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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Helicobacter pylori infects over 50 % of the world’s population, causing gastritis, and in some cases, peptic ulcer disease and gastric cancer. Adherence to the gastric surface occurs primarily through H. pylori outer membrane proteins (HOPs) and is essential for bacterial survival and establishment of infection. The Blood group Antigen-Binding Adhesin (BabA) is the best-characterized attachment protein, mediating adherence by binding to fucosylated carbohydrate structures on the surface of the gastric epithelium. H. pylori is highly adaptable to environmental changes that occur during stomach longterm infection, however little is known about the effect of such changes on the adaptability and functionality of BabA adherence properties. The aim of this study was to evaluate how BabA-mediated binding properties of H. pylori isolates were affected during 20 years of chronic infection. Two H. pylori clinical isolates collected from a single individual, 20 years apart were studied for their Leb-binding properties using a combination of radioimmunoassay (RIA) and in situ histo- and cytochemistry. Our results demonstrated that H. pylori isolated after 20 years of infection had lost its Lewis b binding ability due to a nucleotide deletion in the babA gene, resulting in a translational frame shift and hence, a non-functional BabA protein. We also showed that the non-adherent isolate contains sub-populations of bacteria that express BabA and have therefore maintained the ability to bind to Leb-conjugate and adhere to human gastric mucosa in vitro.   An additional adherence pattern was revealed when H. pylori bacterial cells were applied to human buccal epithelium cells (BEC), with all the isolates demonstrating attachment. These results suggest that H. pylori can express additional binding properties for adherence in the oral cavity which may contribute to re-infection as well as further transmission of the H. pylori infection.

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium. It resides in the gastric mucous layer and epithelial lining of the stomach, often clustering at the junction of epithelial cells. H. pylori colonization usually occurs during childhood, and, when left untreated, generally persists for the host’s lifetime. Persistent H. pylori infection can cause chronic superficial gastritis and gastric duodenal ulcers, which is possibly linked to the development of gastric carcinoma and primary gastric lymphoma, especially of the mucosa-associated lymphoid tissue (MALT) type. It was recently defined as a class 1 carcinogen. The gastric inflammatory response to H. pylori infection is characterized by infiltration of the mucosa by neutrophils, T and B cells, plasma cells and macrophages. This reaction is initially induced by H. pylori attachment, followed by cytokine release by gastric epithelial cells. Epidemiological studies revealed that more than 50% of adults are infected with H. pylori all over the world. However, interestingly, only a subset of individuals develops serious H. pylori-related disease, while most infected individuals show no clinical symptoms. Gastric epithelial cells, like intestinal epithelial cells, express a subset of Toll-like receptors (TLRs) and similar pattern recognition receptors, which are important for the activation of the innate immune system. Bacterial components such as lipopeptides, peptidoglycan, LPS, flagellin, and CpG DNA are the ligands of TLRs. Thus, TLRs in gastric epithelial cells might be able to contribute to innate immune responses to H. pylori infection. However, there is scant knowledge about the mechanisms of innate immune response to acute and chronic H. pylori infection. This study is focused on host cell interaction with H. pylori flagellins, which are major components of the flagellar apparatus, and innate immune responses against them. The flagellins, which are essential for bacterial motility, are important for H. pylori to survive in the stomach mucus during the whole infectious cycle. Flagellins are known to act as the main determinant of many mucosal pathogenic bacteria that mediates proinflammatory signaling, including transcriptional factor NF-B activation via TLR5. In the first part of the study, we investigated the effects of H. pylori flagellins on TLR5 expression, NF-B activation and IL-8 production in various human intestinal and gastric epithelial cell lines by using Western blotting, semi-quantitative RT-PCR and ELISA. IL-8 is a potent neutrophil-activating chemokine expressed by gastric epithelial cells. When we stimulated the cells with the native form of or E. coli-expressed recombinant H. pylori flagellins, FlaA and FlaB, IL-8 was not induced in any case, while S. typhimurium