Objectives:This study aimed to investigate histologic features of children with RAP undergoing upper gastrointestinal endoscopy, after exclusion of common organic disease.Methods:Children referred for endoscopy were prospectively enrolled at a single tertiary center between 2008 and 2010, after obtaining informed consent. Cases were defined as children with clinical diagnosis of RAP, as opposed to controls with suspicion of organic disease. Gastric and duodenal biopsies were analysed by pathologists blinded to indication. Demographic and clinical variables, H. pylori infection, biochemical, endoscopic and pathologic results were compared between cases and controls.Results:A total of 101 children were included after exclusion of villous enteropathy (n = 6) and parasitic, rotaviral or salmonella enteritis (n = 14), resulting in 72 cases and 29 controls. There were no significant differences in demographics, clinical symptoms, H. pylori infection and endoscopic findings between cases and controls. Duodenal eosinophil counts per 5 HPF were significantly increased in cases vs. controls (median (IQR) 86 (62–114) vs. 49 (31–88); p < 0.001) and did not differ regarding age, gender and H. pylori. Intraepithelial lymphocytes per 100 enterocytes were similar in cases vs. controls (19 (15–25) vs. 18 (14–23); p = 0.89) with ≥ 25 in 25% of cases. Duodenal eosinophilia was equally observed in H. pylori negative cases (n = 50) vs. controls (n = 19) (87 (61–119) vs. 53 (36–95); p = 0.01), as well as positive cases (n = 21) vs. controls (n = 10) (85 (55–105) vs. 42 (18–65); p = 0.03). Logistic regression yielded an adjusted odds ratio of 1.23 (95% CI 1.08–1.40) for duodenal eosinophilia in RAP.Conclusions:Children with a clinical diagnosis of RAP have marked duodenal eosinophilia, independent of H. pylori infection, suggesting the role of unknown infectious or allergic triggers in the pathogenesis of functional gastrointestinal disorders in childhood. Further research is needed on the diagnostic and therapeutic benefits of targeting duodenal eosinophilia.

Introduction Functional Recurrent Abdominal Pain (RAP) is a paediatric functional gastrointestinal disorder with poorly investigated pathophysiology. Proposed aetiology varies and the diagnosis is characterised by Rome III criteria. Some studies consider Helicobacter pylori to be a cause of RAP, while others disagree. The aim of this study was to investigate upper gastrointestinal pathology in a cohort of 123 children in Chile with respect to RAP, H pylori infection and other concurrent infection. Methods This blinded retrospective and IRB-approved study analysed biopsies taken from the gastric antrum and body and the duodenum in 123 Chilean children referred to endoscopy (with informed parental consent). Histopathology was evaluated against a clinical database which included symptoms, symptom duration and endoscopy findings. Rome III criteria were used to assign RAP to the relevant cases. All patients had stool microbiology and parasitology. H pylori infection was assessed by serology and histology. In the duodenum, routine histopathology and also eosinophil counts (in 5HPF ×40 magnification), were performed by microscopy. IELs/100 enterocytes were counted. Independently those patients with IELs >25 had serology performed for coeliac disease. Results Overall 105 patients were diagnosed with RAP and 12 patients were able to act as controls, having no symptoms of RAP or concurrent infection. The Rome III diagnosis of RAP was significantly associated with higher IEL counts (>20 in 74 patients) compared to controls (p=0.04). Furthermore, a higher IEL count was also positively associated with parasitic infection (nine with parasites) (p=0.02). Of 16 patients with lymphocytic duodenosis, (>25 IELs per 100 enterocytes) only three were infected by H pylori. One had coeliac disease with positive serology. Antral nodularity was observed in association with lymphoid follicles (p≤0.01) and H pylori infection (p<0.01). 28% in this cohort were positive for H pylori but infection was not significantly associated with RAP (p=0.55) or parasite infection, as concurrent infection was present in only 2 patients (p=0.24). Conclusion From this study, low grade inflammation, manifest by increased IELs, may be associated with RAP and also parasitic infection. H pylori is not associated with parasite infection. However, as eosinophilia was not significantly associated with the condition further investigation is required to elucidate the potential involvement of innate immunity, including mast cells. Furthermore, there is no association between H pylori infection and RAP. Funded by EU CONTENT Project (INCO-CT-2006-032136), CONICYT/BM (RUE #29) and Fondecyt #1100654 (Chile). Competing interests None declared.

