Postnatal inflammation in ApoE-/- mice is associated with immune training and atherosclerosis.

BACKGROUND AND AIMS: Preterm birth is associated with increased risk of cardiovascular disease (CVD). This may reflect a legacy of inflammatory exposures such as chorioamnionitis which complicate pregnancies delivering preterm, or recurrent early-life infections, which are common in preterm infants. We previously reported that experimental chorioamnionitis followed by postnatal inflammation has additive and deleterious effects on atherosclerosis in ApoE-/- mice. Here, we aimed to investigate whether innate immune training is a contributory inflammatory mechanism in this murine model of atherosclerosis. METHODS: Bone marrow-derived macrophages and peritoneal macrophages were isolated from 13-week-old ApoE-/- mice, previously exposed to prenatal intra-amniotic (experimental choriomanionitis) and/or repeated postnatal (peritoneal) lipopolysaccharide (LPS). Innate immune responses were assessed by cytokine responses following ex vivo stimulation with toll-like receptor (TLR) agonists (LPS, Pam3Cys) and RPMI for 24-h. Bone marrow progenitor populations were studied using flow cytometric analysis. RESULTS: Following postnatal LPS exposure, bone marrow-derived macrophages and peritoneal macrophages produced more pro-inflammatory cytokines following TLR stimulation than those from saline-treated controls, characteristic of a trained phenotype. Cytokine production ex vivo correlated with atherosclerosis severity in vivo. Prenatal LPS did not affect cytokine production capacity. Combined prenatal and postnatal LPS exposure was associated with a reduction in populations of myeloid progenitor cells in the bone marrow. CONCLUSIONS: Postnatal inflammation results in a trained phenotype in atherosclerosis-prone mice that is not enhanced by prenatal inflammation. If analogous mechanisms occur in humans, then there may be novel early life opportunities to reduce CVD risk in infants with early life infections.

Coordinate expression loss of GKN1 and GKN2 in gastric cancer via impairment of a glucocorticoid-responsive enhancer.

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression.NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.

Overexpression of IL-11 promotes premalignant gastric epithelial hyperplasia in isolation from germline gp130-JAK-STAT driver mutations.

Expression of the cytokine IL-11 is elevated in human Helicobacter pylori infection and progressively increases with worsening gastric pathology. Additionally, IL-11 is required for tumor development in STAT3-dependent murine models of gastric cancer (GC) and, when administered acutely, causes resolving atrophic gastritis. However, it is unclear whether locally elevated IL-11 ligand expression can, in isolation from oncogenic gp130-JAK-STAT pathway mutations, initiate GC pathogenesis. Here we developed a transgenic mouse model of stomach-specific (keratin 19 promoter) IL-11 ligand overexpression. Keratin 19 promoter-IL-11 transgenic ( K19-IL11Tg) mice showed specific IL-11 overexpression in gastric corpus and antrum but not elsewhere in the gastrointestinal tract or in other tissues. K19-IL11Tg mice developed spontaneous premalignant disease of the gastric epithelium, progressing from atrophic gastritis to TFF2-positive metaplasia and severe epithelial hyperplasia, including adenoma-like lesions in a subset of older (1 yr old) animals. Although locally advanced, the hyperplastic lesions remained noninvasive. H. pylori infection in K19-IL11Tg mice accelerated some aspects of the premalignant phenotype. Finally, K19-IL11Tg mice had splenomegaly in association with elevated serum IL-11, with spleens showing an expanded myeloid compartment. Our results provide direct in vivo functional evidence that stomach-specific overexpression of IL-11, in isolation from germline gp130-JAK-STAT3 genetic drivers, is sufficient for premalignant progression. These findings have important functional implications for human GC, in which frequent IL-11 overexpression occurs in the reported absence of somatic mutations in gp130 signaling components. NEW & NOTEWORTHY We provide direct in vivo functional evidence that stomach-specific overexpression of the cytokine IL-11, in isolation from gp130-JAK-STAT3 pathway mutations, can trigger spontaneous atrophic gastritis progressing to locally advanced epithelial hyperplasia (but not dysplasia or carcinoma), which does not require, but may be accelerated by, concomitant Helicobacter pylori infection.

