Helicobacter pylori Colonization Drives Urokinase Receptor (uPAR) Expression in Murine Gastric Epithelium During Early Pathogenesis.

(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.

Transient and Persistent Gastric Microbiome: Adherence of Bacteria in Gastric Cancer and Dyspeptic Patient Biopsies after Washing.

Helicobacter pylori is a common colonizer of the human stomach, and long-term colonization has been related to development of atrophic gastritis, peptic ulcers and gastric cancer. The increased gastric pH caused by H. pylori colonization, treatment with antibiotics or proton pump inhibitors (PPI) may allow growth of other bacteria. Previous studies have detected non-Helicobacter bacteria in stomach biopsies, but no conclusion has been made of whether these represent a transient contamination or a persistent microbiota. The aim of this study was to evaluate the transient and persistent bacterial communities of gastric biopsies. The washed or unwashed gastric biopsies were investigated by cultivation and microbiota analysis (16S rRNA gene-targeted amplicon sequencing) for the distribution of H. pylori and other non-Helicobacter bacteria. The number of cultured non-Helicobacter bacteria decreased in the washed biopsies, suggesting that they might be a transient contamination. No significant differences in the bacterial diversity were observed in the microbiome analysis between unwashed and washed biopsies. However, the bacterial diversity in biopsies shown H. pylori-positive and H. pylori-negative were significantly different, implying that H. pylori is the major modulator of the gastric microbiome. Further large-scale studies are required to investigate the transient and persistent gastric microbiota.

Inflammation, Immunity, and Vaccines for Helicobacter pylori Infection.

During the last year, a variety of studies have been published that increases our understanding of the basic mechanisms of immunity and inflammation in Helicobacter pylori infection and progression to gastric cancer. Innate immune regulation and epithelial cell response were covered by several studies that contribute with new insights in the host response to H. pylori infection. Also, the adaptive immune response to H. pylori and particularly the role of IL-22 have been addressed in some studies. These advances may improve vaccine development where new strategies have been published. Two major studies analyzed H. pylori genomes of 39 worldwide strains and looked at the protein profiles. In addition, multi-epitope vaccines for therapeutic use have been investigated. Studies on different adjuvants and delivery systems have also given us new insights. This review presents articles from the last year that reveal detailed insight into immunity and regulation of inflammation, the contribution of immune cells to the development of gastric cancer, and understanding mechanisms of vaccine-induced protection.

A Novel Assay for Easy and Rapid Quantification of Helicobacter pylori Adhesion.

BACKGROUND: Reducing adhesion of Helicobacter pylori to gastric epithelial cells could be a new way to counteract infections with this organism. We here present a novel method for quantification of Helicobacter pylori adhesion to cells. METHODS: Helicobacter pylori is allowed to adhere to AGS or MKN45g cells in a 96-well microtiter plate. Then wells are added saponin, which lyses the cells without affecting the bacteria. After addition of alamarBlue(®) (resazurin) and 1- to 2-hour incubation, fluorescence measurements can be used to quantify the number of adherent bacteria. RESULTS: By use of the method, we demonstrate that adhesion of both a sabA and babA deletion mutant of H. pylori is significantly reduced compared to the wild type. CONCLUSION: The method offers a number of applications and may be used to compare the adherence potential of different strains of H. pylori to either cells or different materials or to screen for potential anti-adhesive compounds. The results presented here suggest that this easy and reproducible assay is well suited for quantitative investigation of H. pylori adhesion.

Inflammation, immunity, and vaccines for Helicobacter.

Helicobacter pylori represents the major etiologic agent of gastritis, gastric, and duodenal ulcer disease and can cause gastric cancer and mucosa-associated lymphoid tissue B-cell lymphoma. It is clear that the consequences of infection reflect diverse outcomes of the interaction of bacteria and host immune system. The hope is that by deciphering the deterministic rules--if any--of this interplay, we will eventually be able to predict, treat, and ultimately prevent disease. Over the past year, research on the immunology of this infection started to probe the role of small noncoding RNAs, a novel class of immune response regulators. Furthermore, we learned new details on how infection is detected by innate pattern recognition receptors. Induction of effective cell-mediated immunity will be key for the development of a vaccine, and new work published analyzed the relevance and contribution of CD4 T helper cell subsets to the immune reaction. Th17 cells, which are also induced during natural infection, were shown to be particularly important for vaccination. Cost-efficiency of vaccination was re-assessed and confirmed. Thus, induction and shaping of the effector roles of such protective Th populations will be a target of the newly described vaccine antigens, formulations, and modes of application that we also review here.

Inflammation, immunity, and vaccines for Helicobacter pylori.

Helicobacter pylori infects almost half of the population worldwide and represents the major cause of gastroduodenal diseases, such as duodenal and gastric ulcer, gastric adenocarcinoma, autoimmune gastritis, and B-cell lymphoma of mucosa-associated lymphoid tissue. Helicobacter pylori induces the activation of a complex and fascinating cytokine and chemokine network in the gastric mucosa. Different bacterial and environmental factors, other concomitant infections, and host genetics may influence the balance between mucosal tolerance and inflammation in the course of H. pylori infection. An inverse association between H. pylori prevalence and the frequencies of asthma and allergies was demonstrated, and the neutrophil activating protein of H. pylori was shown to inhibit the allergic inflammation of bronchial asthma. During the last year, significant progress was made on the road to the first efficient vaccine for H. pylori that will represent a novel and very important bullet against both infection and gastric cancer.

