Helicobacter pylori classified as a class 1 carcinogen is a common human pathogen implicated in certain gastrointestinal diseases. Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries. H. pylori infection causes peptic ulcer, duodenitis, gastritis and cancer. The growing resistance of H. pylori to antibiotics used in its treatment as well as other innate limitations of the triple therapy has necessitated a search for alternative treatment from natural sources which could be readily available, less cost effective. The antimicrobial activity of solvents (acetone, ethanol, methanol, chloroform and water) crude extracts of Lippia javanica were investigated against 31 H. pylori strains by the agar well diffusion technique. The minimum inhibitory concentration (MIC) was determined by spectrophotometric analysis at 620 nm using the broth micro dilution method and the rate of kill by broth dilution method. Phytochemical analysis was also performed. H. pylori standard strain NCTC 11638 was included as a positive control. Metronidazole and amoxicillin were used as positive control antibiotics. The ANOVA test was used to analyze the results using SPSS version 17.0. The strains were inhibited by all the extracts with inhibition zones of diameter ranging from 0-36 mm and 0-35 mm for the control antibiotic, clarithromycin. The MIC90 ranged from 0.039- 0.625 mg/mL for acetone; 0.039-1.25mg/mL for methanol, 0.00195-0.313 mg/mL for ethanol; 0.01975-2.5 mg/mL for metronidazole and 0.0048-2.5 mg/mL for amoxicillin. Acetone extract completely inhibited strain PE369C at MIC (0.1 mg/mL) and 2× MIC (0.2 mg/mL) in 18h and at ½× MIC (0.05 mg/mL) in 36h. Strain PE466C was completely inhibited at 4× MIC in 72h. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids and alkaloids. The results indicate that the extracts of the leaves of L. javanica may contain compounds with anti-H. pylori activity and merits further study to identify the compounds.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-09-25T16:51:04Z No. of bitstreams: 1 texto completo.pdf: 950177 bytes, checksum: b83dd272b596a399531e767b4a5521e6 (MD5) / Made available in DSpace on 2018-09-25T16:51:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 950177 bytes, checksum: b83dd272b596a399531e767b4a5521e6 (MD5) Previous issue date: 2018-06-18 / A infecção gástrica pelo Helicobacter pylori afeta até 50% da população mundial e está fortemente associada à doença ulcerosa péptica e ao câncer gástrico. Já existe evidência de que a terapia de erradicação desse patógeno diminua a incidência dessas duas doenças. O segundo e o terceiro Consensos Brasileiros sobre H. pylori indicam a combinação de um inibidor de bomba de prótons (IBP) com amoxicilina e claritromicina como terapia de primeira escolha para o primeiro tratamento e a combinação de IBP com amoxicilina e levofloxacino como alternativa preferencial para o segundo tratamento, mas esses protocolos têm sido criticados pela diminuição recente de sua efetividade em vários pontos do globo. Como existem poucos dados sobre a efetividade dos esquemas de erradicação de H. pylori no Brasil, este estudo buscou por meio de análise de uma coorte retrospectiva composta de pacientes da zona da mata de Minas Gerais, descrever os tratamentos prescritos para erradicação de H. pylori com suas respectivas efetividades e suas correlações com características sociodemográficas. Foram analisados 5.010 prontuários, tendo sido encontrados 314 pacientes com informações completas sobre os tratamentos para erradicação de H. pylori, repartidos em dois períodos (2007 a 2011 e 2013 a 2016). Foram encontradas 264 prescrições de primeiros tratamentos para erradicação de H. pylori, com uso de IBP associado a amoxicilina e claritromicina por sete dias em 88,25% dos casos, com uma taxa de efetividade deste esquema de 54,07%. Não foram encontradas diferenças de efetividade conforme a escolha do IBP e entre os dois períodos analisados. Foram realizadas análises univariada e multivariada e por mineração de dados buscando correlacionar as variáveis idade, sexo, escolaridade, estado civil, naturalidade, local de residência e profissão com a efetividade