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Background & Aims Ablation of gastric inlet patches (GIP) in the cervical esophagus by argon plasma coagulation (APC) can alleviate chronic globus sensations in the throat. We investigated the efficacy of this therapy in a randomized, controlled multicenter trial. Methods Patients with chronic globus sensations and GIP were randomly assigned 1:1 to groups that were treated with APC or a sham procedure (controls). Patients and their referring physicians were blinded to therapy. All patients completed a standardized questionnaire about symptoms before and 3 months after the procedure. Thereafter, control patients were eligible for cross-over therapy. Long-term efficacy was assessed in all patients ≥6 months after APC. Results Improvement of symptoms was reported in 9 (82%) of 11 patients who received APC, compared with 0 (0%) of 10 patients in the control group ( P = .002). Nine (90%) of 10 patients treated with APC had per protocol healing, compared with 0 (0%) of 9 controls ( P Conclusions Ablation of gastric inlet patches appears to be an effective therapy for alleviation of associated globus sensations. This new treatment modality might change the paradigm for treatment of these patients.

BACKGROUND & AIMS: Histamine-producing enterochromaffin-like (ECL) cells play an integrative role in the regulation of acid secretion. Decreased mucosal histamine concentrations and increased levels of interleukin (IL) 1 beta, IL-6, and IL-8 have been detected in the gastric mucosa inflamed with Helicobacter pylori. The aim of this study was to investigate the response of isolated ECL cells to these cytokines. METHODS: Enriched rat gastric ECL cells (85%-95%) were cultured for 2-4 days. RESULTS: Polymerase chain reaction showed IL-1 and IL-6, but not IL-8 receptors, in ECL cell complementary DNA. Positive receptor staining with biotinylated IL-1 beta corresponded to ECL cell enrichment (92%). IL-6 and IL-8 had no effect on histamine secretion. IL-1 beta (2 U/mL) stimulated basal histamine secretion and nitric oxide production within 60 minutes and cyclic guanosine monophosphate production within 20 minutes. Pretreatment for 20 minutes with IL-1 beta (2 U/mL) attenuated gastrin-stimulated histamine secretion by 40%-50%, reversed by the IL-1 receptor antagonist (10 U/ mL). Pretreatment for 20 minutes with IL-1 beta (2 U/mL) completely inhibited gastrin-stimulated (1 nmol/L) histidine decarboxylase activity. IL-1 beta (2 U/mL, 60 minutes) increased lactate dehydrogenase release to 25% of cell content. Cells pretreated with IL- 1 beta did not respond to gastrin after a further 48-hour culture and showed decreased histamine content. CONCLUSIONS: ECL cells appear to express IL-1 receptors. IL-1 beta causes sustained functional impairment of ECL cells in vitro. (Gastroenterology 1997 Feb;112(2):364-75)

Background/Aims: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells. Methods: ECL cells were isolated from rat fundic mucosa to a purity of 90%–95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture. Results: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32–87, inhibited the gastrin-induced calcium signal at 10 −11 mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32–92, and also somatostatin 14 required 100–1000 times higher concentrations (10 −8 mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC 50 ) value of 2 × 10 −12 mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC 50 values of 1 to 5 × 10 −9 mol/L. Conclusions: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.

Background: In vivo studies have suggested an important role for gastric enterochromaffinlike (ECL) cells in mediating acid secretion. Direct evidence for this function is lacking and requires a preparation of highly purified ECL cells. This work investigates the possible role and mechanism of histamine release from the ECL cell in the peripheral regulation of acid secretion, using purified ECL cells from rat fundic mucosa. Methods: A combination of elutriation and density-gradient centrifugation was used to purify rat fundic ECL cells. Enrichment was determined by the presence of acidic vacuoles containing a V type adenosine triphosphatase, electron microscopy, immunostaining, and histamine content and release. Results: ECL cells were enriched at least 65-fold with respect to the fundic epithelium. Gastrin (EC 50 0.2 nmol/L) and cholecystokinin octapeptide (nonsulfated, EC 50 0.04 nmol/L) stimulated histamine release in a time- and dose-dependent manner, suggesting a CCK-B receptor subtype, confirmed by the inhibition of gastrin/CCK stimulation with the CCK-B antagonist L365,260. Somatostatin also inhibited gastrin-mediated histamine release. Single cell imaging showed that gastrin elevated intracellular cytosolic calcium concentration biphasically. Carbachol and the C kinase activator 120-tetradecanoylphorbol-13-acetate also stimulated histamine release. Epinephrine (blocked by propranolol), forskolin, and dibutyryl-5′-cyclic adenosine monophosphate were also effective, implicating a β-adrenergic pathway. The H 3 agonist R-α-methyl-histamine inhibited, whereas the H 3 -antagonist thioperamide potentiated gastrin/CCK stimulated histamine release. Conclusions: These in vitro results support a central role for the ECL cell in the peripheral regulation of gastric acid secretion.

