Test of conditions that affect in vitro production of volatile fatty acids and gases.

In vitro methods have been developed to measure digestibility, but such methods may not accurately reflect gas production or volatile fatty acid (VFA) profiles. The objective of this study was to determine the effect of different in vitro conditions on VFA and gas production. The experimental design was a 4 × 2 × 2 factorial CRD with four replicates. Treatments were four ratios of medium to rumen fluid by volume (5:95, 25:75, 50:50, and 75:25), two concentrations (w/v) of added timothy hay (0.5% or 1%), with or without added sodium acetate (increased initial concentration by 50 mM). Total volume of medium and rumen fluid was 10 mL per tube. Measurements of gas production and VFA were recorded at 0, 4, 16, 24, and 48 h. Statistical analyses used a mixed model including all fixed effects and interactions with tube as a random effect, and time nested within tube. Total gas production increased (P < 0.001) with higher medium proportion. The final pH increased (P < 0.0001) as medium proportion increased. Medium proportion positively affected (P < 0.05) overall average concentration of both acetate production and propionate production. Higher hay concentration increased (P < 0.0001) total gas produced from 0 to 48 h, increased total acetate production (P < 0.01), propionate production (P < 0.001), and decreased pH between 24 and 48 h (P < 0.0001). Sodium acetate addition increased (P < 0.0001) pH between 24 and 48 h. Acetate:propionate (A:P) concentration decreased over time (P < 0.0001). Initial rumen fluid A:P ratio was 3.7 but average A:P ratio of produced VFA started at 2.2 and increased to 2.50 (SE = ±0.51). The A:P ratio differed for VFA produced in vitro compared to initial rumen fluid, but no tested treatments were found to change A:P ratio.

Elevated IL-33 expression is associated with pediatric eosinophilic esophagitis, and exogenous IL-33 promotes eosinophilic esophagitis development in mice.

We tested whether the T helper (Th) type 2 (Th2) cell agonist and allergenic ligand IL-33 was associated with eosinophilic esophagitis (EoE) development in a pediatric cohort and whether IL-33 protein could induce disease symptoms in mice. Biopsies from EoE patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast cells, and epithelial cell markers near blood vessels. Circulating levels of IL-33 were not increased. The time course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model and chronicity of IL-33 expression has been demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 wk and esophageal pathology was evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and upregulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, along with innate lymphoid cell group 2, Th1/17, and M2 macrophage marker genes, were increased after IL-33 application. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with pediatric EoE development and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

Cytokine signalling via gp130 in gastric cancer.

Cytokine signalling pathways that depend on gp130 are dysregulated in several epithelial cancers including gastric cancer. It has been established that blockade of SHP2 activation of MAPK signalling results in hyperactivation of STAT3 resulting in increased cell proliferation, angiogenesis, inflammation and inhibition of both immunocyte and epithelial cell apoptosis. Additionally, key genes regulated downstream of gp130 via MAPK activation such as the stomach-specific tumor suppressor gene tff1 are suppressed, contributing to the oncogenic outcome. The main cytokine driver of gp130 signalling in the stomach is IL-11, with IL-6 having little activity in the antral stomach in which most pathology initiates. IL-11 is up-regulated in both mouse and human gastric cancer and in pre-neoplastic mucosa. A characteristic gene signature specifically associated with IL-11 drive has been observed, although the prognostic value of the signature has not yet been assessed. Infection of human or mouse stomach with Helicobacter pylori, especially that expressing the CagA cytotoxin, produces constitutive MAPK activation, but also activated STAT3 and increases IL-11 expression. The possibility of designing and utilising small molecule inhibitors of either IL-11 or STAT3 activation may be worthwhile in developing new cancer therapeutics.

Augmented gp130-mediated cytokine signalling accompanies human gastric cancer progression.