dos primeiros e segundos tratamentos. Não foram encontradas associações consistentes entre a efetividade dos tratamentos e as variáveis mencionadas. Os dados apresentados demonstraram que, nessa coorte, as taxas de efetividade dos esquemas recomendados pelos Consensos Brasileiros sobre Helicobacter pylori foram inferiores às anteriormente descritas pela literatura brasileira. Mais estudos são necessários para aprimoramento dos esquemas terapêuticos empregados, considerando inclusive a emergência da resistência antimicrobiana do H. pylori. / Helicobacter pylori gastric infection affects almost 50% of the population worldwide. It is strongly associated to peptic ulcer disease and gastric cancer. There is evidence that H. pylori eradication reduces the incidence of both conditions. The second and third Brazilian Consensus on H. pylori indicate the combination of a proton pump inhibitor (PPI) with amoxicillin and clarithromycin as first line therapy for the first treatment and the combination of PPI with amoxicillin and levofloxacin as a preferred alternative for the second treatment. However, these protocols have been criticized recently because a decrease in their effectiveness has been observed worldwide. As we have little data on the H. pylori eradication therapy effectiveness in Brazil, this study has analyzed retrospectively a cohort composed by patients living in southeast Minas Gerais State, southeast Brazil, to describe the rate of H. pylori eradication and its correlation to sociodemographic characteristics. Five thousand and ten medical records were analyzed and 314 patients with complete information on H. pylori eradication therapy, from two periods (2007 to 2011 and 2013 to 2016), were included. Two hundred sixty-four prescriptions were found for first attempt to eradicate H. pylori with an effectivity rate of 54.07% for the scheme using PPIs associated with amoxicillin and clarithromycin for seven days. No statistical differences were observed about treatment effectiveness according to the choice of IBP and between the two analyzed periods. Univariate, multivariate and data mining analyses correlating age, gender, schooling, marital status, naturality, place of residence and profession with the effectiveness of the first and second treatments were performed. No correlations among these features were found. In this cohort, H. pylori eradication rate using the protocols recommended by the Brazilian consensus on Helicobacter pylori were inferior to those previously described by Brazilian literature. More studies are needed to find the best protocols, considering the emerging H. pylori drug resistance.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22384号 / 社医博第106号 / 新制||社||医11(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 妹尾 浩, 教授 古川 壽亮, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

The aim ofthis study was to reveal the presence ofHelicobacter pylori (HP) in the Waldeyers lymphatic tissue in the group ofchildren and adults, along with its possible role in the etiology of tonsillar carcinoma and benign diseases (chronic tonsillitis, OSAS, adenoids). In our study we have confirmed the hypothesis that HP is presented m the Waldeyers lymphatic tissue. as well as in the stomach and that the oropharynx and epipharynx are the an extragastric reservoir ofHP. Mucosa associated lymphatic tissue in the stomach is similar to lymphatic tissue ofWaldeyer ring. These conditions can be very favourable for the survival ofHP and thus can promote inflammation changes and immune changes as well as in the stomach. ln our study, using the real-time PCR method we have detected high incidence ofHP DNA in adenoids and tonsillar tissue. ln the group ofbenign diseases, the most frequent genotypes were CagA­ VacAsIbm1 and CagA-VacAslbm2. In the group of patients with tonsillar carcinoma, the most frequent genotype was CagA-VacAslbml. Genotyping identified strains ofHP showed differences in comparison with the predominant strains which are most frequently found in the stomach. Genotypic analysis ofI-IP strains showed that the less prevalent virulent strains ofHP, known as cagA negative and vacA positive....