Background & Aims Helicobacter pylori infects half of the world's population, thereby causing significant human morbidity and mortality. The mechanisms by which professional antigen-presenting cells recognize the microbe are poorly understood. Methods Using dendritic cells (DCs) from TRIF, MyD88, TLR 2/4/7/9 −/− , and multiple double/triple/quadruple mutant mice, we characterized receptors and pathways mediating innate immune recognition of H pylori . Results We identified a MyD88-dependent component of the DC activation program, which was induced by surface TLRs, with TLR2 and to a minor extent also TLR4 being the exclusive surface receptors recognizing H pylori . A second MyD88-dependent component could be blocked in TLR2/4 −/− DCs by inhibitors of endosomal acidification and depended on intracellular TLRs. We identified TLR9-mediated recognition of H pylori DNA as a principal H pylori –induced intracellular TLR pathway and further showed that H pylori RNA induces proinflammatory cytokines in a TLR-dependent manner. Microarray analysis showed complementary, redundant, and synergistic interactions between TLRs and additionally revealed gene expression patterns specific for individual TLRs, including a TLR2-dependent anti-inflammatory signature. A third component of the DC activation program was primarily composed of type I interferon-stimulated genes. This response was MyD88 and TRIF independent but was inducible by RIG-I–dependent recognition of H pylori RNA. Conclusions These results provide novel comprehensive insights into the mechanisms of H pylori recognition by DCs. Understanding these processes provides a basis for the rational design of new vaccination strategies.

Background & Aims: Recognition of infection leads to induction of adaptive immunity through activation of antigen-presenting cells (APCs). Among APCs, dendritic cells (DCs) have the unique capacity to deliver antigens from the periphery to T cells in secondary lymphoid organs. Methods: We analyzed molecular mechanisms of the Helicobacter pylori–induced APC activation in vitro and investigated the influence of Myd88 signaling on the phenotype of adaptive immunity to H pylori in a murine infection model. Results: The adaptor protein Myd88 mediates Toll-like receptor (TLR), interleukin (IL)-1, and IL-18 signaling. DCs from wild-type, IL-1R−/−, and IL-18−/− mice responded to H pylori with secretion of proinflammatory cytokines and up-regulation of major histocompatibility complex II and costimulatory molecules. In Myd88−/− DCs these processes were impaired profoundly, showing that TLR-dependent H pylori–sensing affects DC activation. Analysis of the H pylori–specific DC transcriptome revealed that large parts of the bacteria-induced transcriptional changes depended on Myd88 signaling, comprising numerous genes involved in crucial steps of immune regulation, such as DC maturation/differentiation, antigen uptake/presentation, and effector cell recruitment/activation. The impaired ability of Myd88−/− DCs, B cells, and macrophages to mount a proinflammatory response to H pylori in vitro was reflected in vivo by reduced gastric inflammation and increased bacterial colonization in Myd88-deficient mice. Furthermore, Helicobacter-specific IgG2c/IgG1 ratios were reduced in Myd88−/− animals, suggesting the involvement of the Myd88-dependent pathway in the instruction of adaptive immunity toward a T helper cell type 1 phenotype. Conclusions: A principal pathway by which DCs sense H pylori and become activated is the TLR-dependent signaling cascade. In vivo, Myd88 signaling affects adaptive immunity to the bacterium.

