Pylori-infected Mice Research Articles

Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice

The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to ≈6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10 10 bacteria, followed after an interval of ≈4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.

Susceptibility to Helicobacter Pylori infection in mice is regulated by STAT6

Susceptibility to Helicobacter Pylori infection in mice is regulated by STAT6

Susceptibility to Helicobacter Pylori infection in mice is regulated by STAT6

Susceptibility to Helicobacter Pylori infection in mice is regulated by STAT6

Neuro-immune interactions in the stomach of H. pylori infected mice: Role of lymphocytes

Neuro-immune interactions in the stomach of H. pylori infected mice: Role of lymphocytes

Neuro-immune interactions in the stomach of H. pylori infected mice: Role of lymphocytes

Neuro-immune interactions in the stomach of H. pylori infected mice: Role of lymphocytes

Helicobacter pylori infection: mechanism of colonization and functional dyspepsia Reduced colonization of gastric mucosa by Helicobacter pylori in mice deficient in interleukin-10.

Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine. Mice deficient in IL-10 production (IL-10-/-mice) develop a spontaneous chronic enterocolitis, suggesting that IL-10 is an important regulator of the mucosal immune response in vivo. The objective of this study was to determine the role of endogenous IL-10 in the host defense against gastric colonization by Helicobacter pylori by using IL-10-deficient mice. The IL-10-/-mice were inoculated intragastrically with a mouse-adapted H. pylori isolate (Sydney Strain 1). Gastric colonization by H. pylori (biopsy urease test and bacterial colony counts), serum levels of H. pylori-specific immunoglobulin (Ig) M, A, G, isotypes of IgG, and the gastric mucosal inflammatory scores were determined 6 weeks after inoculation. Results were compared with those obtained from H. pylori-infected control mice (IL-10+/-mice). The colonization of gastric mucosa by H. pylori was reduced approximately 100-fold (P < 0.0001) in IL-10-/-mice (log10 4.87 +/- 0.26CFU/g tissue) as compared to IL-10+/-mice (log10 6.64 +/- 0.22 CFU/g tissue). Furthermore, IL-10-/-mice infected with H. pylori had significantly higher H. pylori-specific IgA and IgG antibodies in serum (P < or = 0.01), and developed much more severe chronic active gastritis than infected IL-10+/-mice. The median scores of the infiltration of gastric mucosa by mononuclear cells and neutrophils were up to threefold higher in IL-10-/-mice than they were in IL-10+/-mice. Our studies suggest that endogenous IL-10 is an inhibitor of the protective immune response to H. pylori infection. Interleukin-10 participates in the downregulation of H. pylori-induced gastric inflammatory responses, which apparently confers a survival advantage to the organism promoting more effective colonization of gastric mucosa.

Impaired development of HIV-1 gp160-specific CD8(+) cytotoxic T cells by a delayed switch from Th1 to Th2 cytokine phenotype in mice with Helicobacter pylori infection.

Th1 and Th2 cells play a central role in immunoregulation during infection. We show that Helicobacter pylori induces Th1 cytokine responses early (2 weeks) but predominantly Th2 responses later (6 weeks) in infection. The switch is principally mediated by urease-specific CD4(+) T cells, and correlates with a loss of urease-specific high-avidity JNK(+) Th1 and gain of low-avidity JNK(-) (possibly Th2) cells at the later stage of infection, concomitant with a 100-fold higher colonization level of H. pylori at 6 weeks than at 2 weeks that might tolerize high-avidity Th1 cells. Furthermore, differentiation of HIV gp160-specific CD4(+) Th and CD8(+) cytotoxic T lymphocytes (CTL) into effector cells is impaired in 6-week H. pylori-infected mice immunized with vaccinia expressing gp160, and serum IL-12 stimulated by vaccinia infection is barely detectable. Adoptive transfer of urease-specific Th2 cells to mice infected only with gp160-expressing vaccinia abrogates Th1 polarization of the gp120 response, down-modulates virus-specific CTL responses, and delays virus clearance. Therefore, the H. pylori urease-mediated immunoregulation in the switch from JNK(+) Th1 to JNK(-) Th2 phenotype, and the preceding low IL-12 response, are likely critical steps in the impairment of antiviral immunity.

