Membrane-bound ß-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori-infected gastric cancer cells.

ß-catenin has two different cellular functions: intercellular adhesion and transcriptional activity. The E3 ubiquitin ligase Siah1 causes ubiquitin-mediated degradation of the cytosolic ß-catenin and therefore, impairs nuclear translocation and oncogenic function of ß-catenin. However, the effect of Siah1 on the cell membrane bound ß-catenin has not been studied. In this study, we identified that the carcinogenic bacterium H. pylori increased ETS2 transcription factor-mediated Siah1 protein expression in gastric cancer cells (GCCs) MKN45, AGS and Kato III. Siah1 protein level was also noticeably higher in gastric adenocarcinoma biopsy samples as compared to non-cancerous gastric epithelia. Siah1 knockdown significantly decreased invasiveness and migration of H. pylori-infected GCCs. Although, Siah1 could not increase degradation of the cytosolic ß-catenin and its nuclear translocation, it enhanced degradation of the membrane-bound ß-catenin in the infected GCCs. This loss of membrane-bound pool of ß-catenin was not associated with the proteasomal degradation of E-cadherin. Thus, this work delineated the role of Siah1 in increasing invasiveness of H. pylori-infected GCCs.

Review article: the global emergence of Helicobacter pylori antibiotic resistance.

BACKGROUND: Helicobacter pylori is one of the most prevalent global pathogens and can lead to gastrointestinal disease including peptic ulcers, gastric marginal zone lymphoma and gastric carcinoma. AIM: To review recent trends in H. pylori antibiotic resistance rates, and to discuss diagnostics and treatment paradigms. METHODS: A PubMed literature search using the following keywords: Helicobacter pylori, antibiotic resistance, clarithromycin, levofloxacin, metronidazole, prevalence, susceptibility testing. RESULTS: The prevalence of bacterial antibiotic resistance is regionally variable and appears to be markedly increasing with time in many countries. Concordantly, the antimicrobial eradication rate of H. pylori has been declining globally. In particular, clarithromycin resistance has been rapidly increasing in many countries over the past decade, with rates as high as approximately 30% in Japan and Italy, 50% in China and 40% in Turkey; whereas resistance rates are much lower in Sweden and Taiwan, at approximately 15%; there are limited data in the USA. Other antibiotics show similar trends, although less pronounced. CONCLUSIONS: Since the choice of empiric therapies should be predicated on accurate information regarding antibiotic resistance rates, there is a critical need for determination of current rates at a local scale, and perhaps in individual patients. Such information would not only guide selection of appropriate empiric antibiotic therapy but also inform the development of better methods to identify H. pylori antibiotic resistance at diagnosis. Patient-specific tailoring of effective antibiotic treatment strategies may lead to reduced treatment failures and less antibiotic resistance.

The stomach in health and disease.

The stomach is traditionally regarded as a hollow muscular sac that initiates the second phase of digestion. Yet this simple view ignores the fact that it is the most sophisticated endocrine organ with unique physiology, biochemistry, immunology and microbiology. All ingested materials, including our nutrition, have to negotiate this organ first, and as such, the stomach is arguably the most important segment within the GI tract. The unique biological function of gastric acid secretion not only initiates the digestive process but also acts as a first line of defence against food-borne microbes. Normal gastric physiology and morphology may be disrupted by Helicobacter pylori infection, the most common chronic bacterial infection in the world and the aetiological agent for most peptic ulcers and gastric cancer. In this state-of-the-art review, the most relevant new aspects of the stomach in health and disease are addressed. Topics include gastric physiology and the role of gastric dysmotility in dyspepsia and gastroparesis; the stomach in appetite control and obesity; there is an update on the immunology of the stomach and the emerging field of the gastric microbiome. H. pylori-induced gastritis and its associated diseases including peptic ulcers and gastric cancer are addressed together with advances in diagnosis. The conclusions provide a future approach to gastric diseases underpinned by the concept that a healthy stomach is the gateway to a healthy and balanced host. This philosophy should reinforce any public health efforts designed to eradicate major gastric diseases, including stomach cancer.