flagellin (FliC) induced it significantly. H. pylori was able to modulate TLR5 protein expression and NF-B activation in epithelial cells regardless of the presence of flagellins. Having established the finding that H. pylori flagellins have unusually low immune-stimulatory properties, we further investigated to find out possible reasons why H. pylori flagellins are distinct from other flagellins of pathogenic bacteria in terms of immune-stimulatory activity. From amino acid sequence comparisons, we found that some regions in the terminal D0D1 protein domains of H. pylori flagellins are different from flagellins of other pathogenic bacteria. D0D1 is the domain which is known to interact with TLR5 in Salmonella FliC. To examine whether the differences endow H. pylori flagellins with low immune-stimulatory properties, we created several mutated H. pylori flagellins (FlaA and FlaB) by site-directed mutagenesis that contain one to four epitopes of Salmonella flagellin D0D1 domain amino acid sequences. The mutant flagellins expressed both in H. pylori and E. coli were used to determine their influence on TLR5-signaling mediators and cytokines, such as MAPkinases, (ERK, p38), NF-B, IL-8, and MIP-3. Salmonella FliC expressed in E. coli induced activation of p38, IB and NF-B leading to IL-8 and MIP-3 production in gastric epithelial cells. However, none of the H. pylori flagellin mutants activated MAP kinases or induced those cytokines. In a co-immunoprecipitation assay none of the recombinant wild type or mutated H. pylori flagellins showed any direct physical interaction with TLR5, while Salmonella FliC significantly co-precipitated with TLR5. Interestingly, we found H. pylori flagellins bind to the surface of gastric epithelial cells like FliC, although they do not bind to or stimulate TLR5. Based on the physical interaction of H. pylori flagellins and FliC with human gastric epithelial cells, we further analyzed transcriptional regulation by H. pylori flagellin in these host cells using microarray analysis. The result showed that H. pylori flagellins modulate host cell gene expression, and many of the identified regulation events overlap with the genes regulated by FliC. These findings imply that H. pylori flagellins do play a role in gene regulation of host cells probably through still unknown factors or receptors, although they do not trigger TLR5-related signaling pathways. The results of our study suggest that, in addition to the low immune-stimulatory activity of H. pylori LPS, the evolutionary reduction in stimulating activity of H. pylori flagellins on the local innate immune responses in the stomach in vivo might be a further strategy of this chronic mucosal pathogen to evade and minimize deleterious host responses, thereby promoting life-long persistence in the host, and possibly contributing to cancerogenesis. / Helicobacter pylori (H. pylori) ist ein gram-negatives, mikroaerophiles, spiralförmiges Bakterium. Es besiedelt die Schleimschicht und die Epitheloberfläche des Magens, wobei es sich besonders an den Kontaktstellen der Epithelzellen anlagert. Die Kolonisation mit H. pylori erfolgt normalerweise während der Kindheit und bleibt, wenn sie nicht behandelt wird, im allgemeinen während der gesamten Lebenszeit des Wirtes bestehen. Die persistierende H. pylori-Infektion kann chronische oberflächliche Gastritis und Zwöffingerdarmgeschwüre verursachen. Die Infektion kann auch zur Entwicklung von Magenkrebs und Lymphomen des Mukosa-assoziierten Lymphgewebes (MALT-Lymphom) führen. H. pylori ist seit 1994 als Typ I-Karzinogen klassifiziert. Die durch eine H. pylori-Infektion induzierte Entzündungsreaktion der Magenschleimhaut ist charakterisiert durch eine Infiltration der Schleimhaut mit neutrophilen Granulozyten, T- und B-Zellen, Plasmazellen und Makrophagen. Diese Reaktion wird ausgelöst durch die Anheftung von H. pylori gefolgt von der Freisetzung von Cytokinen durch die Magenepithelzellen. Epidemiologische Studien haben ergeben, dass weltweit mehr als 50% aller Erwachsenen mit H. pylori infiziert sind. Jedoch entwickelt interessanterweise nur eine Teilgruppe der infizierten Individuen eine ernsthafte H. pylori-assoziierte Krankheit, während die meisten Infizierten keine klinischen Symptome zeigen. Magenepithelzellen exprimieren, genauso wie Darmepithelzellen, eine Reihe von TOLL-like Rezeptoren (TLRs) und ähnliche Musterekennungsrezeptoren, die wichtig für die Aktivierung des angeborenen Immunsystems sind. Bakterielle Komponenten, wie z. B. Lipopeptide, Peptidoglycan, LPS, Flagellin und CpG-DNA sind die Liganden der TLRs. Auf diese Weise könnten die TLRs in den Magenepithelzellen in der Lage sein, zu der angeborenen Immunreaktion auf eine H. pylori-Infektion beizutragen. Jedoch ist bisher nur wenig über die Mechanismen der angeborenen Immunreaktion