Lack of gastritis and of an adaptive immune response in interferon regulatory factor-1-deficient mice infected with Helicobacter pylori.

To study the role of T cell responses in Helicobacter pylori gastritis, C57BL/6 wild-type and interferon regulatory factor-1-deficient (IRF-1(-/-)) mice were infected with the mouse-adapted H. pylori Sydney strain. Mice lacking the transcription factor IRF-1 are defective in Th1 development and are therefore biased to mount a Th2-type response. After 4 months of infection, C57BL/6 mice developed severe gastritis and atrophy and mounted a Th1-type response towards H. pylori. The Th1 response was abrogated in IRF-1(-/-) mice. This defective Th1 response was associated with the total lack of gastritis and atrophy in IRF-1(-/-) mice despite severe colonization with H. pylori. In addition, IRF-1(-/-) mice did also not develop a Th2 reaction, since they failed to generate H. pylori-specific antibodies and to produce IL-4 in response to H. pylori antigens in vitro. Thus, the transcription factor IRF-1 is necessary for the development of gastritis and atrophy in H. pylori-infected wild-type mice, suggesting a role of Th1 cells in the pathogenesis of H. pylori-associated diseases.

Short-term infection with Helicobacter pylori and 1 week exposure to metronidazole does not enhance gastric mutation frequency in transgenic mice

The aim of this study was to determine whether exposure of Helicobacter pylori-infected mice to metronidazole resulted in the delivery of mutagenic compounds to the gastric epithelium via the oxygen-insensitive NADPH nitroreductase (RdxA) of H. pylori. C57BL/6 transgenic mice containing the lambda/lacI transgene were inoculated with peptone trypsin broth, H. pylori SS1 or SS1-rdxA(-), an SS1-derived mutant in rdxA. Twelve weeks after inoculation, the mice were treated for 7 days with a control solution or with the mouse equivalent of a human dose of metronidazole 1 g od. Three weeks after completion of treatment, the animals were killed and mutations in the target lacI gene assessed by a transgenic mutagenesis assay system. There was no increase in lacI mutations in cells harvested from mice infected with H. pylori and/or exposed to metronidazole. These data suggest that short-term infection with H. pylori and exposure to metronidazole does not enhance the mutation frequency in the gastric cells of mice. Whether chronic infection and/or repeated exposure to metronidazole or other nitroaromatic compounds causes genetic damage to gastric epithelial cells remains to be determined.

Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori.

We previously established a monoclonal antibody (MAb), designated H9, which reacts with the heat shock protein 60 (HSP60) homologue of Helicobacter pylori as well as with other bacterial and human HSP60s. To determine the importance of a cross-reactive epitope on H. pylori HSP60 in H. pylori immunopathogenesis, we performed (i) mapping of an epitope on H. pylori HSP60 recognized by the H9 MAb, (ii) analysis of immunoglobulin G responses of patients with or without H. pylori infection to its epitope region, and (iii) studies of the protective effect of immunization with its epitope region on H. pylori infection in mice. The epitope recognized by the H9 MAb was mapped to the sequence of amino acids 189 to 203 (VEGMQFDRGYLSPYF) on the H. pylori HSP60 molecule. It was confirmed that the synthesized peptide designated pH9 was recognized by the H9 MAb. Enzyme-linked immunosorbent assay analysis showed that patients with H. pylori infection (n = 349) had significantly lower titers of pH9 antibody than did uninfected patients (n = 200) (P < 0.001), but this was not the case with purified H. pylori HSP60 recombinant Escherichia coli GroEL, or recombinant human HSP60. In C57BL/6 mice immunized with the pH9 peptide with Freund's complete adjuvant (FCA), the number of H. pylori organisms colonizing the stomach was significantly lower than that in mice immunized with pCont plus FCA (P < 0.0001) or FCA only (P < 0.005). The results suggest that the immune response to the cross-reactive epitope (pH9 region) on H. pylori HSP60 is unique and might be associated with protection against H. pylori infection.