Postnatal inflammation following intrauterine inflammation exacerbates the development of atherosclerosis in ApoE-/- mice.

Atherosclerosis is a chronic inflammatory disease that has its origins in early life. Postnatal inflammation exacerbates atherosclerosis, but the possible effect of intrauterine inflammation is largely unexplored. Exposure to inflammation in utero is common, especially in infants born preterm, who have increased cardiovascular risk in adulthood. We hypothesised that exposure to inflammation before birth would accelerate the development of atherosclerosis, with the most severe atherosclerosis following exposure to both pre- and postnatal inflammation. Here we studied the effect of prenatal and postnatal inflammation on the development of atherosclerosis by combining established techniques for modelling histological chorioamnionitis and atherosclerosis using apolipoprotein E (ApoE) knockout mice. A single intra-amniotic (IA) injection of lipopolysaccharide (LPS) caused intrauterine inflammation, and increased atherosclerosis at 13 weeks of postnatal age. In mice exposed to postnatal LPS, chorioamnionitis modulated subsequent responses; atherosclerotic lesion size, number and severity were greatest for mice exposed to both intrauterine and postnatal inflammation, with a concomitant decrease in collagen content and increased inflammation of the atherosclerotic plaque. In conclusion, pre- and postnatal inflammation have additive and deleterious effects on the development of atherosclerosis in ApoE knockout mice. The findings are particularly relevant to preterm human infants, whose gestations are frequently complicated by chorioamnionitis and who are particularly susceptible to repeated postnatal infections. Human and mechanistic studies are warranted to guide preventative strategies.

The MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome.

OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1ß and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1ß, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.

STAT3: a critical component in the response to Helicobacter pylori infection.

STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well placed to control inflammatory and oncogenic outcomes in the context of Helicobacter (H.) pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.

TFF2 deficiency exacerbates weight loss and alters immune cell and cytokine profiles in DSS colitis, and this cannot be rescued by wild-type bone marrow.

The trefoil factor TFF2 is a member of a tripartite family of small proteins that is produced by the stomach and the colon. Recombinant TFF2, when applied intrarectally in a rodent model of hapten colitis, hastens mucosal healing and reduces inflammatory indexes. Additionally, TFF2 is expressed in immune organs, supporting a potential immunomodulatory and reparative role in the bowel. In this study we confirm that TFF2 is expressed in the colon and is specifically enriched in epithelial cells relative to colonic leukocytes. TFF2-deficient, but not TFF1-deficient, mice exhibit a more severe response to acute or chronic dextran sulfate (DSS)-induced colitis that correlates with a 50% loss of expression of TFF3, the principal colonic trefoil. In addition, the response to acute colitis is associated with altered expression of IL-6 and IL-33, but not other inflammatory cytokines. While TFF2 can reduce macrophage responsiveness and block inflammatory cell recruitment to the colon, the major role in limiting the susceptibility to acute colitis appears to be maintenance of barrier function. Bone marrow transfer experiments demonstrate that leukocyte expression of TFF2 is not sufficient for prevention of colitis induction but, rather, that the gastrointestinal epithelium is the primary source of TFF2. Together, these findings illustrate that epithelial TFF2 is an important endogenous regulator of gut mucosal homeostasis that can modulate immune and epithelial compartments. Because of its extreme stability, even in the corrosive gut lumen, TFF2 is an attractive candidate as an oral therapeutic scaffold for future drug development in the treatment of inflammatory bowel disease.

Perinatal inflammation: a common factor in the early origins of cardiovascular disease?

