D. Brent Polk

Helicobacter pylori: gastric cancer and beyond

Helicobacter pylori is the dominant species of the human gastric microbiome, and colonization causes a persistent inflammatory response. H. pylori-induced gastritis is the strongest singular risk factor for cancers of the stomach; however, only a small proportion of infected individuals develop malignancy. Carcinogenic risk is modified by strain-specific bacterial components, host responses and/or specific host–microbe interactions. Delineation of bacterial and host mediators that augment gastric cancer risk has profound ramifications for both physicians and biomedical researchers as such findings will not only focus the prevention approaches that target H. pylori-infected human populations at increased risk for stomach cancer but will also provide mechanistic insights into inflammatory carcinomas that develop beyond the gastric niche.

Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and beta-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCbetaI and PKCepsilon. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

Mechanisms of Probiotic Action : Implications for Therapeutic Applications in Inflammatory Bowel Diseases

Probiotics are defined as nonpathogenic living microorganisms, including some commensal bacterial flora, which have beneficial effects on host health and disease prevention and/or treatment. Clinical trials have shown beneficial effects of probiotics on several human diseases, such as inflammatory bowel diseases (IBDs), which are among the most‐studied diseases testing probiotics as a potential therapy. However, a significant question regarding clinical use of probiotics is the mechanism underlying the wide range of actions. Studies discussed in this review suggest 3 distinct cellular and molecular mechanisms for probiotic regulation in IBD therapy: 1) Probiotics block pathogenic bacterial effects by producing bactericidal substances and competing with pathogens and toxins for adherence to the intestinal epithelium; 2) Probiotics regulate immune responses by enhancing the innate immunity and modulating pathogen‐induced inflammation via toll‐like receptor‐regulated signaling pathways; and 3) Probiotics regulate intestinal epithelial homeostasis by promoting intestinal epithelial cell survival, enhancing barrier function, and stimulating protective responses. Probiotics modulate host cell signaling pathways, including Akt, mitogen‐activated protein kinases, and nuclear factor‐&kgr;B to mediate these intestinal epithelial functions. It is hoped that developing a mechanistic understanding of probiotic action will provide the rationale to support the development of new hypothesis‐driven studies to define the clinical efficacy in preventive, adjunctive, or alternative treatments for IBD.

Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes

BACKGROUND & AIMS Derivatives of 5-aminosalicylic acid (mesalamine) represent a mainstay in inflammatory bowel disease therapy, yet the precise mechanism of their therapeutic action is unknown. Because tumor necrosis factor (TNF)-alpha is important in the pathogenesis of inflammatory bowel disease, we investigated the effect of mesalamine on TNF-alpha-regulated signal transduction and proliferation in intestinal epithelial cells. METHODS Young adult mouse colon cells were studied with TNF-alpha, epidermal growth factor, or ceramide in the presence or absence of mesalamine. Proliferation was studied by hemocytometry. Mitogen-activated protein (MAP) kinase activation and IkappaBalpha expression were determined by Western blot analysis. Nuclear transcription factor kappaB (NF-kappaB) nuclear translocation was determined by confocal laser immunofluorescent microscopy. RESULTS The antiproliferative effects of TNF-alpha were blocked by mesalamine. TNF-alpha and ceramide activation of MAP kinase were inhibited by mesalamine, whereas epidermal growth factor activation of MAP kinase was unaffected. TNF-alpha-stimulated NF-kappaB activation and nuclear translocation and the degradation of Ikappa-Balpha were blocked by mesalamine. CONCLUSIONS Mesalamine inhibits TNF-alpha-mediated effects on intestinal epithelial cell proliferation and activation of MAP kinase and NF-kappaB. Therefore, it may function as a therapeutic agent based on its ability to disrupt critical signal transduction events in the intestinal cell necessary for perpetuation of the chronic inflammatory state.

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria-derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40s effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

Recurrent abdominal pain : Symptom subtypes based on the Rome ii Criteria for Pediatric functional gastrointestinal disorders

Objectives Recurrent abdominal pain (RAP) is a common childhood complaint rarely associated with organic disease. Recently, the Pediatric Rome Criteria were developed to standardize the classification of pediatric functional gastrointestinal disorders (FGIDs) using a symptom-based approach. The authors tested the hypothesis that most patients with childhood RAP could be classified into one or more of the symptom subtypes defined by the Pediatric Rome Criteria. Methods Using a prospective longitudinal design, new patients with RAP (n = 114) were studied at a tertiary care childrens medical center. Before the medical evaluation, parents completed a questionnaire about their child, assessing symptoms defined by the Pediatric Rome Criteria. Results Of the 107 children for whom medical evaluation revealed no organic etiology for pain, 73% had symptom profiles consistent with the Pediatric Rome Criteria for one of the FGIDs associated with abdominal pain (irritable bowel syndrome, 44.9%; functional dyspepsia,15.9%; functional abdominal pain, 7.5%; abdominal migraine, 4.7%) Conclusions This study provides the first systematic empirical evidence that RAP, originally defined by Apley, includes children whose symptoms are consistent with the symptom criteria for several FGIDs defined by the Rome criteria. The pediatric Rome criteria may be useful in clinical research to (1) describe the symptom characteristics of research participants who meet Apleys broad criteria for RAP, and (2) select patients with particular symptom profiles for investigation of potential biologic and psychosocial mechanisms associated with pediatric FGIDs.

