

The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12(-/-) p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H. pylori. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H. pylori than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (10(6) versus 10(5) CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (10(4) to 10(5) CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, approximately 10(3) CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with H. pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.     

Development of high-grade lymphoma in Helicobacter pylori-infected C57BL/6 mice

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. Mice with H. pylori infection develop severe gastritis and atrophic changes in their stomachs after 6 months. We followed H. pylori-infected animals for 13 months to find out whether dysplasia, carcinoma or lymphoma developed. Six-week-old C57BL/6 mice were infected with the CagA-positive and VacA-positive H. pylori mouse-passaged strain 119/95, fed a low antioxidant diet, and kept in microisolated cages. Histopathological changes were examined after 13 months' infection. All H. pylori-inoculated mice (n = 5) developed a gastric squamous papilloma with nagging of the lamina muscularis after 13 months. Three out of five animals developed high-grade B-cell lymphoma derived from a MALT lymphoma at the squamous-corpus border with manifestations also in the liver, spleen and kidney. There was a suspicion of local gastric lymphoma in the two remaining mice but with no significant changes in the liver, spleen or kidney. The normal control mice showed no pathological changes in any of these organs. It is concluded that this mouse model with infection by the CagA-positive, vac-toxin-producing H. pylori strain 119/95 is suitable for use in the study of lymphoma development and also development of squamous cell papilloma with proliferative features.     

Effects of Nonsteroidal Anti-Inflammatory Drugs on Helicobacter pylori-Infected Gastric Mucosae of Mice: Apoptosis, Cell Proliferation, and Inflammatory Activity

Helicobacter pylori and nonsteroidal anti-inflammatory drugs (NSAIDs) are two well-known important causative factors of gastric damage. While H. pylori increases apoptosis and the proliferation of gastric epithelial cells and is an important factor in peptic ulcer and gastric cancer, NSAIDs induce cell apoptosis and have antineoplastic effects. We investigated the effects of NSAIDs (a nonselective cyclooxygenase [COX] inhibitor [indomethacin] and a selective COX-2 inhibitor [NS-398]) on the apoptosis and proliferation of gastric epithelial cells and gastric inflammation in H. pylori-infected mice. C57BL/6 mice were sacrificed 8 weeks after H. pylori SS1 inoculation. Indomethacin (2 mg/kg) or NS-398 (10 mg/kg) was administered subcutaneously once daily for 10 days before sacrifice. The following were assessed: gastric inflammatory activity, gastric COX protein expression by Western blotting; gastric prostaglandin E(2) levels by enzyme immunoassay, apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and cell proliferation by Ki67 immunostaining. Compared to the controls, H. pylori infection and/or NSAID treatment increased COX-1 and COX-2 protein expression. Gastric prostaglandin E(2) levels, apoptotic index, cell proliferation index, neutrophil activity, and the degree of chronic inflammation were all increased by H. pylori infection, and these effects were significantly decreased by indomethacin treatment. However, NS-398 treatment after H. pylori infection did not induce a significant reduction, although it did result in a tendency to decrease. These results show that NSAIDs can reverse the increased apoptosis and proliferation of epithelial cells and inflammatory activity in the stomachs of H. pylori-infected mice and that, like COX-2 activation, COX-1 induction contributes to the change of gastric mucosal cell turnover and inflammation induced by H. pylori infection.