Cardiovascular disease continues to be the leading cause of global morbidity and mortality. Traditional risk factors account for only part of the attributable risk. The origins of atherosclerosis are in early life, a potential albeit largely unrecognized window of opportunity for early detection and treatment of subclinical cardiovascular disease. There are robust epidemiological data indicating that poor intrauterine growth and/or prematurity, and perinatal factors such as maternal hypercholesterolaemia, smoking, diabetes and obesity, are associated with adverse cardiovascular intermediate phenotypes in childhood and adulthood. Many of these early-life risk factors result in a heightened inflammatory state. Inflammation is a central mechanism in the development of atherosclerosis and cardiovascular disease, but few studies have investigated the role of overt perinatal infection and inflammation (chorioamnionitis) as a potential contributor to cardiovascular risk. Limited evidence from human and experimental models suggests an association between chorioamnionitis and cardiac and vascular dysfunction. Early life inflammatory events may be an important mechanism in the early development of cardiovascular risk and may provide insights into the associations between perinatal factors and adult cardiovascular disease. This review aims to summarise current data on the early life origins of atherosclerosis and cardiovascular disease, with particular focus on perinatal inflammation.

Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

IL-11 is a parietal cell cytokine that induces atrophic gastritis.

BACKGROUND AND AIMS: IL-is important in gastric damage, mucosal repair and gastric cancer progression. We analysed IL-11 expression in H.pylori infected mouse stomach, the site of gastric IL-11 expression in mice and humans, and the effect of exogenous IL-11 on gastric mucosal homeostasis. METHODS: IL-11 protein was localised in mouse and human stomach. The impact of chronic, exogenous IL-11 on normal mouse stomach was examined histologically and transcriptionally by microarray, confirmed by mRNA and protein analysis. Functional impact of IL-11 on gastric acid secretion was determined. RESULTS: In mice infected with H.pylori, IL-11 was increased in fundic mucosa with temporal expression similar to IL-1b. IL-11 protein was localised predominantly to parietal cells in mouse and human stomach. Application of exogenous IL-11 to resulted in fundic parietal and chief cell loss, hyperplasia, mucous cell metaplasia and inflammation. Coincident with cellular changes were an increased gastric pH, altered parietal cell ultrastructure and altered gene expression, particularly genes involved in immune response and ion transport which could result in compromised acid secretion. We confirmed that a single dose of IL-11 effectively ablated the gastric response to histamine. CONCLUSIONS: IL-11 is a parietal cell cytokine that blocks gastric acid secretion, likely via reducing expression of parietal cell ion transport genes, CCKb and histamine H2 receptors. IL-11 expression is increased in H. pylori infected mouse stomach and treatment of wild type mice with IL-11 induced changes in the gastric fundic mucosa reminiscent of chronic atrophic gastritis, a precursor to gastric cancer.

Targeting Host Sulphonyl Urea Receptor 2 Can Reduce Severity of Helicobacter pylori Associated Gastritis.

Background and Aims: While most Helicobacter pylori-infected individuals remain asymptomatic throughout their lifetime, in a significant proportion, the resulting severe chronic gastritis drives the development of gastric cancer. In this study, we examine a new therapeutic target, a host potassium channel regulatory subunit, SUR2 (encoded by ABCC9), with potential to protect against H pylori-associated diseases. Methods: SUR2 gene (ABCC9) expression in human gastric biopsies was analyzed by quantitative polymerase chain reactions. Helicobacter-infected mice were administered the SUR2-channel agonists, pinacidil and nicorandil, then gastric tissues analyzed by histology, immunohistochemistry and quantitative polymerase chain reaction, and splenic tissues by enzyme-linked immunosorbent assays. In vitro studies were performed on human and mouse macrophages, human gastric epithelial cells and mouse splenocytes. Results: ABCC9 expression in human and mouse stomachs is downregulated with H pylori infection. Treatment of Helicobacter-infected mice with SUR2 channel modulators, pinacidil or nicorandil, significantly reduced gastritis severity. In gastric epithelial cells, nicorandil-induced opening of the SUR2 channel increased intracellular K+ and prevented H pylori-mediated Ca2+ influx and downstream pro-inflammatory signaling. Conclusion: SUR2 is a novel host factor that regulates Helicobacter pathogenesis. Pharmacological targeting of SUR2 provides a potential approach for reducing the severity of H pylori-associated gastritis, without eradicating infection.