Synaptic deficits in layer 5 neurons precede overt structural decay in 5xFAD mice.

Synaptic decay and neurodegeneration are hallmarks of Alzheimer's disease that are thought to precede dementia. Recently, we have reported that the first signs of neuritic dystrophy in a new transgenic mouse model of familial Alzheimer's disease (FAD) called the "5xFAD" are axonal dystrophy followed by loss of spines on basal dendrites. The 5xFAD mouse has profound loss of layer 5 neurons by 12months, and these initial structural insults appear between 4 and 6months of age. Here, we test, for the first time, if synaptic failure of layer 5 neurons in the 5xFAD mouse precedes these structural changes. We used longitudinal, in vivo two-photon fluorescence imaging of bigenic 5xFAD/YFP mice to assess the overall structural stability of layer 5 neurons in young mice (age less than 14weeks). We found these neurons to be structurally and morphologically sound. In parallel, we used in vitro, whole-cell patch clamp electrophysiology of layer 5 pyramidal neurons, from mice aged 8-12weeks, to reveal significant pre- and postsynaptic defects in these cells. Thus our data suggest that layer 5 neurons in the 5xFAD mouse model have synaptic deficits at an early time point, before any overt structural dystrophy, and that such synaptic failure, with co-temporal biochemical changes, may be an early step in neuronal loss.

A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence.

Helicobacter pylori causes a lifelong infection and provides a model of bacterial adaptation and persistent colonization. Adenosine is an anti-inflammatory mediator that limits tissue damage during inflammation. We studied the role of adenosine in the T-cell-mediated regulation of gastritis and bacterial persistence. After 4 h of activation, human T helper (Th) cells increased A(2A) adenosine receptor (A(2A)AR) mRNA level (sevenfold). A(2A)AR was the predominant subtype expressed in resting and stimulated gastric or peripheral Th cells. Stimulation with ATL313, an A(2A)AR agonist, increased cyclic AMP (cAMP) accumulation and reduced interleukin-2 (IL-2) production by 20-50%. ATL313 also attenuated tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production, which was inhibited by an A(2A)AR antagonist. Infection of IL-10-deficient mice with H. pylori is cleared spontaneously due to the marked inflammation. Administration of ATL313 during infection reduced gastritis and pro-inflammatory cytokine responses while bacterial load increased. In contrast, infection of A(2A)AR-deficient mice enhanced gastritis. Thus, A(2A)AR limits the pro-inflammatory effects of Th cells and favor chronic Helicobacter infection.

Effectiveness of an experimental consensus phytase in improving dietary phytate-phosphorus utilization by weanling pigs.

Consensus phytase is a new biosynthetic, heat-stable enzyme derived from the sequences of multiple homologous phytases. Two experiments were conducted to determine its effectiveness, relative to inorganic P and a mutant enzyme of Escherichia coli phytase (Mutant-EP), in improving dietary phytate-P availability to pigs. In Exp. 1, 36 pigs (3 wk old, 7.00 +/- 0.24 kg of BW) were fed a low-P corn-soybean meal basal diet plus consensus phytase at 0, 250, 500, 750, 1,000, or 1,250 U/kg of feed for 5 wk. Plasma inorganic P concentration, plasma alkaline phosphatase activity, bone strength, and overall ADG and gain:feed ratio of pigs were improved (P < 0.05) by consensus phytase in both linear (R2 = 0.20 to 0.70) and quadratic (R2 = 0.30 to 0.70) dose-dependent fashions. In Exp. 2, 36 pigs (4 wk old, 9.61 +/- 0.52 kg BW) were fed the basal diet + inorganic P at 0.1 or 0.2%, consensus phytase at 750 or 450 U/kg of feed, Mutant-EP at 450 U/kg of feed, or 225 U consensus + 225 U Mutant-EP/kg of feed. Pigs fed 750 U of consensus phytase or 450 U of Mutant-EP/kg feed had plasma inorganic concentrations and bone strength that fell between those of pigs fed 0.1 or 0.2% inorganic P. These two measures were 16 to 29% lower (P < 0.05) in pigs fed 450 U of consensus phytase/kg of feed than those of pigs fed 0.2% inorganic P. Plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed Mutant-EP vs. consensus phytase at 450 U/kg at wk 2 and 3. In conclusion, the experimental consensus phytase effectively releases phytate P from the corn-soy diet for weanling pigs. The inorganic P equivalent of 750 U of consensus phytase/kg of feed may fall between 0.1 and 0.2%, but this requires further determination.

