Culture and successful eradication of Helicobacter pylori from heterotopic gastric mucosa.

Helicobacter pylori colonizes the gastric mucosa of humans and can cause chronic gastritis, peptic ulcer disease, gastric cancer or mucosa-associated-lymphoid tissue (MALT) lymphoma. Here, we report the case of a 61-year-old male patient who presented with tickle of the throat, globus sensation and heartburn. In an esophagogastroduodenoscopy subpharyngeal localized heterotopic gastric mucosa (HGM), reflux esophagitis and a chronic gastritis were diagnosed. HGM and stomach were H. pylori positive as proven by culture and histopathological examination. After eradication therapy with a proton pump inhibitor (PPI), amoxicillin and clarithromycin followed by PPI treatment, the patient reported clinical improvement and the histopathological changes in the HGM due to H. pylori infection improved, too. This case report demonstrates that culture and susceptibility testing of H. pylori using established protocols succeeds not only from tissue samples of the stomach but also from heterotopic gastric mucosa. Eradication therapy may not only improve typical H. pylori associated discomforts of the stomach but also extragastric signs and symptoms of H. pylori infection.

[S3-guideline "Helicobacter pylori and gastroduodenal ulcer disease"]. / S3-Leitlinie "Helicobacter pylori und gastroduodenale Ulkuskrankheit" der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (DGVS).

This guideline updates a prior concensus recommendation of the German Society for Digestive and Metabolic Diseases (DGVS) from 1996. It was developed by an interdisciplinary cooperation with representatives of the German Society for Microbiology, the Society for Pediatric Gastroenterology and Nutrition (GPGE) and the German Society for Rheumatology. The guideline is methodologically based on recommendations of the Association of the Scientific Medical Societies in Germany (AWMF) for providing a systematic evidence-based consensus guideline of S 3 level and has also implemented grading criteria according to GRADE (Grading of Recommendations Assessment, Development and Evaluation). Clinical applicability of study results as well as specifics for Germany in terms of epidemiology, antibiotic resistance status, diagnostics and therapy were taken into account.

[Pathogenesis, diagnostics and treatment of Helicobacter pylori infection]. / Pathogenese, Diagnostik und Therapie der Helicobacter-pylori-Infektion.

More than one-half of the world population is infected with Helicobacter pylori. Of those, approx. 500,000 die from gastric carcinoma every year. Ulcer disease, gastricatrophy and the rare MALT lymphoma are other sequelae of H. pylori infection. H. pylori possesses an array of virulence factors that include urease, flagellar motility, adhesins, the vacuolating cytotoxin VacA and the protein CagA. The gene encoding CagA is located on the cag pathogenicity island, comprising 29 genes the majority of which encodes components of a type IV secretion system capable of translocating CagA into epithelial cells where it interferes with cellular signal transduction processes. A number of diagnostic tests for H. pylori infection require gastroendoscopy. These include the biopsy urease test, histology, culture with susceptibility testing, and molecular detection methods such as fluorescent in situ hybridization. Non-invasive tests that do not require endoscopy include the (13)C urea breath test, H. pylori stool antigen ELISA and serology. The latter is unsuitable for treatment follow-up, since antibody titres persist up to a year after successful treatment. When patients have never been treated for H. pylori infection, biopsy urease test and histology are usually sufficient for diagnosis. In patients where endoscopy is not required, H. pylori infection can be reliably detected by (13)C urea breath test, stool antigen ELISA or serology. Patients who have under gone one or more unsuccessful cycles of eradication therapy in most cases harbour H. pylori resistant to one or several antibiotics. In these patients, culture and antibiotic susceptibility testing are indicated. Patients who have never been treated for H. pylori infection usually harbour susceptible strains. In such patients, classic "Italian" or "French" triple therapies may achieve eradication in >90% of cases. In the case of treatment failure, second-line antibiotic treatment regiments (rescue therapy) are used, optimally guided by susceptibility data.

Helicobacter pylori eradication and gastric ulcer healing--comparison of three pantoprazole-based triple therapies.