Immune response to an 18-kilodalton outer membrane antigen identifies lipoprotein 20 as a Helicobacter pylori vaccine candidate.

Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.

Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection.

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.

The effect of sofalcone on indomethacin‐induced gastric ulcers in a Helicobacter pylori‐infected gnotobiotic murine model

Sofalcone has been reported to exert anti-ulcer and gastroprotective actions, but its exact mechanism of action remains unknown. In our laboratory, we found that indomethacin-induced gastric ulcers become worse when associated with Helicobacter pylori infection. We employed the H. pylori-infected gnotobiotic murine model to examine the effect of sofalcone on indomethacin-induced gastric ulcers in the presence of H. pylori infection. In vitro experiments were also done to evaluate the effects of sofalcone on H. pylori growth, adherence of H. pylori to the MKN45 cells (a human gastric epithelial cell line), and these cells' IL-8 production in the presence of H. pylori. We found that sofalcone produced a significant improvement in ulcer size as well as a substantial reduction in the number of H. pylori colonies in H. pylori-infected gnotobiotic mice. In vitro sofalcone has a significant bacteriocidal effect against H. pylori and can also significantly prevent adherence of this bacterium to MKN45 cells, thus remarkably reducing IL-8 production of these cells in response to stimulation by H. pylori. Our results suggest that sofalcone can improve ulcer healing by the mechanisms mentioned above.

Induction of follicular gastritis following postthymectomy autoimmune gastritis in Helicobacter pylori-infected BALB/c mice.

Helicobacter pylori is the major causative agent of chronic antral gastritis and is thought to be involved in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (MALToma) developing in the human stomach. The aim of this study was to clarify whether corporal autoimmune gastritis (AIG), which is known to decrease acidity due to destruction of parietal cells, predisposes mice to H. pylori infection, thereby leading to MALToma-like pathology. BALB/c mice in which AIG had been induced by thymectomy 3 days after birth (AIG mice) were used. The AIG mice were orally administered mouse-adapted H. pylori at the age of 6 weeks and were examined histologically and serologically after 2 to 12 months. The results were compared with those obtained from uninfected AIG mice and infected normal mice. Germinal centers were induced in the corpus in 57% of the H. pylori-infected AIG mice, which elicited anti-H. pylori antibody responses in association with upregulation of interleukin-4 (IL-4) mRNA. In these mice, parietal cells remained in the corpus mucosa. These findings were in contrast to those with the uninfected AIG mice: fundic gland atrophy due to disappearance of parietal cells associated with upregulation of gamma interferon, but not IL-4, mRNA and no germinal center formation in the corpus. These observations suggest that AIG alters the infectivity of H. pylori, leading to MALToma-like follicular gastritis, at an early stage after H. pylori infection.

Factors affecting the validity of the 13C-urea breath test for in vivo determination of Helicobacter pylori infection status in a mouse model.

The mouse model using a human isolate of Helicobacter pylori is being widely accepted as an economical means of studying gastric infection. A noninvasive monitoring method would be useful for repeated testing to establish the time course of infection and the efficacy of treatments. In this study, we describe factors that affected interpretation of 13C urea breath test results for the assessment of H. pylori infection status in this model. Female C57B1/6 mice that underwent gavage with H. pylori or saline were breath-tested using 50 micrograms of 13C urea at intervals up to 2 months after inoculation. The generation of 13CO2 (excess delta 13CO2) by infected mice was compared to that of uninfected controls. The effects of diet, fasting, and coprophagy on the reliability of the 13C urea breath test were quantitated. Both commercial and synthetic mouse diets exhibited marked in vitro urease activity. A minimum fasting time of 13 hours prior to breath testing significantly reduced this dietary contribution to excess delta 13CO2 values. The coprophagic tendency of the mice caused spuriously high excess delta 13CO2 counts in the breath of both control and H. pylori-infected mice. Although the dietary contribution to spuriously high values of excess delta 13CO2 in mice breath-tested for H. pylori infection was reduced by fasting, the high nonspecific urease activity generated by coprophagy severely limited the reliability of the urea breath test in the assessment of H. pylori infection status.

Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes

Helicobacter pylori is a Gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes. In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma. The pathogenesis is in part caused by the immunological response. In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes. Mucosal T-lymphocytes are producing IFN-γ thus increasing mucosal inflammation and mucosal damage. A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H. pylori by humans. Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H. pylori infected mice. Antioxidants, including carotenoids, have anti-inflammatory effects. The aim of the present study was to investigate whether dietary antoxidant induced modulation of H. pylori in mice affected the cytokines produced by H. pylori specific T-cells. We found that treatment of H. pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation. These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-γ to a Th1/Th2-response with IFN-γ and IL-4. To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.

Murine splenocytes induce severe gastritis and delayed-type hypersensitivity and suppress bacterial colonization in Helicobacter pylori-infected SCID mice.

The goal of this study was to evaluate the role of host immunity in gastritis and epithelial damage due to Helicobacter pylori. Splenocytes from H. pylori-infected and uninfected C57BL/6 mice were adoptively transferred to H. pylori-infected and uninfected severe combined immunodeficient (SCID) mice. Transfer was verified by flow cytometry, and all mice were evaluated for the presence of delayed-type hypersensitivity (DTH) by footpad inoculation with sterile H. pylori sonicate and for humoral immunity by enzyme-linked immunosorbent assay. The severity of gastritis and gastric epithelial damage was quantified histologically, epithelial proliferation was determined by proliferating cell nuclear antigen staining, and colonization was quantified by culture. C57BL/6 mice, but not nonrecipient SCID mice, developed moderate gastritis in response to H. pylori. In contrast, recipient SCID mice developed severe gastritis involving 50 to 100% of the gastric mucosa and strong DTH responses not present in C57BL/6 mice. DTH, but not serum anti-H. pylori immunoglobulin G, correlated with adoptive transfer, gastritis, and bacterial clearance. Severe gastritis, but not bacterial colonization, was associated with epithelial metaplasia, erosions, and an elevated labeling index. This study demonstrates that (i) adaptive immunity is essential for development of gastritis due to H. pylori in mice, (ii) T-cell-enriched lymphocytes in SCID mice induce DTH and gastritis, which is more severe than donor gastritis, and (iii) the host inflammatory response, not direct bacterial contact, causes epithelial damage. The greater severity of gastritis in recipient SCID mice than in donor C57BL/6 mice suggests that gastritis is due to specific T-cell subsets and/or the absence of regulatory cell subsets in the transferred splenocytes.

The Authors Respond to Dr. Taylor

We concur overall with Taylor's presentation of what is known about the dynamics of Helicobacter pylori infection in childhood (1). but we would like to emphasize that many more questions than answers remain. Because H. pylori infection is not normally detectable at onset, information about incidence must be inferred from age-specific prevalence or repeated screening for prevalent infection over time (2). The infection does not appear to confer lasting immunity, and currently, there is no way to determine whether uninfected individuals have been infected in the past (3). Thus, the average duration of naturally acquired infections and the proportion that are self-limiting is not known for any population or age group. Few studies have examined the infection status of asymptomatic individuals over time. Prospective studies that observe infection status at frequent time intervals have been particularly rare; thus, detailed information regarding the natural history of the infection, peak age ranges for acquisition, and factors that may differentially influence acquisition, persistence, and recurrence is limited. Taylor speculates that antibiotics and characteristics of H. pylori strains may account for spontaneous elimination of infection in children. Emerging evidence suggests that factors that influence the ecology of the gastric niche or the immune response may also influence persistence. In recent laboratory studies, feeding H. pylori-infected mice algae meals with a high antioxidant concentration induced immune changes associated with reduced bacterial load (4). This finding complements an observed inverse association between consumption of fruits and vegetables and H. pylori prevalence in Colombian children (5). We would expect the age-related peaks in acquisition and elimination to vary considerably across popu-

Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice.