Epidermal growth factor receptor–stimulated intestinal epithelial cell migration requires phospholipase C activity

BACKGROUND & AIMS Ulceration of intestinal mucosa is rapidly followed by enterocyte migration via restitution. The aim of this study was to investigate signaling mechanisms of epidermal growth factor (EGF) receptor-stimulated monolayer restitution in a mouse intestinal epithelial cell line. METHODS EGF-stimulated cell migration was determined using a wound model in the presence of agonists and/or antagonists of tyrosine kinase, phospholipase C, phosphatidylinositol 3-kinase, or protein kinase C. The tyrosine phosphorylation state of the EGF receptor, phosphatidylinositol phospholipase C gamma1 (PLCgamma1), focal adhesion kinase, and cellular lysates was determined by immunodetection. RESULTS EGF stimulated cell migration twofold at 4, 8, and 24 hours. Inhibition of EGF receptor tyrosine kinase activity, phospholipase C, or phosphatidylinositol 3-kinase attenuated EGF-induced intestinal cell migration. Pretreatment of cells with phorbol 12-myristate 13-acetate, known to down-regulate protein kinase C expression, blocked EGF-induced cell migration. Increased tyrosine phosphorylation of the EGF receptor and PLCgamma1 was detected within 5 minutes after wounding. CONCLUSIONS EGF-stimulated intestinal cell migration requires intact EGF receptor tyrosine kinase, phospholipase, and protein kinase C activities. PLCgamma1 may be a key regulatory molecule in the initial EGF receptor signal transduction pathway of EGF-stimulated cell migration.

Histopathology of Ulcerative Colitis in Initial Rectal Biopsy in Children

Definitive histologic diagnosis of ulcerative colitis relies upon mucosal architectural distortion and inflammation in the appropriate clinical setting. Although crypt branching, atrophy, and loss are usually present in first biopsies from adults with ulcerative colitis, it has been our impression that features of chronicity are often lacking in first biopsies from children. To test this hypothesis, initial rectal biopsies and follow-up biopsies and/or colonic resections from 53 children (age 15 months to 17 years) and 38 adults (age 21–76 years) with ulcerative colitis were examined in a blinded fashion for villiform surface, crypt atrophy, branching crypts, lamina propria inflammation, crypt abscesses, cryptitis, and basal plasma cells. Mucosal architecture was classified as normal, focally abnormal, or diffusely abnormal. Medical records were reviewed for confirmatory evidence of ulcerative colitis and for duration of symptoms before biopsy. In 87 of 91 biopsies, the lamina propria contained a mixed inflammatory infiltrate. Crypt abscesses and cryptitis were common in both groups. Initial biopsies from children were less likely to show diffuse architectural abnormalities (17 of 53, 32.1%) compared with biopsies from adults (22 of 38, 57.9% p <0.05). Duration of symptoms before diagnosis was significantly shorter in children (mean 17.5 weeks) compared with adults (mean 54.9 weeks). In summary, initial rectal biopsies from children with ulcerative colitis are less likely to show diagnostic mucosal architectural distortion than biopsies from adults. This difference may be related to a shorter duration of symptoms before biopsy.

p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF‐stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen‐activated protein kinase, we show that p38 is required for efficient EGF‐induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin‐1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand‐stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.

Bmp signaling is required for intestinal growth and morphogenesis

Intestinal growth, morphogenesis, differentiation, and homeostasis are regulated by reciprocal interactions between the epithelium and the underlying mesenchymal stroma. The identification of BMPR1A mutations in patients with Juvenile Polyposis implicates Bmp signaling as an important mediator of these interactions. To test this hypothesis, we inhibited Bmp signaling in the mouse proximal intestine by transgenic misexpression of the BMP antagonist, noggin, using regulatory elements of the fatty acid binding protein (Fabp1) gene. This leads to abnormal villus morphogenesis, stromal and epithelial hyperplasia, and ectopic crypt formation. The resulting intestinal histopathology resembles that seen in human Juvenile Polyposis. Misexpression of noggin in the large intestine gives a similar abnormal phenotype in this region of the gut. Analysis of gene expression in the transgenic small intestine raises the possibility that Hedgehog and Pdgf signaling play a role in the development of the Juvenile Polyposis‐like phenotype. Developmental Dynamics 235:1563–1570, 2006.