Charles A. Dangler

Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.

BACKGROUND & AIMS Hypergastrinemia occurs frequently in association with acid suppression and Helicobacter infection, but its role in the progression to gastric atrophy and gastric cancer has not been well defined. METHODS The effects of hypergastrinemia, and possible synergy with Helicobacter felis infection, were investigated in insulin-gastrin (INS-GAS) transgenic mice. RESULTS INS-GAS mice initially showed mild hypergastrinemia, increased maximal gastric acid secretion, and increased parietal cell number but later progressed to decreased parietal cell number and hypochlorhydria. Development of gastric atrophy was associated with increased expression of growth factors, heparin-binding epidermal growth factor and transforming growth factor alpha. At 20 months of age, INS-GAS mice showed no evidence of increased enterochromaffin-like cell number, but instead exhibited gastric metaplasia, dysplasia, carcinoma in situ, and gastric cancer with vascular invasion. Invasive gastric carcinoma was observed in 6 of 8 INS-GAS mice that were >20 months old. Helicobacter felis infection of INS-GAS mice led to accelerated (< or = 8 mo) development of intramucosal carcinoma (85%), with submucosal invasion (54%) and intravascular invasion (46%; P < or = 0.05). CONCLUSIONS These findings support the unexpected conclusion that chronic hypergastrinemia in mice can synergize with Helicobacter infection and contribute to eventual parietal cell loss and progression to gastric cancer.

Concurrent enteric helminth infection modulates inflammation and gastric immune responses and reduces helicobacter-induced gastric atrophy.

Helicobacter pylori is causally associated with gastritis and gastric cancer. Some developing countries with a high prevalence of infection have high gastric cancer rates, whereas in others, these rates are low. The progression of helicobacter-induced gastritis and gastric atrophy mediated by type 1 T-helper cells may be modulated by concurrent parasitic infection. Here, in mice with concurrent helminth infection, helicobacter-associated gastric atrophy was reduced considerably despite chronic inflammation and high helicobacter colonization. This correlated with a substantial reduction in mRNA for cytokines and chemokines associated with a gastric inflammatory response of type 1 T-helper cells. Thus, concurrent enteric helminth infection can attenuate gastric atrophy, a premalignant lesion.

Processing and proliferative effects of human progastrin in transgenic mice.

Incompletely processed gastrins have been postulated to play a role in growth of the gastrointestinal tract, but few studies have examined the effects of progastrin on mucosal proliferation in vivo. Human gastrin gene expression and progastrin processing were therefore studied in transgenic mice containing a human gastrin (hGAS) minigene, and compared to processing in mice bearing an insulin gastrin (INS-GAS) transgene that overexpresses amidated gastrin. Progastrin processing was studied using region-specific antisera and radioimmunoassays, biosynthetic labeling, immunoprecipitation, and HPLC. Proliferative effects due to overexpression of processed and unprocessed gastrin in INS-GAS and hGAS mice, respectively, were determined using routine histology and BrdU incorporation. The pancreatic islets of INS-GAS mice were able to produce carboxyamidated G-17, resulting in a twofold elevation of serum amidated gastrin, marked thickening of the oxyntic mucosa, and an increased BrdU labeling index (LI) of the gastric body. In contrast, livers of adult hGAS mice expressed abundant human gastrin mRNA and human progastrin but were unable to process this peptide to the mature amidated form, resulting in markedly elevated serum progastrin levels and normal amidated gastrin levels. Nevertheless, there was a marked increase in the BrdU labeling index of the colon in hGAS mice (LI 7.46+/-1.90%), as well as in INS-GAS mice (LI 6.16+/-1.17%), compared to age-matched, wild type control mice (LI 4.01+/-0.98%, P < 0.05). These studies suggest that incompletely processed gastrin precursors may contribute to colonic mucosal proliferation in vivo.

Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection

BACKGROUND & AIMS Infection with Helicobacter pylori uniformly leads to a chronic superficial gastritis that may progress to atrophic gastritis, a premalignant process. A mouse model of Helicobacter felis infection was used to study possible genetic determinants of the response to infection. METHODS Three inbred mouse strains with known secretory phospholipase A2 (sPLA2) genotypes [BALB/c (+/+), C3H/HeJ (+/+), and C57BL/6 (-/-)] were orally infected with H. felis and examined longitudinally using routine histology, immunocytochemistry, electron microscopy, proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and Northern and Western blot studies. RESULTS Only the C57BL/6 strain showed increased gastric fundic proliferation and apoptosis in response to infection. In addition, the C57BL/6 mouse showed a marked loss of parietal and chief cells, along with a marked expansion of an aberrant gastric mucous cell lineage that stained positive for spasmolytic polypeptide. In contrast, no significant change in these cell types was observed in BALB/c and C3H/HeJ strains. Increased expression of sPLA2 was observed in BALB/c and C3H/HeJ after H. felis infection, whereas sPLA2 expression was absent in C57BL/6 mice. CONCLUSIONS H. felis infection leads to increased apoptosis and altered cellular differentiation in the C57BL/6 mouse, a strain that lacks gastric sPLA2 expression. Because sPLA2 has been identified recently as the MOM1 (modifier of MIN) locus that influences polyp formation in the colon, these studies suggest that sPLA2 may also influence the gastric epithelial response to Helicobacter infection.

Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation

Gastrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Glys contribute to colonic mucosal proliferation in vivo.

Host and microbial constituents influence helicobacter pylori-induced cancer in a murine model of hypergastrinemia

BACKGROUND & AIMS Helicobacter pylori cag(+) strains and high-expression host interleukin 1beta (IL-1beta) polymorphisms augment the risk for intestinal-type gastric adenocarcinoma, a malignancy that predominates in males. We examined the effects of an H. pylori cancer-associated determinant (cagE), IL-1beta, and host gender in a transgenic hypergastrinemic (INS-GAS) murine model of gastric carcinogenesis. METHODS Male and female INS-GAS mice infected with wild-type H. pylori, an H. pylori cagE(-) mutant, or H. felis were killed 2-24 weeks postchallenge. Gastric injury was scored from 0 to 4, and mucosal IL-1beta levels were quantified by ELISA. RESULTS Male INS-GAS mice infected with H. pylori uniformly developed atrophy, intestinal metaplasia, and dysplasia by 6 weeks and carcinoma by 24 weeks. Mucosal IL-1beta concentrations increased 12 weeks following Helicobacter challenge, but levels then decreased by 24 weeks. Inactivation of cagE delayed the progression to carcinoma, but neoplasia ultimately developed in all males infected with the H. pylori mutant. In contrast, none of the H. pylori-infected female mice developed cancer, and injury scores, but not IL-1beta levels, were significantly higher in males compared with females. CONCLUSIONS H. pylori infection induces gastric adenocarcinoma in an experimental mouse model of disease. Cancer is restricted to males and loss of cagE temporally retards but does not abrogate pathologic progression. Mucosal levels of IL-1beta increase prior to the development of gastric cancer but are not related to gender. The INS-GAS model is effective for investigating discrete host-microbial interactions that culminate in gastric cancer within the context of biologic conditions induced by H. pylori.

Cutting Edge: Typhlocolitis in NF-κB-Deficient Mice

Activation of inflammatory gene expression by the transcription factor NF-κB is a central pathway in many inflammatory disorders, including colitis. Increased NF-κB activity has been linked with development of colitis in humans and animal models, thus it was unexpected when NF-κB-deficient mice developed spontaneous typhlocolitis. To further characterize this finding, we induced typhlocolitis in rederived NF-κB-deficient mice using intragastric infection with Helicobacter hepaticus. At 6 wk postinfection (PI), severe colitis with increased type 1 cytokine expression was seen in infected mice that lacked the p50 subunit of NF-κB and were also heterozygous for the p65 subunit of NF-κB(p50−/−p65+/−). Mice lacking the p50 subunit alone (p50−/−) were less severely affected, and wild-type mice and p65+/− mice were unaffected. T cell development in NF-κB-deficient mice was normal. These data indicate that p50 and p65 subunits of NF-κB have an unexpected role in inhibiting the development of colitis.