Background & Aims: Release of serotonin from mucosal enterochromaffin cells triggered by luminal substances is the key event in the regulation of gut motility and secretion. We were interested to know whether nasal olfactory receptors are also expressed in the human gut mucosa by enterochromaffin cells and whether their ligands and odorants present in spices, fragrances, detergents, and cosmetics cause serotonin release. Methods: Receptor expression was studied by the reverse-transcription polymerase chain reaction method in human mucosal enterochromaffin cells isolated by laser microdissection and in a cell line derived from human enterochromaffin cells. Activation of the cells by odorants was investigated by digital fluorescence imaging using the fluorescent Ca2+ indicator Fluo-4. Serotonin release was measured in culture supernatants by a serotonin enzyme immunoassay and amperometry using carbon fiber microelectrodes placed on single cells. Results: We found expression of 4 olfactory receptors in microdissected human mucosal enterochromaffin cells and in a cell line derived from human enterochromaffin cells. Ca2+ imaging studies revealed that odorant ligands of the identified olfactory receptors cause Ca2+ influx, elevation of intracellular free Ca2+ levels, and, consequently, serotonin release. Conclusions: Our results show that odorants present in the luminal environment of the gut may stimulate serotonin release via olfactory receptors present in human enterochromaffin cells. Serotonin controls both gut motility and secretion and is implicated in pathologic conditions such as vomiting, diarrhea, and irritable bowel syndrome. Thus, olfactory receptors are potential novel targets for the treatment of gastrointestinal diseases and motility disorders.

Background & Aims: Helicobacter pylori colonizes the human gastric mucosa of >50% of the world’s population. Most of the patients have no overt clinical symptoms. However, the infection is invariably associated with the development of active chronic gastritis, leading in some cases to the development of peptic ulcer disease, distal gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. In contrast to most other pathogens, infection with H pylori persists lifelong, but reasons for the persistence remain obscure. CD4-positive T cells are crucial for bacterial elimination but are inhibited by H pylori. We aimed to identify the factor responsible for suppression of T-cell response and characterize this inhibitory effect on a cellular and molecular level. Methods: Using size-exclusion chromatography, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and a spectrophotometric enzyme assay, we identified the secreted -glutamyl transpeptidase of H pylori (HPGGT) as the factor responsible for inhibition of T-cell proliferation. Results: Mutagenesis of HPGGT in different H pylori strains completely abrogated this inhibitory effect. Recombinantly expressed HPGGT protein showed full antiproliferative activity. Site-directed mutagenesis and application of the GGT inhibitor acivicin revealed that inhibition of T cells depends on catalytic activity of HPGGT. Cell cycle analysis of human T cells indicated that HPGGT was necessary and sufficient to induce G1 arrest. Reduced levels of c-Myc and phosphorylated c-Raf protein suggest the disruption of Ras-dependent signaling by HPGGT. Conclusions: GGT is a novel immunosuppressive factor of H pylori inhibiting Tcell proliferation by induction of a cell cycle arrest in the G1 phase.

Background & Aims: Helicobacter pylori infects more than half of the world's population. In contrast to most other pathogens, the microbe persists for the virtual life of its host. It is unclear why the immune system is unable to eliminate the infection, but recent studies suggested that CD4 + /CD25 + /Foxp3 + regulatory T cells may be involved in this process. Methods: By using a mouse model of infection and gastric biopsies from 108 patients, we performed a detailed descriptive and functional characterization of the Helicobacter -induced CD25 + /Foxp3 + T-cell response. Results: In C57BL/6 mice, H pylori induced a marked gastric Foxp3 + T-cell response, which increased over several months together with the severity of inflammation, until a stable homeostatic situation became established. Accordingly, in Helicobacter -infected patients, but not in uninfected individuals, large numbers of gastric Foxp3 + T cells were detected immunohistochemically. To define the functional in vivo relevance of this response, CD25 + cells were depleted systemically in mice by using an anti-CD25 monoclonal antibody (PC61). Already 4 weeks after infection, PC61-treated mice, but not untreated animals, developed a severe gastritis with heightened cytokine expression and increased numbers of mucosal T cells, B cells, and macrophages. This was accompanied by increased titers of H pylori -specific IgG1 and IgG2c antibodies in the sera of PC61-treated mice. This increased gastric inflammatory response in CD25-depleted mice was associated with reduced bacterial loads. Conclusions: CD25 + /Foxp3 + T cells actively participate in the immune response to H pylori . In vivo depletion of these cells in infected mice leads to increased gastric inflammation and reduced bacterial colonization.