Molecular typing of strains among the highly diverse population of Helicobacter pylori (H. pylori) is an important approach for both basic and clinical studies. Genomic DNAs prepared from 18 gastric biopsy specimens, 21 H. pylori clinical isolates obtained from gastric biopsy specimens, and five isolates collected from a single patient at weekly intervals, were subjected to a combined single- and double-stranded conformational polymorphism (SDSCP) assay. The results showed that 19 of 21 isolates tested were discriminated by SDSCP analysis. SDSCP analysis of five H. pylori isolates collected from the same patient at different times resulted in five identical profiles, suggesting the reproducibility of the method. When DNA preparations from 18 gastric biopsy specimens were subjected to SDSCP analysis, 18 unique profiles were generated that matched those of their corresponding cultured H. pylori isolates from each patient. For comparison, polymerase chain reaction (PCR)-based restriction fragment length polymorphism analysis yielded only nine profiles for 20 strains. The data suggest that SDSCP analysis may be an effective and reliable method for differentiation of H. pylori strains directly from gastric biopsy specimens without requiring isolation of the organisms by culture.

Background: The prevalence of H. pylori is greater than 50% in low development countries. In children, infection with H. pylori produces effects such as low height, impairment in growth especially in pubertal age. Polymerase Chain Reaction has a high sensitivity and specificity of 89.6% and 100% respectively, compared to other invasive methods. The objective of this study is to determine the frequency of patients with presence of H. pylori in asymptomatic children in a rural community in the north of Peru, using PCR technique in stool samples. Methods: A cross-sectional study with a convenience sampling was performed, resulting in 147 children aged between 6-14 years old of the district of San Pablo in Cajamarca, Peru. Using stool samples H. pylori DNA was obtained, then PCR amplification was done, and sequencing those with positive results, in an agreement with Macrogen, Seoul - Korea. Results: It was found a frequency of H. pylori positive cases of 21.1% with a CI 95% of 14.8 to 28.6. It was found a frequency (p=0.01) of positive for H. pylori among males (29% IC95: 19.1- 40.5) compared to women (12.7%; IC95: 6.0-22.7). There was no statistically significant differences between those positive and negative patients with H. pylori compared to age (p = 0.57), presence of overweight (p = 0.09), or excreta disposal (p = 0.71). It was not found significance difference among other variables. Conclusion: Our study found a high frequency of H. pylori determined by a molecular test with high sensitivity and specificity, demonstrating the feasibility of its implementation in rural communities. It can be considered as a tool for surveillance in areas of high prevalence of H. pylori infection. / Tesis

Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

Cette thèse présente la découverte de nouveaux inhibiteurs de l’amidotranférase ARNt-dépendante (AdT), et résume les connaissances récentes sur la biosynthèse du Gln-ARNtGln et de l’Asn-ARNtAsn par la voie indirecte chez la bactérie Helicobacter pylori. Dans le cytoplasme des eucaryotes, vingt acides aminés sont liés à leur ARNt correspondant par vingt aminoacyl-ARNt synthétases (aaRSs). Ces enzymes sont très spécifiques, et leur fonction est importante pour le décodage correct du code génétique. Cependant, la plupart des bactéries, dont H. pylori, sont dépourvues d’asparaginyl-ARNt synthétase et/ou de glutaminyl-ARNt synthétase. Pour former le Gln-ARNtGln, H. pylori utilise une GluRS noncanonique nommée GluRS2 qui glutamyle spécifiquement l’ARNtGln ; ensuite, une AdT trimérique, la GatCAB corrige le Glu-ARNtGln mésapparié en le transamidant pour former le Gln-ARNtGln, qui lira correctement les codons glutamine pendant la biosynthèse des protéines