Introduction Iron deficiency is the most common nutritional disorder globally. There is increasing evidence from clinical and population studies for a role of H pylori infection in the aetiology of iron deficiency. Rodent models of Helicobacter infection are needed to investigate causal links to iron deficiency in the host. The aim of this study was to investigate the effects of gastric Helicobacter infection on host iron metabolism gene expression in hypergastrinaemic INS-GAS mice. Methods Male transgenic INS-GAS mice were inoculated with H felis or broth only by oral gavage. Uninfected genetic background FVB/N mice were used as controls. Infected mice and uninfected controls were sacrificed at 9 months post-inoculation. Gastric corpus mucosa was stored for total RNA extraction and analysis by quantitative real time PCR. The level of expression of target genes was assessed as relative abundance compared to Gapdh control gene by the ΔΔCt method. Serum ferritin protein levels were measured by ELISA. Results H felis-infected INS-GAS mice had a significant reduction in hepcidin transcript levels in the gastric mucosa compared to uninfected INS-GAS mice and FVB/N controls (p < 0.03). The reduction in hepcidin hormone expression in H felis infected mice is likely to result in increased expressions of genes which control the influx and efflux of iron in gastrointestinal cells. Divalent metal transporter 1 (DMT1) which is negatively controlled by hepcidin is expressed on the apical membrane of gastrointestinal cells and absorbs reduced ferrous iron (Fe2+) from the lumen. There was a significant increase (p < 0.02) in Dmt1 gastric transcript levels in H felis-infected INS-GAS compared to uninfected INS-GAS mice or FVB/N controls. Other iron metabolism gene expression levels were also significantly increased with gastric H felis infection, such as Ferroportin 1 (p < 0.05), Transferrin receptor 1 (p < 0.003) and Lipocalin 2 (p < 0.001). Serum ferritin levels were significantly reduced (p < 0.001) in H felis-infected INS-GAS mice compared to uninfected controls at 9 months. Conclusion This study demonstrates the interplay between hepcidin transcript levels and the expression of host iron metabolism genes. Previous studies have shown that chronic gastric H felis infection in INS-GAS mice results in decreased serum transferrin saturation. This study investigates the molecular basis for previous observations and finds that chronic gastric Helicobacter infection results in a significant reduction in gastric hepcidin gene expression levels which coincides with significant increases in gene expression of several proteins implicated in host iron metabolism.

Introduction Acute H pylori infection is associated with transient hypochlorhydria of variable duration. Hypochlorhydria in H pylori-associated atrophy has a role in iron deficiency (ID) through changes in the physiology of iron absorption. The role of hypochlorhydria in H pylori-associated ID in childhood has not been investigated. The aims were to evaluate the association between H pylori-associated hypochlorhydria and ID in children. Methods A total of 123 children were prospectively enrolled in the study. Any child with peptic ulceration, or who received antacids, PPIs, H2 antagonists, and antibiotics in the previous 4 weeks was excluded. Blood was taken for complete blood count and iron profile. Gastric biopsies were taken for H. pylori determination by histology and rapid urease test. H pylori status was defined as +ve if either test was positive. Gastric juice was obtained at the beginning of the endoscopy avoiding water insufflation. Duodenal biopsies were taken for exclusion of coeliac disease (CD). Stool samples were collected for parasitology/microbiology. Of the 123 children, 2 with CD, 8 with non-specific duodenal inflammation (DI), 5 with lost blood analysis and 9 with parasitic infections (2 with DI) were excluded. Results Of the remaining 101 children 31 were H pylori +ve and 10 were hypochlorhydric (pH>4). There were no differences in demographic characteristics with regard to infection status, or gastric pH. In H pylori +ve children with pH >4 (n=6) serum iron (median (IQR): 60.2 (45–66) μg/dl) and transferrin saturation levels percentage (16.6 (15–19)) were significantly lower (p<0.01) than H pylori positive children (n=25) with pH≤4 (iron 113.2 (91–132); transferrin saturation 32.6 (24–38)). No differences in ferritin or TIBC were observed. In H pylori negative children with pH>4 (n=4) both iron (119 (101–137)), and transferrin saturation (36.4 (31–41)), were not significantly different from children with pH≤4 (n=66) (iron 98.7 (80–122); transferrin saturation 32.2 (25–40)). Conclusion Low serum iron and transferrin in childhood H pylori infection is associated with hypochlorhydria. In uninfected children, hypochlorhydria was not associated with altered serum iron parameters, indicating a combination of H pylori infection and/or inflammation and hypochlorhydria has a role in the aetiology of iron deficiency. Even though H pylori-associated hypochlorhydria may reflect a transient period during acute gastritis, it alters iron homeostasis with clinical impact in developing countries with a high H pylori prevalence. Funded by EU CONTENT Project (INCO-CT-2006-032136) and CONICYT/BM (RUE #29).