Background & aims: Although Helicobacter pylori is recognized by the human immune system, the bacteria are not eliminated and lead to a chronic inflammation of the gastric mucosa. Methods: We investigated the interaction of H pylori with human lymphocytes. T and B lymphocytes were isolated from H pylori –infected patients and stimulated with anti-CD3/CD28 or interleukin-6. Results: Proliferation of lymphocytes was abolished on co-incubation with different H pylori strains (1–5 bacteria/cell) or with protein extracts of culture supernatants. Inhibition of proliferation was independent of known virulence factors. The factor is a protein or protein complex with an apparent molecular weight between 30 and 60 kilodaltons, clearly distinct from VacA. Although antigen-specific activation of T cells (as shown by nuclear factor of activated T cells [NFAT]-activation, interferon-γ production, and CD25 or CD69 up-regulation) remained intact, cell-cycle analysis showed that S-phase entry of T cells was inhibited completely by H pylori . Consequently, stimulated T cells arrested in the G1 phase of the cell cycle. Western blot analysis showed markedly reduced phosphorylation of the retinoblastoma protein (pRb), suggesting inhibition of G1 cyclin-dependent kinase activity. In line with this, activities of cyclin D3 and cyclin E were down-regulated, and levels of the cyclin-dependent kinase inhibitor p27 Kip1 were increased. Mouse embryonic fibroblasts deficient in p27 showed a decrease in H pylori –induced inhibition of cell proliferation, suggesting a central role for p27 in mediating H pylori –induced G1 arrest. Conclusions: Induction of cell-cycle arrest in lymphocytes may be of major significance for the chronic persistence of bacteria in the human stomach.

Background & Aims: Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa. Previous studies have shown that the proinflammatory cytokine interleukin (IL)-1β present during chronic gastritis inhibits histamine synthesis in ECL cells and leads to sustained functional impairment. This study investigated the effects of IL-1β on ECL cell apoptosis and the related signal-transduction mechanisms. Methods: ECL cells were isolated by pronase digestion and a combination of elutriation, gradient centrifugation, and 48-hour culture (purity ≥90%). Apoptosis was measured by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling reaction and cell death detection enzyme-linked immunosorbent assay. Results: IL-1β (100 pg/mL) increased the rate of programmed cell death 2–3 fold in ECL cells after 24 hours of incubation (total of 12%–14%). This effect was completely inhibited by the NF-κB inhibitor, proteasome inhibitor I, and the nitric oxide synthase inhibitor (iNOS) N G -monomethyl-L-arginine (10 −4 mol/L), but not by the caspase 3 inhibitor, Asp-Glu-Val-Asp-CHO. Western blot analysis, reverse-transcription polymerase chain reaction (PCR), and in situ PCR showed that IL-1β induced gene expression (after 2–4 hours) and protein synthesis (6–18 hours) of the iNOS isoform in ECL cells. Bax protein expression was increased in response to IL-1β. In contrast, bcl-2 gene expression was increased in response to basic fibroblast growth factor, which has been shown to counteract IL-1β– induced apoptosis. Conclusions: These data suggest that IL-1β induces programmed cell death in isolated rat ECL cells via activation of NF-κB, iNOS, and the Bax protein. GASTROENTEROLOGY 2000;118:515-524

Four Membrane-Associated Antigens of Helicobacter pylori Suitable for Diagnosis of Both Infection and Eradication. Petra Voland, II Medical Dept, Munich Germany; David Weeks, VAGLAI-IS and Univ of CA, Los Angeles, Los Angeles, CA; Dino Vaira, Dept of Medicine, Orsola Hosp, Bologna Italy; Nina Neumayer, Dept of Medicine II, Munich Germany; George Sachs, VAGLAHS and Univ of CA, Los Angeles, Los Angeles, CA; Christian Pdnz, Oept of Medicine II, Teeh Univ, Munich Germany