H. pylori infection accounts for most cases of gastric cancer, but the initiating events remain unclear. The principal H. pylori pathogenicity-associated CagA protein disrupts intracellular SHP-2 signalling pathways including those used by the IL-6 family cytokines, IL-6 and IL-11. Imbalanced IL-6 family cytokine signalling in the gp130(757FF) mouse model of gastric cancer arising from hyperactivation of oncogenic STAT3 after altered SHP-2 : ERK1/2 signalling produces dysplastic antral tumours preceded by gastritis and metaplasia. In a cohort of patient gastric biopsies with known H. pylori and CagA status, we investigated whether (i) STAT3 and ERK1/2 activation is altered in H. pylori-dependent gastritis; (ii) these profiles are more pronounced in CagA+ H. pylori infection; and (iii) the expression of pro-inflammatory cytokines that activate STAT3 and ERK 1/2 pathways is associated with progression to gastric cancer. IL-6, IL-11, and activated STAT3 and ERK1/2 were quantified in antral biopsies from gastritic stomach, metaplastic tissue, and resected gastric cancer tissues. We observed significantly increased STAT3 and ERK1/2 activation (p = 0.001) in H. pylori-dependent gastritis, which was further enhanced in the presence of CagA+ H. pylori strains. Of known gastric ligands that drive STAT3 activation, IL-6 expression was increased after H. pylori infection and both IL-6 and IL-11 were strongly up-regulated in the gastric cancer biopsies. This suggests a mechanism by which IL-11 drives STAT3 activation and proliferation during gastric cancer progression. We addressed this using an in vitro approach, demonstrating that recombinant human IL-11 activates STAT3 and concomitantly increases proliferation of MKN28 gastric epithelial cells. In summary, we show increased STAT3 and ERK1/2 activation in H. pylori-dependent gastritis that is likely driven in an IL-6-dependent fashion. IL-11 expression is associated with adenocarcinoma development, but not gastritic lesions, and we identify a novel mechanism for IL-11 as a potent inducer of proliferation in the human gastric cancer setting.

Differentiation of the Gastric Mucosa IV. Role of trefoil peptides and IL-6 cytokine family signaling in gastric homeostasis.

Gastric trefoil peptides mediate mucosal repair by stimulating cell migration, inhibiting apoptosis and inflammation, and likely augmenting the barrier function of mucus. One of these, tff1, is a gastric-specific tumor suppressor gene, which when repressed is associated with gastric cancer progression. IL-6 family cytokines play an important role in maintaining gastric homeostasis by regulating tff1 and other mediators of mucosal proliferation, inflammation, angiogenesis, and apoptosis. In this review the signaling cascades downstream of the common IL-6 cytokine family coreceptor gp130 that contribute to control of this homeostasis are described, as are the pathological outcomes of imbalancing these pathways.

Reciprocal changes in trefoil 1 and 2 expression in stomachs of mice with gastric unit hypertrophy and inflammation.

H+/K+-ATPase beta-subunit-deficient mice (129/Sv background) display numerous pathologies in the stomach. Expression of the mutation in BALB/cCrSlc mice results in the development of an aberrant 'mucus-rich' cell population. 'Mucus-rich' cells have been described in stomachs of mice with autoimmune gastritis, a disease mediated by CD4+ T cells. Other pathological features of autoimmune gastritis are similar to those in H+/K+ beta-deficient mice and include a mononuclear cell infiltrate in the gastric mucosa, non-functional or absent parietal cells, depletion of zymogenic cells, hypergastrinaemia, and gastric unit hypertrophy caused by immature cell hyperplasia. The present study investigates further the aberrant gastric 'mucus-rich' cell lineage and analyses the mRNA expression of mucus cell products TFF1 and TFF2. 'Mucus-rich' cells stained for both acidic and neutral mucins, and with a TFF2-specific antibody. Stomachs from both models expressed decreased TFF1 mRNA and reciprocally increased TFF2 mRNA. The involvement of gastrin in regulating trefoil mRNA expression was also investigated using gastrin-deficient mice. In contrast to previous findings, gastrin did not positively regulate TFF1 mRNA expression, but there was possible augmentation of TFF2. Additionally, a clear role for inflammation was established involving both polymorphonuclear and mononuclear cells in these models, and a link was found between mucosal hypertrophy and increased interleukin-11 (IL-11) expression.