sur les ribosomes. La formation de l’Asn-ARNtAsn est similaire à celle du Gln-ARNtGln, et utilise la même GatCAB et une AspRS non-discriminatrice. Depuis des années 2000, la GatCAB est considérée comme une cible prometteuse pour le développement de nouveaux antibiotiques, puisqu’elle est absente du cytoplasme de l’être humain, et qu’elle est encodée dans le génome de plusieurs bactéries pathogènes. Dans le chapitre 3, nous présentons la découverte par la technique du « phage display » de peptides cycliques riches en tryptophane et en proline, et qui inhibent l’activité de la GatCAB de H. pylori. Les peptides P10 (CMPVWKPDC) et P9 (CSAHNWPNC) inhibent cette enzyme de façon compétitive par rapport au substrat Glu-ARNtGln. Leur constante d’inhibition (Ki) est 126 μM pour P10, et 392 μM pour P9. Des modèles moléculaires ont montré qu’ils lient le site actif de la réaction de transmidation catalysée par la GatCAB, grâce à la formation d’une interaction π-π entre le résidu Trp de ces peptides et le résidu Tyr81 de la sous-unité GatB, comme fait le A76 3’-terminal de l’ARNt. Dans une autre étude concernant des petits composés contenant un groupe sulfone, et qui mimiquent l’intermédiaire de la réaction de transamidation, nous avons identifié des composés qui inhibent la GatCAB de H. pylori de façon compétitive par rapport au substrat Glu-ARNtGln. Cinq fois plus petits que les peptides cycliques mentionnés plus haut, ces composés inhibent l’activité de la GatCAB avec des Ki de 139 μM pour le composé 7, et de 214 μM pour le composé 4. Ces inhibiteurs de GatCAB pourraient être utiles pour des études mécanistiques, et pourraient être des molécules de base pour le développement de nouvelles classes d’antibiotiques contre des infections causées par H. pylori. / This thesis describes the discovery of inhibitors of a tRNA-dependent amidotransferase (AdT) and summarizes the present state of our knowledge about the two-step biosynthesis of Gln-tRNAGln and Asn-tRNAAsn in Helicobacter pylori. In eukaryotic cytoplasm, twenty amino acids (aa) are generally attached to their cognate tRNAs by twenty corresponding aminoacyl-tRNA synthetases (aaRSs). These enzymes have a high specificity, and their function is important to the proper decoding of mRNA. However, in a number of bacteria including H. pylori, GlnRS and/or AsnRS are absent. To synthesize Gln-tRNAGln, H. pylori first uses a noncanonical GluRS2 which is specific for tRNAGln to form Glu-tRNAGln; then the trimeric AdT (GatCAB) transforms Glu-tRNAGln into Gln-tRNAGln which is proper for protein biosynthesis. In a similar manner, the biosynthesis of Asn-tRNAAsn also takes place in H. pylori by using the same GatCAB and a canonical nondiscriminating AspRS. The widespread use of these indirect pathways among prominent human pathogens, and their absence in the mammalian cytoplasm, identify AdT as a promising target for the development of new and highly specific antimicrobial agents. By using phage display, we discovered several cyclic peptides rich in tryptophan and proline that inhibit H. pylori GatCAB. Peptides P10 (CMPVWKPDC) and P9 (CSAHNWPNC) are competitive inhibitors of GatCAB with respect to its substrate Glu-tRNAGln. The inhibition constants (Ki) of P10 and P9 are 126 and 392 μM, respectively. Their docking models revealed that they bind to the transamidation active site of GatB via π-π stacking interactions with Tyr81, as does the 3’-terminal A76 of tRNA. We also discovered two small dipeptide-like sulfone-containing inhibitors of H. pylori GatCAB by mimicking the intermediate of its transamidation reaction. Although they are much smaller than the cyclic peptides mentioned above, they are competitive inhibitors of GatCAB with respect to GlutRNAGln, with Ki values of 139 μM for compound 7 and 214 μM for compound 4. These inhibitors could be useful not only to study the reaction mechanisms of GatCAB, but also could be lead compounds for the development of a new class of antibiotics to treat infections caused by H. pylori.