Introduction Iron deficiency (ID) is the most common nutritional disorder globally. There is increasing evidence from clinical and population studies for a role of H pylori infection in the aetiology of ID. Rodent models of Helicobacter infection need to be refined to investigate any causal links to ID. The aim of this study is to examine haematological parameters during the acute phase of Helicobacter infection in mice fed on iron-replete and iron deficient diets. Methods Five-week-old female SPF C57Bl/6 mice were fed on iron deficient (2–6 ppm) or iron-replete (48 ppm) diets 3 weeks before oral gavage with H felis. Infected mice and uninfected controls were sacrificed at 4 and 8 weeks post-inoculation. H felis infection was confirmed by urease test and histology. Blood was collected and haematological data were assessed by automated counting machine. Results Red blood cell count (RBC) and mean corpuscular haemoglobin concentration were similar in groups on both diets regardless of H felis status at 4 and 8 weeks. In uninfected mice total haemoglobin levels were significantly decreased in iron deficient mice at 4 (p<0.01) and 8 weeks (p=0.05). This decrease was abrogated by H felis infection in dietary replete groups due to a slight increase (NS) in haemoglobin. A dichotomy emerges between iron deficient and replete groups for parameters which reflect size and haemoglobin content of erythrocytes. The haematocrit test, mean corpuscular volume (MCV) and mean haemoglobin concentration (MHC) were significantly decreased (p<0.001) in H felis infected and control mice on iron deficient diet compared to matched groups fed on iron-replete diet. A significant increase (p<0.01) in red cell distribution width (RDW) in iron deficiency suggests erythrocytes have an increased variation in cell volume. H felis infection made no difference to the levels observed. White blood cell (WBC) counts were significantly increased at 4 weeks (p<0.03) and 8 weeks (p<0.001) in mice fed on iron defiecient diet, regardless of H felis status. Conclusion Iron deficiency in this mouse model results in reduced MCH indicating an increased percentage of hypochromic red cells and reduced MCV indicating microcytosis. Increased WBC counts as a result of ID, suggests a higher susceptibility to the effects of inflammation and infection. All other parameters measured showed no difference due to H felis status in either diet cohort. This suggests that H felis has no impact on haematological parameters of iron status in the acute stage of the infection. Other markers of iron status such as serum iron, TIBC and serum ferritin are needed to explore further the impact of Helicobacter infection.

Introduction H pylori cagA+ 48GX strain with a functional cag PAI was isolated from a patient in Guangxi Province, PRC, an area of high incidence of gastric cancer. Inoculation of 48GX into gerbils for 4 weeks resulted in recovery of 12.2 and 12.3 output strains from two gerbils, with RAPD profiles and glmM sequence identical to 48GX, and variable ability to activate IL-8 transcription in gastric epithelial cells. The aims of the study were to recover output strains from gerbils inoculated with 12.3 to evaluate stability of cag PAI function, and to examine the effects of long-term infection with 12.2 and 12.3 on pathology. Methods Male Mongolian gerbils were orally infected with either 12.2 or 12.3 output strains. Gerbils colonised with strain 12.3 were sacrificed at 6 and 21 weeks to recover output strains. At 60 weeks post-infection 12.2 and 12.3 infected gerbils and controls were sacrificed. Gastric tissue was taken for pathology. Plasma gastrin was measured by radioimmunoassay. Ability of strains to induce IL-8 transcription was determined by an IL-8 reporter assay in L5F11 epithelial cells. Results Six week isolates (41.1 and 41.11) from two gerbils infected with strain 12.3 induced lower IL-8 transcription than the input strain. In contrast, 21-week isolates (36.1–36.9) showed variability in inducing IL-8 transcription: four stimulated IL-8 transcription equivalent to, or greater than, the parent strain, whereas five had minimal, or reduced, IL-8 transcription relative to 12.3 input strain. At 60 weeks, gross morphological changes and increased mucosal thickness were evident in 12.2 and 12.3 infected gerbils compared to controls. Infection with 12.2 and 12.3 increased plasma gastrin (p<0.001) relative to uninfected gerbils (142.7+15.7 and 117.3+12.6 vs 18+3.17 pM, respectively). Infection with both strains was associated with extensive intramucosal and submucosal inflammation, mucous metaplasia, cystic and foveolar hyperplasia. Gastric carcinoids were evident in two gerbils with hypergastrinaemia, infected with 12.2 (gastrin 194 pM), and 12.3 (gastrin 168 pM) strains. Dysplasia was evident in 4/12 and 5/12 gerbils infected with 12.2 and 12.3 strains, respectively. Overall 11/25 (44%) of gerbils infected with 48GX output strains had gastric dysplasia or carcinoids at 60 weeks post-infection. Conclusion These studies identify a new gerbil colonising cag PAI+ H pylori strain which induces severe pathology. They also demonstrate the functional instability of the cag PAI in vivo in the gerbil model, and provide multiple output strains for analysis of in vivo genomic modifications in H pylori.