The Helicobacter pylori SS1 mouse model was used to characterize the development of resistance in H. pylori after treatment with metronidazole monotherapy and to examine the effect of prior exposure to metronidazole on the efficacy of a metronidazole-containing eradication regimen. Mice colonized with the metronidazole-sensitive H. pylori SS1 strain were treated for 7 days with either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole once a day or three times per day (TID). In a separate experiment, H. pylori-infected mice were administered either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole TID for 7 days, followed 1 month later by either peptone trypsin broth or the mouse equivalent of 20 mg of omeprazole, 250 mg of clarithromycin, and 400 mg of metronidazole twice a day for 7 days. At least 1 month after the completion of treatment, the mice were sacrificed and their stomachs were cultured for H. pylori. The susceptibilities of isolates to metronidazole were assessed by agar dilution determination of the MICs. Mixed populations of metronidazole-resistant and -sensitive strains were isolated from 70% of mice treated with 400 mg of metronidazole TID. The ratio of resistant to sensitive strains was 1:100, and the MICs for the resistant strains varied from 8 to 64 micrograms/ml. In the second experiment, H. pylori was eradicated from 70% of mice treated with eradication therapy alone, compared to 25% of mice pretreated with metronidazole (P < 0.01). Mice still infected after treatment with metronidazole and eradication therapy contained mixed populations of metronidazole-resistant and -sensitive isolates in a ratio of 1:25. These results demonstrate that H. pylori readily acquires resistance to metronidazole in vivo and that prior exposure of the organism to metronidazole is associated with failure of eradication therapy. H. pylori-infected mice provide a suitable model for the study of resistance mechanisms in H. pylori and will be useful in determining optimal regimens for the eradication of resistant strains.

Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.

Helicobacter pylori infection in humans is a major risk factor for peptic ulcer, but studies on the relation between H. pylori infection and gastric pathology are limited due to a deficiency of convenient animal models resembling this infection in humans. We studied the effects of inoculation of conventional BALB/c mice with CagA and VacA positive (type I) H. pylori or CagA and VacA negative H. pylori (type II) strains on gastric secretion and healing of chronic acetic acid-induced ulcers in mouse stomachs. The ulcer area, gastric blood flow, plasma interleukin (IL)-1beta and IL-12, as well as plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy samples were also taken for assessment of the presence of viable H. pylori using a rapid urease test, H. pylori-culture and the RT-PCR analysis of the signal for H. pylori CagA. Gastric acid and pepsin secretion was reduced by over 50% immediately after H. pylori inoculation and accompanied by a significant increment in plasma gastrin and fall in gastric luminal somatostatin content observed over all test days, particularly in mice infected with type I H. pylori. The area of ulcers in vehicle-treated controls decreased significantly starting from day 2 after ulcer induction and then continued to decline for a further 14 days to heal almost completely after 28 days. In contrast, the ulcers were present until day 28 in all mice infected with type I or type II H. pylori strains, being significantly larger, especially with type I H. pylori infection. The gastric blood flow at the ulcer margin and ulcer crater in vehicle-treated mice gradually increased with decreasing ulcer size, after 14 and 28 days reaching a value which was not significantly different from that in vehicle-administered mice. In contrast, the gastric blood flow in type I H. pylori and, to a lesser extent, in type II H. pylori infected mice was significantly lower than in vehicle controls, both at the margin and at the crater of ulcers at all tested days. Histological changes such as oedema or congestion of surface epithelium were found after 7 days whereas mucosal inflammatory infiltration appeared after 14 days with a further increase after 28 days, especially in type I H. pylori and to a lesser extent in type II H. pylori infected mice. Plasma IL-1beta and IL-12 were significantly elevated at all tested days of ulcer healing and their increments were significantly higher in type I than in type II H. pylori infection. Conventional mice with gastric ulcers can be successfully infected by both toxigenic and nontoxigenic H. pylori strains, and this infection causes an immediate suppression of gastric secretion and markedly delays the healing of ulcers due to the fall in mucosal microcirculation in the ulcer region, cytokine release and an impairment in the gastrin-somatostatin link that appears to be independent of gastritis and more pronounced with infection of toxigenic than nontoxigenic strains.