AIM: To study the efficacy of three pantoprazole-based triple therapy regimens for the eradication of Helicobacter pylori infection and gastric ulcer healing. METHODS: In an open, multi-centre, randomized study, 519 H. pylori-positive patients with active gastric ulcer were randomized to receive pantoprazole (40 mg) (P) and two of three antibiotics: clarithromycin (500 mg) (C), metronidazole (500 mg) (M) or amoxicillin (1000 mg) (A). Triple therapy (PAC, PCM, PAM) was administered twice daily for 7 days, followed by pantoprazole until the ulcer had healed. Antrum and corpus biopsies were taken to determine the pattern of gastritis, to assess the H. pylori status and to determine the strain susceptibility to antibiotics, and from the ulcer margins and base to exclude malignancy. Scores based on the Sydney system were used to categorize the gastritis phenotypically. RESULTS: The H. pylori eradication rates for the per protocol (intention-to-treat) analysis were 89% (67%) for PAC, 83% (68%) for PCM and 76% (60%) for PAM, with a significant difference between PAC and PAM. Healing rates after 4 weeks were 91% for PAM, 90% for PCM and 88% for PAC (per protocol analysis). The eradication rates were lower in patients in whom strains resistant to any antibiotic used in the triple therapies were detected. Successful eradication [odds ratio, 5.2 (3.3; 8.3)] and the ulcer size (< 15 mm) were significant predictors for healing after 4 weeks. The regimens showed a comparable safety profile and compliance. CONCLUSIONS: Pantoprazole-based triple therapies are effective in the eradication of H. pylori infection in gastric ulcer patients, as reported in previous similar sized studies in duodenal ulcer patients. Successful eradication and an ulcer size of < 15 mm are the best predictors of gastric ulcer healing after 4 weeks.

Antibiotic-associated diarrhea: incidence, risk factors of antibiotics and patients, pathophysiology and differential diagnosis--an interdisciplinary approach to a common problem.

Antibiotic-associated diarrhea (AAD) is a common complication of antibiotic treatment, most often seen in non-hospitalised patients. In principle, such diarrhea can be triggered by any antibiotic. An interdisciplinary working group discussed the different aspects of AAD in view of its gastroenterological, microbiological, paediatric, general medical and pharmaceutical implications, also in consideration of the position of patients and health insurance funds. The incidence, risk factors of antibiotics and patients, the pathophysiology of the various types of AAD and the differential diagnosis are reviewed.

Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level.

The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pylori regulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 microM NiCl(2) resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptional ureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pylori urease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.

The role of Helicobacter pylori virulence factors in interleukin production by monocytic cells.

Helicobacter pylori infection results in chronic gastritis, which is initiated by the release of cytokines like interleukin (IL)-12 and IL-8 from mononuclear cells, and IL-8 from gastric epithelial cells. The severity of gastritis is influenced both by host factors and by bacterial factors such as the Cag proteins and the vacuolating cytotoxin VacA. Amounts of IL-12 and IL-8 produced by monocytic THP-1 cells differed considerably between the eight H. pylori isolates tested, but in contrast to H. pylori-induced IL-8 production by gastric epithelial cells, did not correlate to the Cag and VacA types of the strains. Apparently, in addition to Cag and VacA, other bacterial factors determine the extent in which H. pylori induced IL production in monocytes.

Diagnosis of small intestinal bacterial overgrowth in patients with cirrhosis of the liver: poor performance of the glucose breath hydrogen test.

BACKGROUND/AIMS: Small intestinal bacterial overgrowth is known to occur in association with cirrhosis of the liver and studies are needed to assess its pathophysiological role. The glucose breath hydrogen test as an indirect test for small intestinal bacterial overgrowth has been applied to patients with cirrhosis but has not yet been validated against quantitative culture of jejunal secretion in this particular patient population. METHODS: Forty patients with cirrhosis underwent glucose breath hydrogen test and jejunoscopy. Jejunal secretions were cultivated quantitatively for aerobe and anaerobe microorganisms. RESULTS: Small intestinal bacterial overgrowth was detected by culture of jejunal aspirates in 73% of patients, being associated with age and the administration of acid-suppressive therapy. The glucose breath hydrogen test correlated poorly with culture results, sensitivity and specificity ranging from 27%-52% and 36%-80%, respectively. CONCLUSIONS: In patients with cirrhosis, the glucose breath hydrogen test correlates poorly with the diagnostic gold standard for small intestinal bacterial overgrowth. Until other non-invasive tests have been validated, studies addressing the role of small intestinal bacterial overgrowth in patients with cirrhosis should resort to microbiological culture of jejunal secretions.

Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori.

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun.

Helicobacter pylori is an etiological agent in the development of mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma. Patients infected with H. pylori carry a 3-6-fold increased risk of developing cancer compared with uninfected individuals. H. pylori strains expressing the cytotoxin-associated antigen A (CagA) are more frequently associated with the development of neoplasia than cagA-negative strains. However, the molecular mechanism by which H. pylori causes neoplastic transformation remains unclear. Here we report that exposure of gastric epithelial cells to H. pylori induces activation of the transcription factor activator protein 1. Activation of the proto-oncogenes c-fos and c-jun is strongly induced. We show that H. pylori activates the ERK/MAP kinase cascade, resulting in Elk-1 phosphorylation and increased c-fos transcription. H. pylori strains that do not express CagA or that are mutated in cag genes encoded by the CagI pathogenicity island do not induce activator protein 1, MAP kinase activity, or c-fos or c-jun activation. Proto-oncogene activation may represent a crucial step in the pathomechanism of H. pylori induced neoplasia.

Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation.

BACKGROUND: The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. AIM: To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. METHODS: A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. RESULTS: In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. CONCLUSIONS: Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples.

Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp).

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

High prevalence of antibodies to calreticulin of the IgA class in primary biliary cirrhosis: a possible role of gut-derived bacterial antigens in its aetiology?

BACKGROUND: In a preliminary study we showed that antibodies to the endoplasmic reticulum protein calreticulin (CR) occur in primary biliary cirrhosis (PBC) and autoimmune hepatitis type 1 (AIH). Since anti-CR antibodies have also been found in patients with infectious diseases, we investigated their prevalence and immunoglobulin classes in patients with various hepatic and intestinal diseases, hoping to get some information on a possible relationship between an infectious trigger and the induction of a certain class of anti-CR antibodies. METHODS: Sera were tested for anti-CR antibodies of the IgA, IgG, and IgM class by Western blotting, using CR isolated from human liver: in autoimmune liver diseases (primary biliary cirrhosis (PBC) (n = 86) and autoimmune hepatitis (AIH) type 1 (n = 57)), alcoholic liver cirrhosis (ALC) (n = 32), viral liver infections (acute hepatitis A (n = 8), acute hepatitis B (n = 20), and chronic hepatitis C (n = 28)), and intestinal diseases (Crohn disease (CD) (n = 30), acute yersiniosis (n = 26)). Sera from 100 healthy individuals served as negative controls. RESULTS: The most prominent finding was the high prevalence of anti-CR antibodies of the IgA class and the similarity in the anti-CR antibody class pattern in PBC (IgA, 62%; IgG, 43%; IgM, 55%) and yersiniosis (IgA, 62%; IgG, 39%; IgM, 42%). Class IgA anti-CR antibodies also occurred frequently in ALC (IgA, 44%; IgG, 41%; IgM, 19%). In contrast, in AIH anti-CR antibodies were predominantly of class IgG (IgA, 28%; IgG, 60%; IgM, 33%). In hepatitis A anti-CR antibodies were absent. In the other diseases they had a low prevalence and were mostly of class IgG (acute hepatitis B: IgA, 0%; IgG, 15%; IgM, 0%; chronic hepatitis C: IgA, 7%; IgG, 21%; IgM, 0%; CD: IgA, 13%; IgG, 20%; IgM, 13%). Of the healthy individuals 7% had anti-CR antibodies exclusively of class IgG. CONCLUSIONS: The high prevalence of anti-CR antibodies of class IgA in patients with PBC and yersiniosis as well as in alcoholic liver disease reflects a reactivity of the gut-associated immune system and could imply that a still undefined gut-derived bacterial (?) agent may trigger PBC.

Proteins encoded by the cag pathogenicity island of Helicobacter pylori are required for NF-kappaB activation.

Helicobacter pylori is the etiological agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. The difference in virulence between individual strains is reflected in their ability to induce interleukin-8 (IL-8) secretion from gastric epithelial cells. It has been shown that virulence is associated with the presence of a bacterial gene cluster (a pathogenicity island). We have recently demonstrated that H. pylori-mediated IL-8 secretion requires activation of the transcription factor NF-kappaB. Here, we show that NF-kappaB induction requires six membrane proteins encoded within the pathogenicity island.

A secreted/shed product of Helicobacter pylori activates transcription factor nuclear factor-kappa B.

Helicobacter pylori is an etiologic agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. Exposure of gastric epithelial cells to H. pylori induces secretion of the cytokine IL-8, which plays a pivotal role in the immunopathogenesis of H. pylori infections. Isolated Helicobacter strains differ in their virulence and in their ability to induce cytokine production. High degrees of virulence correlate with enhanced IL-8 production. However, the molecular mechanism of this variance in Helicobacter pathogenicity remains poorly understood. Here we show that H. pylori-mediated IL-8 secretion requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in a gastric epithelial cell line. Several H. pylori strains which fail to induce IL-8 secretion do not activate NF-kappaB, while all IL-8-inducing strains activate the transcription factor. Moreover, the antioxidant curcumin, which inhibits NF-kappaB activation, also completely suppresses IL-8 induction by H. pylori. NF-kappaB activation is not mediated by LPSs, since purified H. pylori LPS had no effect on gastric epithelial cells. In contrast, both IL-8 secretion and NF-kappaB activation require a secreted H. pylori product, which is not secreted by strains mutated in picB/cagE, a recently identified putative transport protein.