Helicobacter pylori infection as a model for gastrointestinal immunity and chronic inflammatory diseases.

Approximately 50% of humanity is infected with Helicobacter pylori. It is a life-long infection that elicits a marked host inflammatory response; however, natural infection fails to yield protective immunity. Rather than providing protection, the chronic inflammatory response associated with natural infection contributes to tissue damage and the pathogenesis of gastroduodenal disease, including atrophic gastritis, peptic ulcer, and gastric cancer. While bacterial factors are important triggers of inflammation, many subjects infected with strains bearing putative virulence factors remain free from disease. Recent genetic studies have implicated the host's immune and inflammatory responses, suggesting that disease results from an interaction between bacterial and environmental factors in genetically susceptible hosts. Other digestive diseases, including celiac disease and inflammatory bowel disease, mimic this paradigm, where it appears that luminal triggers only manifest disease in subjects with the right combination of host and environmental factors. Since infection with H. pylori is relatively common, it is possible to study the impact of a specific etiologic agent on the pathogenesis of disease in humans. This approach has illuminated the complexity of the pathogenic mechanisms, but the advances achieved to date may provide some hints regarding the pathogenesis of chronic inflammatory diseases elsewhere in the digestive tract.

Gastrointestinal food allergies: do they exist?

Gastrointestinal (GI) symptoms are often attributed to adverse reactions to foods (ARF), but it is not always clear whether such reactions are caused by food allergy. A reaction to food proteins that is mediated by immunologic events is referred to as food allergy or food hypersensitivity. One of the most common types of food allergy is the IgE-mediated immediate hypersensitivity reaction to foods, which can give rise to dermatologic and respiratory tract symptoms in addition to GI complaints. Other GI forms of food allergy include food protein-induced enterocolitis or gastroenteropathy, celiac disease, and some cases of eosinophilic gastroenteritis. Because most patients complaining of adverse reactions to food have non-immune mechanisms for their complaints, it is important to distinguish the various types of ARF, as their management may differ substantially. Recent advances in the field of food allergy provide opportunities to improve diagnostic methods and develop new modalities for management that will complement the current practice of allergen avoidance.

Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D.

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.

Virulence and pathogenicity of Helicobacter pylori.

Bacterial and host response factors play significant roles in the pathogenicity of H. pylori-related disease manifestations. The complete DNA sequences for two H. pylori strain genomes have been published. The differences in the sequences between these two unrelated strains may enable clinicians to identify rapidly other conserved and potentially virulent genes and products. Whether these two DNA sequences are sufficient representation of the H. pylori genetic heterogeneity is unknown. The host immune response and the cascade of events that occurs with H. pylori infection are being clarified rapidly. Understanding the role of this gastric bacterium in apoptosis and cellular proliferation would enable clinicians to understand its relationship to ulcerogenesis and gastric malignancy. Piecing together many observations related to H. pylori would result in understanding the interaction of H. pylori factors and host responses that lead to the variety of disease manifestations associated with this chronic infection. The development of animal models with H. pylori and other Helicobacter species has set the stage in which in vitro observations can be tested in the in vivo model.

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis.

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.

Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14.

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.

Myofibroblasts. II. Intestinal subepithelial myofibroblasts.

Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c-kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohn's disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na(+) diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-beta), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells.

Myofibroblasts. I. Paracrine cells important in health and disease.

Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have a similar appearance and function regardless of their tissue of residence. Through the secretion of inflammatory and anti-inflammatory cytokines, chemokines, growth factors, both lipid and gaseous inflammatory mediators, as well as extracellular matrix proteins and proteases, they play an important role in organogenesis and oncogenesis, inflammation, repair, and fibrosis in most organs and tissues. Platelet-derived growth factor (PDGF) and stem cell factor are two secreted proteins responsible for differentiating myofibroblasts from embryological stem cells. These and other growth factors cause proliferation of myofibroblasts, and myofibroblast secretion of extracellular matrix (ECM) molecules and various cytokines and growth factors causes mobility, proliferation, and differentiation of epithelial or parenchymal cells. Repeated cycles of injury and repair lead to organ or tissue fibrosis through secretion of ECM by the myofibroblasts. Transforming growth factor-beta and the PDGF family of growth factors are the key factors in the fibrotic response. Because of their ubiquitous presence in all tissues, myofibroblasts play important roles in various organ diseases and perhaps in multisystem diseases as well.

Regulation of the mucosal immune response.

Infectious diseases continue to exact an extensive toll on populations living closest to the equatorial regions of the globe. A substantial proportion of these infections gain access to the host via the mucosal tissues. Thus, the development of new vaccines that enhance mucosal immunity is considered to be of paramount importance in order to prevent or limit the impact of these infections. Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive amounts of inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the unique properties of mucosal immune responses and focus on recent advances that have significantly influenced our understanding of the regulation of immune and inflammatory responses following infection.

Comparison of biopsy and serological methods of diagnosis of Helicobacter pylori infection and the potential role of antibiotics.

OBJECTIVE: Endoscopic biopsy and serological methods were compared for their ability to detect Helicobacter pylori infection in patients undergoing upper gastrointestinal endoscopy at a state university hospital. METHODS: Subjects were characterized on the basis of gastrointestinal symptoms, endoscopic findings, socioeconomic and demographic features, and the use of certain medications, tobacco, and alcohol. Current infection was detected in gastric antral specimens by rapid urease testing, histopathology, and bacterial culture. Serum levels of IgG to H. pylori were measured by ELISA. RESULTS: Of 240 subjects, 115 (47.9%) were currently infected as determined by rapid urease testing, histopathology, and/or culture results, whereas 63.3% had elevated anti-H. pylori IgG levels (p < 0.001). This difference in the prevalence of current infection and seropositivity was preserved when the study population was analyzed according to age, race, gender, and other characteristics. Prior use of antibiotics was associated with a significant reduction in the frequency of H. pylori infection. CONCLUSIONS: Serological evidence of H. pylori infection was consistently greater than the prevalence of infection documented by biopsy methods in this study, suggesting suppression or recent clearance of infection. Further studies are needed to examine the factors that may affect the detection of H. pylori infection.

The effect of class II major histocompatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage.

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as "bait" to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-gamma, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-gamma. IFN-gamma increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II-negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.

Rotavirus infection of cultured intestinal epithelial cells induces secretion of CXC and CC chemokines.

BACKGROUND & AIMS: Rotaviruses are the major cause of pediatric gastroenteritis worldwide. The target cell of rotavirus infection is the mature enterocyte of the small intestine. Recently, intestinal epithelial cells have been shown to produce chemoattractant mediators in response to cytokine stimulation and bacterial infection, suggesting a potentially important role of epithelial cells in initiating immune responses. In this study, the production of chemokines by cultured intestinal epithelial cells after rotavirus infection was investigated. METHODS: Two human intestinal epithelial cell lines (HT29 and Caco-2) were infected with sucrose-purified rotavirus (strain SA114F) and assayed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay for chemokine expression. Virus-like particles and inactivated rotavirus were used to test the importance of viral attachment and replication. RESULTS: Increased messenger RNA expression and secretion of immunoreactive interleukin 8, growth-related peptide alpha, and RANTES (regulated upon activation, normal T cell expressed and secreted) were detected in rotavirus-infected cells. Chemokine production was time and dose dependent and required viral replication. CONCLUSIONS: Rotavirus infection induces the expression of a subset of chemokines in intestinal epithelial cells. These data support the hypothesis that chemokine secretion by enterocytes may play a role in the initiation and modulation of the immune response to rotavirus infection.

Cell-specific expression of RANTES, MCP-1, and MIP-1alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). Production of MCP-1 and MIP-1alpha was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-gamma), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-gamma treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1alpha as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-gamma and by the secretion of eosinophil chemokines.