Regulation of gastric epithelial cell development revealed in H(+)/K(+)-ATPase beta-subunit- and gastrin-deficient mice.

The gastric H(+)/K(+)-ATPase is essential for normal development of parietal cells. Here we have directly assessed the role of the H(+)/K(+)-ATPase beta-subunit (H/K-beta) on epithelial cell development by detailed quantitation of the epithelial cell types of the gastric mucosa of H/K-beta-deficient mice. H/K-beta-deficient mice had a 3.1-fold increase in the number of immature cells per gastric unit; however, the numbers of surface mucous and parietal cells were similar to those in the gastric units of wild-type mice. The effect of elevated gastrin levels in the H/K-beta-deficient mice was determined by producing mice that are also deficient in gastrin. We demonstrated that the increased production of immature cells and resulting hypertrophy is caused by the overproduction of gastrin. However, the depletion of zymogenic cells, which is another feature of H/K-beta-deficient mice, is independent of hypergastrinemia. Significantly, parietal cells of H/K-beta- and gastrin-deficient mice had abnormal secretory membranes and were devoid of resting tubulovesicular membranes. Together these data suggest a homeostatic mechanism limiting the number of immature cells that can develop into end-stage epithelial cells and indicate a direct role for H/K-beta in the development of mature parietal cells.

Requirements for autoimmune responses to mouse gastric autoantigens.

Autoimmune gastritis, in which the H+/K(+)-ATPase of parietal cells is the major antigen, is one of the most common autoimmune diseases. Here we examined if specific properties of the H+/K(+)-ATPase or parietal cells are involved in rendering them autoimmune targets. The model antigens beta-galactosidase and ovalbumin (OVA) were expressed in parietal cells of transgenic mice. On experimental induction of autoimmune gastritis by neonatal thymectomy, autoantibodies to beta-galactosidase developed in mice expressing beta-galactosidase in parietal cells, a response that was independent of either the response to the gastric H+/K(+)-ATPase or gastric inflammation. In contrast, mice that expressed OVA in parietal cells did not exhibit an antibody response to OVA after thymectomy. However, increasing the frequency of anti-OVA T lymphocytes in OVA-expressing mice resulted in autoantibodies to OVA and gastritis. These studies indicate that parietal cells can present a variety of antigens to the immune system. Factors such as the identity and expression level of the autoantigen and the frequency of autoreactive T cells play a role in determining the prevalence and outcome of the particular immune response. In addition, as not all mice of a particular genotype displayed autoimmunity, random events are involved in determining the target of autoimmune recognition.

Gastric H(+),K(+)-adenosine triphosphatase beta subunit is required for normal function, development, and membrane structure of mouse parietal cells.

BACKGROUND & AIMS: Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. METHODS: Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. RESULTS: The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. CONCLUSIONS: The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.

Autoimmune gastritis results in disruption of gastric epithelial cell development.

We have investigated the underlying basis of the lesion in murine autoimmune gastritis, a model of the human disease pernicious anemia. The disease is mediated by T lymphocytes and characterized by selective depletion of parietal and zymogenic cells from the gastric unit (gland) together with gastric epithelial cell hyperplasia. The gastric units of gastritic stomachs contained 2.3-fold more cells than normal and accumulated rapidly dividing, short-lived gastric epithelial stem cells and mucous neck cells. Most of these immature cells failed to differentiate into end-stage cells but rather appeared to die by apoptosis. We also found no correlation between anti-parietal cell autoantibody titers and the degree of gastric pathology, providing further evidence that autoantibodies do not play a direct role in the pathogenesis of gastritis. Taken together, the normal developmental pathways of the gastric mucosa are disrupted in autoimmune gastritis, resulting in an amplification of immature cell types. The differentiation of these immature cells appears to be blocked, contributing to depletion of end-stage cells. This scenario provides an explanation for depletion of not only parietal cells but also zymogenic cells even though they are not directly targeted by the immune system.