Interaction of Helicobacter pylori (strain 151) and Campylobacter coli with human peripheral polymorphonuclear granulocytes.

Helicobacter pylori associated gastritis is characterized by dense mucosal inflammatory infiltrations with predominantly neutrophilic granulocytes, together with a local and systemic immune response. Nevertheless, the natural course of the infection is chronic in nature, and active phagocytosis of H. pylori by mucosal granulocytes was only rarely observed. The aim of the present study was to investigate with electronmicroscopic methods the interaction of H. pylori with freshly harvested human peripheral granulocytes, with Campylobacter coli as control organism. Bacteria, either untreated or opsonized with complement or antiserum, were coincubated with phagocytes for up to 120 min. After defined time periods the following parameters were electronmicroscopically evaluated: i) internalization of bacteria., ii) morphological characteristics of bacteria and phagocytes, iii) decrease of lysosomes, and iv) by use of myeloperoxidase staining, the characteristics of phagolysosomal fusion. In the absence of complement, both organisms were internalized to a comparable extent. However, in contrast to C. coli, remarkable amounts of H. pylori cells remained extracellularly attached even after 120 min of coincubation, as well as internalized bacteria remained morphologically largely unimpaired. If complement was present, internalization and morphological destruction of H. pylori cells were significantly enhanced. The latter was characterized by rounding and swelling of H. pylori cells. It was already apparent in the extracellular space, and therefore probably induced by a complement effect, rather than by tee phagocytic action. Decrease of lysosomes, in general paralleled the degree of microbial uptake. Myeloperoxidase staining experiments furthermore showed an obviously regular consumption of lysosomal granules. However, if complement opsonization was excluded, lysosomal degranulation was not accompanied by a corresponding degradation of H. pylori cells, the latter indicating an at least partial resistance to phagocyte caused microbicidal mechanisms. In most of those cases ingested H. pylori cells were, in contrast to C. coli, surrounded by a rather "tight" phagosome. A possible explanation for this phenomenon could be a "leakage" of the phagosomal membrane, possibly caused by membranotoxic ammonia produced by the organism. If such an impairment of the phagocytic action would occur in vivo, it could lead to an impaired cellular defense, and therefore contribute to the chronic course of H. pylori infections.

Serodiagnosis of Helicobacter pylori infections with an enzyme immunoassay using the chromatographically purified 120 kilodalton protein.

A membrane-associated 120 kDa protein of Helicobacter pylori with known species-specificity was isolated and used in an enzyme immunoassay (EIA) for the detection of Helicobacter pylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis of Helicobacter pylori infections: an EIA using sonicated whole Helicobacter pylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92%) compared with the EIA using a whole cell sonicate of a single Helicobacter pylori strain as antigen (60.7%) and the immunoblot (90.2%). The sensitivity was 96%, 100% and 92%, respectively. Sera of three control patients reacted strongly in all three methods, indicating possible Helicobacter pylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis of Helicobacter pylori infections.

Cationic Yersinia antigen-induced chronic allergic arthritis in rats. A model for reactive arthritis in humans.

Cationic antigens are known to have considerable arthritogenic potential in experimental systems. During a systematic search for suitable, naturally occurring candidates an intracellular protein was isolated from the ribosomal pellet of Yersinia enterocolitica 0:3, a bacterial strain associated with reactive arthritis in humans. The protein is highly cationic, contains two 19-kD polypeptide chains linked by a disulfide bond, and reveals a strong tendency for spontaneous aggregation. It is suggested to be a nucleic acid binding protein. We tested this antigen for its ability to induce arthritis after intra-articular challenge in preimmunized rats. An acute inflammatory phase followed by transition to chronicity was observed both by technetium-99m scintigraphy and from histology. Massive polymorphonuclear leucocyte infiltration of the synovium was seen early on and fibrosis and thickening of the joint capsule occurred in later stages. Control groups showed no evidence of inflammation. Western blot and ELISA analysis of unselected sera from Yersinia enterocolitica 0:3-infected patients revealed antibodies to the antigen in the majority of cases, whereas healthy individuals rarely reacted. This is the first report of a naturally occurring cationic antigen capable of inducing immunologic tissue injury; it justifies the speculation that cationic antigens from prokaryotic cells could trigger reactive arthritis in humans.