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection.

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.

Delineation of Campylobacter concisus genomospecies by amplified fragment length polymorphism analysis and correlation of results with clinical data.

Campylobacter concisus has been as frequently isolated from human diarrhea as the important enteropathogen Campylobacter jejuni, but it also occurs in the feces of healthy individuals. The role of C. concisus in human disease has been difficult to determine, since the species comprises at least two phenotypically indistinguishable but genetically distinct taxa (i.e., genomospecies) that may vary in pathogenicity. We examined 62 C. concisus strains by amplified fragment length polymorphism (AFLP) profiling and correlated the results with clinical data. All C. concisus strains gave unique AFLP profiles, and numerical analysis of these data distributed the strains among four clusters. The clustering was of taxonomic significance: two clusters contained, respectively, the type strain (of oral origin) and a reference strain (from diarrhea) of each of the known genomospecies. Genomospecies 2 strains were more frequently isolated from immunocompetent patients and/or patients without concomitant infections that presented with fever, chronic diarrhea, and gut inflammation than was genomospecies 1, clustering with the type strain of oral origin. Bloody diarrhea was recorded only with C. concisus genomospecies 2 infections. We identified two additional C. concisus genomospecies: genomospecies 3 comprised a single strain from an immunocompetent patient, and genomospecies 4 contained five isolates from severely immunodeficient patients, i.e., organ transplantation recipients or those with hematological malignancies. All genomospecies 4 strains were of the same protein profile group and failed to react with a C. concisus species-specific PCR assay based on 23S rRNA gene sequences: the taxonomic position of this group requires closer investigation. Campylobacter concisus is genetically and taxonomically diverse and contains at least four distinct genomospecies that may exhibit differences in their spectra of virulence potential.

Characterization and subgrouping of Campylobacter concisus strains using protein profiles, conventional biochemical testing and antibiotic susceptibility.

OBJECTIVE: To characterize and subgroup clinical strains of Campylobacter concisus isolated from patients with gastrointestinal disease. METHODS: A total of 109 C. concisus isolates from 98 patients obtained between June 1997 and December 1998 were analysed using protein profiles, conventional biochemical tube tests, ApiCampy, and susceptibility patterns by Neosensitabs and E-test. RESULTS: Two groups were identified by using protein profiles. One resembled the ATCC 33237 type strain of oral origin, and a second group differing from it, particularly in the high molecular weight zone. Considerable diversity exists in the lower molecular range of the gels, also within assigned subgroups. Biochemical testing showed differences between the groups in the ability to reduce nitrate, ApiCampy testing also yielded differences between the two assigned groups, although reactions were highly heterogeneous. Resistance to erythromycin, ciprofloxacin, ampicillin, ceftriaxone and tetracycline occurred in 3%, 13%, 7%, 11% and 0% of the isolates when using Neosensitabs. The E-test yielded comparable results 7%, 5%, 0%, 2% and 3%, respectively. CONCLUSION: Results indicate that C. concisus can be assigned to two broad groups based on differences in protein profiles. No distinct phenotypic marker was identified. Susceptibility patterns are not suitable for discrimination between the two assigned groups. Further studies using a polyphasic approach including the application of genetic methods are needed to assess the complex taxonomy of this potential pathogen.

Gastric inflammatory markers and interleukins in patients with functional dyspepsia, with and without Helicobacter pylori infection.

Helicobacter pylori is the most important cause of gastritis, peptic ulcers and the development of gastric cancer. The chronic active inflammation is dominated by neutrophils, macrophages, lymphocytes and plasma cells. Several interleukins (IL-8, IL-10 and IFN-gamma) are involved in the inflammatory process in the gastric mucosa. The aim of this study was to investigate the gastric inflammation in patients with functional dyspepsia. Fifty-three consecutive patients were included and antral biopsies were obtained for histology, culture and immunohistochemistry. The sections were examined for the interleukins IL-4, IL-6, IL-8, IL-10 and IFN-gamma as well as for the cell markers CD4, CD8, CD14, Cd19, CD25 and CD30. Only CD4 and CD19 were significantly increased in patients with increased gastric inflammation and increased density of H. pylori. However, several of the examined markers (IFN-gamma, IL-8, IL-10 and CD14) showed a non-significant trend to be increased in patients with extensive gastric inflammation and high density of H. pylori. Therefore, an arbitrary index (IM11) for all the 11 immunological markers was made as an average value for each of the four morphological groups. For the four morphologically different groups of patients the values were 0.49, 0.77, 0.86 and 1.25, respectively. Significant increases in the index from none to moderate antral inflammation as well as the density of H. pylori were found (p<0.001). By using an index of inflammatory markers trends can be summarized and thereby significant which may be of importance when gastric inflammation is investigated in children and patients with functional dyspepsia.

Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori.

Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.