Novel Helicobacter species isolated from rhesus monkeys with chronic idiopathic colitis.

Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys. Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea. Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses. Colonic tissues were cultured for Salmonella spp. and Shigella spp.; all results were negative. Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative. The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV. Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture. Micro-aerobic cultures from all eight monkeys incubated at 37 degrees C and 42 degrees C revealed pinpoint or spreading colonies on antibiotic-containing media. Bacteria were identified as gram-negative, oxidase positive and urease negative. Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified. One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin. Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp. By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella. This is the first report describing the isolation of novel Helicobacter spp. from inflamed colons of rhesus monkeys. Studies are needed to determine whether these novel Helicobacter spp. play a causal role in the initiation and progression of chronic colitis in macaques. Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man.

Mice lacking inducible nitric oxide synthase develop spontaneous hypercholesterolaemia and aortic atheromas

Nitric oxide (NO) has been implicated in various aspects of the atherogenic process and has been shown to possess both protective and cytotoxic properties. Recently, increased expression of inducible nitric oxide synthase (iNOS) has been detected in atherosclerotic lesions, although there is no consensus as to its pathogenetic significance [1,2]. In this longitudinal study we show that iNOS plays an important protective role in the atherogenic process. Indirect systolic blood pressure was measured by photoplethysmography in unanesthetized mice fed either a basal or high salt diet, and found to be significantly higher in iNOS-deficient mice than in wild type controls at three months of age (P=0.038 (basal diet) and P=0.0005 (high salt diet)). In addition, relative to controls, the iNOS-deficient mice had significantly elevated serum cholesterol levels at 3, 9 and 12 months of age (P=0.0017, 0.0001 and 0.0002 for the respective ages) as well as a significantly higher incidence of atherosclerotic plaques. These findings suggest that iNOS targeted mutant mice, historically used as an animal model to investigate the role of nitric oxide in the inflammatory response [3,4], may also serve as a model for the study of cholesterol homeostasis and atherogenesis.

Isolation and Characterization of a Helicobacter sp. from the Gastric Mucosa of Dolphins, Lagenorhynchus acutus and Delphinus delphis

ABSTRACT Gastric ulcerations in dolphins have been reported for decades. Some of these lesions were associated with parasitic infections. However, cases of nonparasitic gastric ulcers with no clearly defined etiology also have been reported in wild and captive dolphins. Considerable speculation exists as to whether dolphins haveHelicobacter-associated gastritis and peptic ulcer disease. The stomachs of seven stranded Atlantic white-sided dolphins,Lagenorhynchus acutus, and 1 common dolphin,Delphinus delphis, were assessed for the presence ofHelicobacter species. Novel Helicobacterspecies were identified by culture in the gastric mucosa of two of the eight dolphins studied and by PCR in seven of the eight dolphins. The gram-negative organisms were urease, catalase, and oxidase positive. Spiral to fusiform bacteria were detected in gastric mucosa by Warthin Starry staining. Histopathology revealed mild to moderate diffuse lymphoplasmacytic gastritis within the superficial mucosa of the main stomach. The pyloric stomach was less inflamed, and bacteria did not extend deep into the glands. The lesions parallel those observed inHelicobacter pylori-infected humans. Bacteria from two dolphins classified by 16S rRNA analysis clustered with gastric helicobacters and represent a novel Helicobacter sp. most closely related to H. pylori. These findings suggest that a novel Helicobacter sp. may play a role in the etiopathogenesis of gastritis and gastric ulcers in dolphins. To our knowledge this represents the first isolation and characterization of a novel Helicobacter sp. from a marine mammal and emphasizes the wide host distribution and pathogenic potential of this increasingly important genus.