auf eine akute und chronische H. pylori-Infektion bekannt. Diese Studie befasst sich mit den Zellinteraktionen mit und den Antworten des Wirtsimmunsystems auf H. pylori-Flagelline, stark exprimierte Proteine des Flagellenapparats. Der Flagellenapparat ist essentiell für die Fähigkeit der Bakterien, im Magenschleim (Mukus) beweglich zu sein, und befähigt die Bakterien dazu, während des gesamten Infektionszyklus im Mukus und an der Magenmukosa zu überleben. Flagellin ist für viele pathogene Bakterien im Intestinaltrakt oder auch in der Lunge des Säuger-Wirts ein sehr wichtiger Faktor, der durch Bindung an TLR5 proinflammatorische Signalvorgänge, einschließlich der Aktivierung des Transkriptionsfaktors NF-B, herbeiführt. Im ersten Teil der vorliegenden Studie haben wir die Wirkungen von H. pylori-Flagellinen (FlaA, FlaB) auf TLR5-Expression, NF-B-Aktivierung und IL-8-Produktion in verschiedenen menschlichen Darm- und Magenepithelzelllinien mittels Western Blot, semi-quantitativer RT-PCR und ELISA untersucht. IL-8 ist ein hochwirksames Neutrophilen-aktivierendes Chemokin, welches von den Magenepithelzellen sezerniert wird und als ein Marker für die Zellaktivierung durch H. pylori, seine löslichen Produkte und Kontrollen diente. Nach Stimulation verschiedener Epithelzellen und humaner Makrophagen mit nativen oder in E. coli rekombinant hergestellten H. pylori-Flagellinen FlaA und FlaB wurde in keinem Fall IL-8 gebildet, während S. typhimurium-Flagellin (FliC) IL-8-Bildung und -Sekretion in signifikanter Menge induzierte. H. pylori war in der Lage, TLR5-Protein-Expression und die NF-B-Aktivierung in Epithelzellen zu modulieren, unabhängig von dem Vorhandensein von Flagellinen. Nachdem wir gezeigt hatten, dass H. pylori-Flagelline ungewöhnlich geringe immunstimulierende Eigenschaften besitzen, setzten wir unsere Untersuchungen fort, um mögliche Gründe herauszufinden, warum H. pylori-Flagelline sich von anderen Flagellinen pathogener Bakterien hinsichtlich der das Immunsystem stimulierenden Aktivitäten unterscheiden. Bei Vergleichen von Aminosäuresequenzen fanden wir heraus, dass einige Regionen in den terminalen D0D1-Domänen der H. pylori-Flagelline sich von Flagellinen anderer pathogener Bakterien unterscheiden. D0D1 ist der Funktionsbereich des Flagellins, von dem bekannt ist, dass er bei Salmonellen-FliC mit TLR5 interagiert. Um zu untersuchen, ob diese Unterschiede für die geringe immunstimulierende Wirkung von H. pylori-Flagellinen verantwortlich sind, generierten wir durch eine zielgerichtete Mutagenese mehrere mutierte H. pylori-Flagelline (FlaA und FlaB), die ein bis vier Epitope der Aminosäuresequenzen der D0D1-Domäne des Salmonella-Flagellins enthielten. Die mutierten Flagelline, die sowohl in H. pylori als auch in E. coli exprimiert wurden, wurden genutzt, um ihren Einfluss auf TLR5-Signal-Mediatoren und Cytokine, wie z. B. MAP-Kinasen (ERK, p38), NF-B, IL-8 und MIP-3α herauszufinden. In E. coli exprimiertes Salmonella-FliC bewirkte die Aktivierung von p38, IB, NF-B und ERK und führte zur Produktion von IL-8 und MIP-3α in den Magenepithelzellen. Im Gegensatz dazu aktivierte keine der H. pylori-Flagellinmutanten MAP-Kinasen oder induzierte diese Cytokine. Wir konnten durch Koimmunpräzipitationstechniken zeigen, dass wildtypische oder mutierte H. pylori-Flagelline nicht physisch mit TLR5 interagieren, während Salmonella-FliC spezifisch an TLR5 bindet. Interessanterweise fanden wir heraus, dass H. pylori-Flagelline wie FliC an die Oberfläche verschiedener humaner Epithelzellen binden, obwohl sie nicht TLR5 stimulieren oder an TLR5 binden. Basierend auf der physischen Interaktion von H. pylori-Flagellinen und FliC mit menschlichen Magenepithelzellen haben wir weiterhin die Transkriptionsregulation durch H. pylori-Flagellin in den Wirtszellen mit Hilfe der Microarray-Analyse untersucht. Die Ergebnisse zeigten, dass H. pylori-Flagelline die Gene der Wirtszelle modulieren, und viele der identifizierten Regulationsereignisse überschnitten sich mit den durch FliC regulierten Genen. Diese Ergebnisse implizieren, dass H. pylori-Flagelline doch eine Rolle bei der Genregulierung von Wirtszellen spielen, wahrscheinlich durch noch unbekannte Faktoren und Rezeptoren, obwohl sie keine mit TLR5 in Zusammenhang stehenden Signaltransduktionsketten auslösen. Die Resultate unserer Studien lassen darauf schließen, dass zusätzlich zu der das Immunsystem nur gering stimulierenden Aktivität von H. pylori-LPS die evolutionäre Reduzierung der stimulierenden Aktivität von H. pylori-Flagellinen auf lokale angeborene Immunreaktionen im Magen in vivo eine weitere Strategie dieses chronischen Schleimhautpathogens sein könnte, um schädliche Wirtsreaktionen zu verhindern und zu minimieren und hierdurch seine lebenslange Persistenz, jedoch auch die Krebsentstehung im Wirt zu fördern.