Background. Human and rat fundic mucosa contain Enterochromaffinlike(ECL) cells. We have previously shown that these cells undergo apoptosis in the presence of TNF-a, a cytokine that is released during H. pylori infection in the gastric mucosa. Of further interest is basic FGF which is detected in ECL cell tumors. The aim of our current study was to elucidate the mechanisms of TNF-a induced apoptotis in ECL-cells and the role of bFGF during this reponse. MethodslResults. ECL-cells were isolated from rat fundic mucosa to a purity> 90% and cultured up to 24 hr. Western Blotting showed a strong signal from unphosphorylated lKBa at 0 min. After 5 min, the signal was decreased and after 10 min, unphosphorylated lKBa-was no longer detectable, indicating that IKBa was strongly phosphorylated, which in tum leads to the activation of NFKB. To determine NFKB-activation more directly, we performed electromobility-shift assays (EMSA) using cell extracts pretreated with vehicle for I hr, then treated with TNF-a (25 ng/mL) or vehicle for another hour. The EMSAs revealed a specific activation of the NFKB subunits p50 and p65. In order to investigate the functional role of NFKB in the mediation of pro-apoptotic TNF-a effects, we measured ECL-cell apoptosis (programed cell death) after preincubation (I hr) with the specific NFKB-inhibitors MGl32 (5 mM) and PSI (6 mM) and subsequent incubation with TNF-a (25 ng/mL, I hr). Cells incubated without inhibitors and/or TNF-a served as controls. Using the TUNEL technique, TNF-a was found to cause apoptosis in 16% ofECL cells compared to a basal rate of6%. MGI32 and PSI alone did not induce apoptosis (total of 5-8% of ECL cells), but were able to inhibit TNF-a-induced apoptosis completely. For MG132, these results were repeated using an ELISA-assay based upon the detection of nucleosomes which also showed complete inhibition ofTNF-a effects. The observations were verified using the EMSA technique mentioned above. To elucidate the role of basic FGF, we tested bFGF effects on TNF-a induced apoptosis. Using the ELISA, we found that bFGF at 10-100 ng/mL completely blocked TNF-a induced cell death. Conclusion. TNF-a induced apoptosis of ECL cells is mediated by phosphorylation of IKBa and subsequent activation of NFKB. bFGF inhibits TNF-a induced ECL cell death, which may also playa role for the persistence of ECL cells during chronic gastric inflammation (DFG Pr 41112-2).

Background. Human and rat fundic mucosa contain Enterochromaffinlike(ECL) cells. We have previously shown that these cells undergo apoptosis in the presence of TNF-a, a cytokine that is released during H. pylori infection in the gastric mucosa. Of further interest is basic FGF which is detected in ECL cell tumors. The aim of our current study was to elucidate the mechanisms of TNF-a induced apoptotis in ECL-cells and the role of bFGF during this reponse. MethodslResults. ECL-cells were isolated from rat fundic mucosa to a purity> 90% and cultured up to 24 hr. Western Blotting showed a strong signal from unphosphorylated lKBa at 0 min. After 5 min, the signal was decreased and after 10 min, unphosphorylated lKBa-was no longer detectable, indicating that IKBa was strongly phosphorylated, which in tum leads to the activation of NFKB. To determine NFKB-activation more directly, we performed electromobility-shift assays (EMSA) using cell extracts pretreated with vehicle for I hr, then treated with TNF-a (25 ng/mL) or vehicle for another hour. The EMSAs revealed a specific activation of the NFKB subunits p50 and p65. In order to investigate the functional role of NFKB in the mediation of pro-apoptotic TNF-a effects, we measured ECL-cell apoptosis (programed cell death) after preincubation (I hr) with the specific NFKB-inhibitors MGl32 (5 mM) and PSI (6 mM) and subsequent incubation with TNF-a (25 ng/mL, I hr). Cells incubated without inhibitors and/or TNF-a served as controls. Using the TUNEL technique, TNF-a was found to cause apoptosis in 16% ofECL cells compared to a basal rate of6%. MGI32 and PSI alone did not induce apoptosis (total of 5-8% of ECL cells), but were able to inhibit TNF-a-induced apoptosis completely. For MG132, these results were repeated using an ELISA-assay based upon the detection of nucleosomes which also showed complete inhibition ofTNF-a effects. The observations were verified using the EMSA technique mentioned above. To elucidate the role of basic FGF, we tested bFGF effects on TNF-a induced apoptosis. Using the ELISA, we found that bFGF at 10-100 ng/mL completely blocked TNF-a induced cell death. Conclusion. TNF-a induced apoptosis of ECL cells is mediated by phosphorylation of IKBa and subsequent activation of NFKB. bFGF inhibits TNF-a induced ECL cell death, which may also playa role for the persistence of ECL cells during chronic gastric inflammation (DFG Pr 41112-2).