Introduction Gastric infection with Helicobacter in transgenic hypergastrinaemic INS-GAS mice has been shown to accelerate the development of gastric cancer. Both H pylori and gastrin transactivate the EGF receptor (EGFR) in an autocrine or paracrine manner, respectively. The aims of the study were to examine the effects of treatment with an EGFR inhibitor in H felis infected INS-GAS mice. Methods Male INS-GAS mice were orally infected with H felis. Six weeks post-infection, infected and uninfected controls were fed either control, or EKB-569 (EGF inhibitor) supplemented food. Mice were sacrificed at 9 months post-infection. At post mortem gastric tissue was taken for pathology, analysis of RNA, proteins and DNA. Mucosal thickness and gastric epithelial cell proliferation (Mib1), apoptosis (active caspase 3), parietal cell density (H+K+ ATPase), B cell density (CD20) and Aicda (activation-induced cytidine deaminase, a DNA editing enzyme) were assessed immunohistologically. Gene expression was determined by quantitative RT-PCR. Results H felis infection resulted in significant increases in corpus mucosal thickness, Mib1 positive epithelial cells, active caspase 3 positive epithelial cells, and reduced number of parietal cells relative to uninfected controls. EKB-569 had no effect on epithelial proliferation, apoptosis, parietal cell number and mucosal thickness. Infection was associated with increased levels of transcripts for Cox-2, Egfr, Hb-egf, γ-Ifn (1.5–3.5-fold increase, p<0.05), and a marked increase in transcripts of CD20 and Aicda (17 and 18-fold increase, respectively, p<0.001). Transcript levels of Cox-2 and Hb-egf were not altered by EKB-569 treatment and Egfr transcripts were significantly increased (p<0.05). In contrast, EKB-569 induced a marked reduction (p<0.002) in Aicda transcripts, but not in gastric CD20 transcript levels. In EKB-569 treated infected mice there was a strong correlation between Aicda and CD20 mRNA expression (r=0.73), which was absent in untreated infected mice (r=0.02), implying a non-B cell origin of Aicda in untreated mice. Immunohistology confirmed marked upregulation of Aicda in the hyperplastic epithelial cells of the corpus mucosa of infected untreated mice compared to controls. Conclusion In this study, EKB-569 did not affect gastric pathology, but markedly reduced Helicobacter induced aberrant expression of Aicda, a DNA editing enzyme, shown to induce TP53 mutations in human gastric epithelial cells in vitro.

greater risk for infection in chronically stressed subordinate animals, which are known to develop more severe coronary artery disease.These findings in a prospective randomized trial provide justification for a randomized, double blind study evaluating coronary artery atherosclerosis development in H. pylori infected and uninfected animals (Supported by CA082312).

More than 50% of the world population is infected by Helicobacter pylori nowdays. Although in most cases it doesn't causes any symptoms it's a significant risk factor in the development of gastric ulcer or gastric carcinoma. Elimination of the bacteria can be attained by the combination of at least three drugs and it may also causes several side effects. It's beyond doubt that a new drug showing good efficacy without side effects is needed. Kinase inhibitors have proved their usefulness in the treatment of cancer inhibiting cell proliferation via inhibition of intracellular signaling system. Moreover host cell kinases can be involved in viral or bacterial infections, this way human kinases may be the targets of the treatment. In our research we are investigating kinase inhibitors and developing novel compounds for host cell inhibition. The point of our strategy (called chemistry driven drug development) is to identify active compounds in cellular assay and identify cellular targets afterwards. Initially, we are using the Nested Chemical Library™ (NCL) technology for hit finding and lead optimization. Core of the NCL is Chemical Validation Library (CVL) which can be used for hit finding in the preliminary screen. 313 compounds of CVL were tested and focused libraries were prepared around the validated hit molecules of the first screen. After three optimization cycle lead compounds were identified. From one of the best lead compounds we have prepared a special derivative containing a linker which can be used to attach to an affinity column for target fishing in chemistry driven drug development. Target fishing is an efficient proteomic method in seeking cellular drug targets based on affinity chromatography, 2D electrophoresis and MS-MS protein identification.