Fong, Yu Hang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 102-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Ho Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 103-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

H. pylori has been classified by World Health Organization as type I carcinogen for its association with gastric cancer. Among its suggested pathological pathways is oxidative stress, which may reduce the bioavailability of dietary antioxidants. The main objectives of this thesis were to estimate the prevalence of H. pylori infection in Ontario and to assess its effect on the bioavailability of two main dietary antioxidants, vitamins E and C. To estimate the prevalence of H. pylori infection, a volume of 10 ul of plasma was aliquoted from stored blood of 1306 adults from Ontario. The blood samples belonged to controls of a population-based study of colorectal cancer. The overall weighted seroprevalence of H. pylori was 23.1% (95% CI: 17.7-29.5) with males having higher infection rates than females. Seroprevalence of the infection increased with age and number of siblings. Being non-white, born outside Canada and immigrating at an age greater than 20 years increased risk for H. pylori infection. An inverse association with seroprevalence was found for education and alcohol consumption. In order to investigate whether H. pylori positive compared to H. pylori negative subjects have lower changes in plasma concentrations of ascorbic acid and alpha tocopherol when supplemented with these vitamins, H. pylori negative (n=32) and H. pylori positive (n=27) volunteers received vitamin C (500 mg) and alpha tocopherol (400 IU) supplementation daily for 28 days. H. pylori infection status was determined by 13C urea breath test. Post supplementation plasma ascorbic acid and alpha tocopherol were significantly higher than pre supplementation concentrations in both groups. The changes in plasma ascorbic acid and alpha tocopherol were not significantly different between H. pylori negative and positive groups (ascorbic acid: 13.97±16.86 vs. 20.87±27.66, p=0.76; alpha tocopherol: 15.52±9.4 vs. 14.47±15.77; p=0.39 for H. pylori negative and positive groups respectively). The weighted seroprevalence of H. pylori infection was 23.1%. Age, sex, ethnicity, place of birth, age at immigration, education and alcohol consumption were factors associated with the infection prevalence in the population studied. In addition, we found no effect of H. pylori infection on the bioavailability of vitamins E and C.

H. pylori has been classified by World Health Organization as type I carcinogen for its association with gastric cancer. Among its suggested pathological pathways is oxidative stress, which may reduce the bioavailability of dietary antioxidants. The main objectives of this thesis were to estimate the prevalence of H. pylori infection in Ontario and to assess its effect on the bioavailability of two main dietary antioxidants, vitamins E and C. To estimate the prevalence of H. pylori infection, a volume of 10 ul of plasma was aliquoted from stored blood of 1306 adults from Ontario. The blood samples belonged to controls of a population-based study of colorectal cancer. The overall weighted seroprevalence of H. pylori was 23.1% (95% CI: 17.7-29.5) with males having higher infection rates than females. Seroprevalence of the infection increased with age and number of siblings. Being non-white, born outside Canada and immigrating at an age greater than 20 years increased risk for H. pylori infection. An inverse association with seroprevalence was found for education and alcohol consumption. In order to investigate whether H. pylori positive compared to H. pylori negative subjects have lower changes in plasma concentrations of ascorbic acid and alpha tocopherol when supplemented with these vitamins, H. pylori negative (n=32) and H. pylori positive (n=27) volunteers received vitamin C (500 mg) and alpha tocopherol (400 IU) supplementation daily for 28 days. H. pylori infection status was determined by 13C urea breath test. Post supplementation plasma ascorbic acid and alpha tocopherol were significantly higher than pre supplementation concentrations in both groups. The changes in plasma ascorbic acid and alpha tocopherol were not significantly different between H. pylori negative and positive groups (ascorbic acid: 13.97±16.86 vs. 20.87±27.66, p=0.76; alpha tocopherol: 15.52±9.4 vs. 14.47±15.77; p=0.39 for H. pylori negative and positive groups respectively). The weighted seroprevalence of H. pylori infection was 23.1%. Age, sex, ethnicity, place of birth, age at immigration, education and alcohol consumption were factors associated with the infection prevalence in the population studied. In addition, we found no effect of H. pylori infection on the bioavailability of vitamins E and C.