The mucosal immunity of the gastrointestinal (GI) tract is constituted by a complex, highly specialized and dynamic system of immune components that aim to protect the gut from external threats. The sustained exposure of the mucosal immune system of the GI tract to an enormous number of lumen antigens, requires the constant upkeep of a highly regulated balance between initiation of immune responses against harmful agents and the generation of immune tolerance towards innocuous antigens. Therefore, the regulatory component is key to preserve tissue homeostasis and a normal functioning of the system. Indeed, defective regulatory responses lead to the development of pathological conditions, including unresolved infections, and inflammatory diseases. In this study, we aim to elucidate novel mechanisms involved in host-pathogen interactions during Helicobacter pylori and Clostridium difficile infections. Indeed, this work integrates preclinical in vivo and in vitro experimental approaches together with a bioinformatics pipeline to identify and characterize novel regulatory mechanisms and molecular targets of the mucosal immune system during enteric infections. Firstly, we identified a novel regulatory mechanism during H. pylori infection mediated by a specific subset of IL10-producing tissue resident macrophages. Secondly, we employed an ex vivo H. pylori co-culture with bone marrow derived macrophages, that together with a global transcriptomic analysis and a bioinformatics pipeline, lead to the discovery of promising regulatory genes based on expression kinetics. Lastly, we characterized the innate inflammatory responses induced during the course of C. difficile infection and identified IL-1ß, and its subsequent induction of neutrophil recruitment, as a key mediator of C. difficile-induced effectors responses. The characterized regulatory mechanisms in this work show promise to lead the generation of new host-centered therapeutics through the modulation of the immune response as promising alternative treatments for infectious diseases. / Doctor of Philosophy / The immune system is responsible for protecting the human body from external threats. To achieve this goal, it must differentiate between harmless and harmful agents to only fight the latter. To combat these dangerous agents, the immune system induces highly controlled, inflammatory processes that aim to eliminate the external threat while minimizing the damage of human tissues and organs. The gastrointestinal tract is exposed to an enormous number of molecules, mostly harmless molecules from both the ingested food and the beneficial bacteria inhabiting the gut, but also from harmful bacteria and agents, only separated from the internal body structures by a thin layer called the epithelial barrier of the gut. The immune system responsible for the protection of the gastrointestinal tract includes an important regulatory component critical to maintain a proper gut function. This regulatory component regulates the generation of inflammatory processes to fight the dangerous agents, while blocking the responses against the inoffensive agents and preventing excessive tissue damage to maintain the integrity of the epithelial barrier. Indeed, a failure in the regulatory component results in severe consequences for the body's health, such as the inability to resolve infections. In this study, we aim to investigate the interaction between the human body and the enteric bacteria Helicobacter pylori and Clostridium difficile, to bring new insights in the regulatory component of the immune system of the gut. Moreover, the new mechanisms discovered in the regulatory system, might allow the development of new treatments for infectious diseases.

The gastrointestinal mucosal immune system has the daunting task of maintaining immune homeostasis by eliminating potentially harmful microorganisms and limiting tissue injury while inducing tolerogenic responses to luminal antigens including innocuous food, commensal bacteria and self-antigens. This carefully orchestrated system depends on elaborate down-regulating mechanisms that mediate and maintain a state of tolerance under normal conditions. Changes in such delicate balance are linked to the development of gastrointestinal pathology as well as systemic disease states. Despite the rapid increase in our appreciation of the gastrointestinal immune system, there is still a major disconnect between the description of how mucosal immune responses are organized and controlled and an insufficient mechanistic understanding of how such responses shape and influence disease outcome and pathogenesis. By using model enteric microorganisms Helicobacter pylori and Clostridium difficile, this dissertation presents a systematic effort to generate novel mechanistic hypothesis based on computational predictions and experimentally elucidate the mechanisms of action underlying mucosal immune responses and pathology in the gut. In this thesis I present i) an overview on mucosal immunology and the need to develop novel therapeutics that limit the pathogenic effects of invading bacteria while maintaining their protective functions, ii) the role of miRNAs in the modulation of immune responses to enteric pathogens, iii) the mechanisms by which Helicobacter pylori is able to limit effector inflammatory responses required for bacterial clearance thus favoring tolerance over immunity, iv) intracellular mechanisms of immune evasion that contribute to bacterial persistence and chronic infection. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling, immunoinformatics and experimental immunology holds enormous potential for discovering unforeseen targets and developing novel vaccines and cures for infectious, allergic and immune-mediated diseases. / Ph. D.