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.

IL-33 promotes gastric tumour growth in concert with activation and recruitment of inflammatory myeloid cells.

Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130 F/F mouse model of GC. Expression of IL-33 (and it's cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130 F/F /Il33 -/- mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

Choroid plexus-derived extracellular vesicles exhibit brain targeting characteristics.

The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface.

A novel gastrokine, Gkn3, marks gastric atrophy and shows evidence of adaptive gene loss in humans.

BACKGROUND & AIMS: Gastrokines are stomach mucus cell-secreted proteins; 2 gastrokines are known, GKN1 and GKN2. Gastrokine expression is lost in gastric cancer, indicating a possible function in tumor suppression. We have identified a third gastrokine gene in mammals. METHODS: Gkn3 was characterized by studies of molecular structure, evolutionary conservation, and tissue expression as well as transcriptional/translational outcome in mouse genetic models of gastric pathology. The functional consequences of Gkn3 overexpression were evaluated in transfected cell lines. RESULTS: Gkn3 encodes a secreted (approximately 19 kilodalton) protein that is co-expressed with trefoil factor (Tff)2 in the distal stomach and discriminates a Griffinia simplicifolia lectin (GS)-II-positive mucus neck cell (MNC) subpopulation in the proximal stomach. In humans, widespread homozygosity for a premature stop codon polymorphism, W59X, has likely rendered GKN3 non-functional. Population genetic analysis revealed an ancestral GKN3 read-through allele that predominates in Africans and indicates the rapid expansion of W59X among non-Africans during recent evolution. Mouse Gkn3 expression is strongly up-regulated in (Tff2-deficient) gastric atrophy, a pre-cancerous state that is typically associated with Helicobacter pylori and marks a non-proliferative, GS-II positive lineage with features of spasmolytic polypeptide-expressing metaplasia (SPEM). Gkn3 overexpression inhibits proliferation in gastric epithelial cell lines, independently of incubation with recombinant human TFF2 or apoptosis. CONCLUSIONS: Gkn3 encodes a novel, functionally distinct gastrokine that is overexpressed and might restrain epithelial cell proliferation in gastric atrophy. Spread of the human GKN3 stop allele W59X might have been selected for among non-Africans because of its effects on pre-neoplastic outcomes in the stomach.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS: Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS: We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS: In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS: TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Chromosome-wide identification of novel imprinted genes using microarrays and uniparental disomies.

Genomic imprinting refers to a specialized form of epigenetic gene regulation whereby the expression of a given allele is dictated by parental origin. Defining the extent and distribution of imprinting across genomes will be crucial for understanding the roles played by imprinting in normal mammalian growth and development. Using mice carrying uniparental disomies or duplications, microarray screening and stringent bioinformatics, we have developed the first large-scale tissue-specific screen for imprinted gene detection. We quantify the stringency of our methodology and relate it to previous non-tissue-specific large-scale studies. We report the identification in mouse of four brain-specific novel paternally expressed transcripts and an additional three genes that show maternal expression in the placenta. The regions of conserved linkage in the human genome are associated with the Prader-Willi Syndrome (PWS) and Beckwith-Wiedemann Syndrome (BWS) where imprinting is known to be a contributing factor. We conclude that large-scale systematic analyses of this genre are necessary for the full impact of genomic imprinting on mammalian gene expression and phenotype to be elucidated.

Altered gp130 signalling ameliorates experimental colitis via myeloid cell-specific STAT3 activation and myeloid-derived suppressor cells.

STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130(757F/F) mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3(flox); gp130(757F/F) LysMcre/STAT3(flox)). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130(757F/F) mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines.

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells. METHODS: Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2'-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed. RESULTS: ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia. CONCLUSIONS: NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

Loss of gastrokine-2 drives premalignant gastric inflammation and tumor progression.

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.