CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A infecÃÃo pelo Helicobacter pylori acomete mais da metade da populaÃÃo mundial, sendo esta bactÃria reconhecida como carcinÃgeno do grupo I pela OrganizaÃÃo Mundial de SaÃde-OMS. Familiares em primeiro grau de pacientes com cÃncer gÃstrico tÃm um maior risco de desenvolver cÃncer gÃstrico. Avaliamos a prevalÃncia de lesÃes precursoras do cÃncer gÃstrico e do Helicobacter pylori nos familiares em primeiro grau de pacientes com cÃncer gÃstrico, quando comparado a controles sem histÃria familiar. Cento e quatro familiares foram recrutados à partir de 40 casos de cÃncer gÃstrico tipo nÃo-cÃrdia e foram comparados com cento e dezoito controles, nÃo havendo diferenÃas estatisticamente significantes entre os dois grupos com relaÃÃo a idade, sexo, tabagismo, etilismo e condiÃÃes socioeconÃmicas garantindo a homogeneidade da amostra. Todos os pacientes foram submetidos a avaliaÃÃo endoscÃpica e biÃpsias seguindo o protocolo de Sydney. A anÃlise histopatolÃgica foi realizada pÃr um Ãnico patologista experiente e mascarado quanto a origem das amostras. Ainda que a prevalÃncia da atrofia e da metaplasia intestinal tenha ocorrida de forma similar nos dois grupos, a associaÃÃo destas lesÃes foi mais encontrada nos familiares que nos controles (p=0,021). A metaplasia intestinal tipo incompleta foi mais significante nos familiares (p=0,001), assim como a displasia (p=0,025). O padrÃo de gastrite encontrado nos familiares foi o de pangastrite associada a presenÃa de folÃculos linfÃides, padrÃo este jà definido como o de fenotÃpico de maior risco para a carcinogÃnese gÃstrica. NÃo houve diferenÃa estatisticamente significante entre os dois grupos com relaÃÃo a prevalÃncia do H. pylori , porÃm a topografia da infecÃÃo envolvendo antro e corpo foi maior nos familiares (p=0,001). De acordo com os resultados obtidos neste estudo, encontramos que familiares de pacientes com cÃncer gÃstrico tÃm uma maior prevalÃncia de alteraÃÃes histopatolÃgicas, estando estas alteraÃÃes confinadas a presenÃa do Helicobacter pylori / Infection by Helicobacter pylori, a bacterial species classified by WHO as being carcinogenic (group I) affects more than half the world population. First-degree relatives to patients with gastric cancer are at increased risk of developing gastric cancer. The present study evaluated the prevalence of precursor lesions of gastric cancer and infection by Helicobacter pylori in first-degree relatives to patients with gastric cancer as compared to controls with no family history of gastric cancer. One hundred four first-degree relatives to 40 patients with noncardiac gastric cancer were enrolled in the study and compared to 108 controls. The groups were statistically homogenous in terms of age. All patients were submitted to endoscopic evaluation and biopsy as described in the Sydney protocol. The histopathological analysis was carried out by a single, experienced pathologist blinded to the origin of the samples. Although the prevalence of atrophy and intestinal metaplasia was similar for the two groups, association with these lesions was more common among relatives than controls (p=0.021). Incomplete intestinal metaplasia was also more significant among relatives (p=0.001), as was displasia (p=0.025). The group of relatives presented a pattern of pangastritis associated with lymphoid follicles characteristic of increased risk for gastric carcinogenesis. There was no statistically significant difference between the groups with regard to the prevalence of H. pylori, though infection involving body and antrum was more prevalent among relatives (p=0.001). Our findings suggest that relatives to patients with gastric cancer present a greater prevalence of histopathological changes associated with the presence of H. pylori

No Brasil, cerca de 65% da população é infectada por H. pylori, bactéria associada à patogênese de gastrite, úlcera péptica e considerada fator de risco de câncer gástrico. A transmissão da bactéria parece ocorrer de pessoa para pessoa, nas formas oral-oral, gástrica-oral e fecal-oral, e a cavidade bucal pode ser importante neste processo de transmissão. Os objetivos deste estudo foram: pesquisar a presença de H. pylori na cavidade bucal de pacientes dispépticos e identificar as possíveis cepas existentes. Para tanto, 43 pacientes com dispepsia funcional do Ambulatório de Estômago do Departamento de Gastroenterologia, Disciplina de Gastroenterologia Clínica FMUSP foram selecionados para o estudo. Todos realizaram exame de endoscopia digestiva alta e coleta de fragmento da mucosa gástrica para pesquisa de H. pylori através de teste da urease. A presença de H. pylori no estômago foi também evidenciada pelo teste respiratório com 14C ou sorologia, e em 30 pacientes foi identificado H. pylori no estômago. Foram coletadas 144 amostras da cavidade bucal: 43 de saliva, 43 de dorso de língua, 43 de placa supra-gengival e 15 de placa sub-gengival. A detecção de H. pylori das amostras bucais realizou-se através de PCR utilizando os primers espécie-específicos: P1/P2, Urease A/B e primers para investigar os genótipos cagA e vacA (m1, m2, s1a, s1b, s2). Não foi possível detectar o microorganismo em nenhuma amostra da cavidade bucal. Esse estudo mostrou que a cavidade bucal de pacientes dispépticos não representa reservatório para a bactéria. / Helicobacter pylori (H. pylori) infection is very prevalent in Brazil, infecting almost 65% of our population, being associated with peptic ulcer disease, gastric cancer, lymphoma and functional dyspepsia. The aim of this study was to evaluate the presence of this bacterium in the oral cavity of epigastric pain syndrome (functional dyspepsia) patients and assess the frequency of cagA and vacA genotypes of oral H. pylori. All 43 epigastric pain syndrome outpatients, who entered the study, were submitted to upper gastrointestinal endoscopy to rule out organic diseases. H. pylori infection was confirmed by rapid urease test, urea breath test and sorology, and thirty patients harbored H. pylori in the stomach. 144 samples of the oral cavity were collected: 43 samples of saliva, 43 of the tongue dorsum and 43 of supragingival dental plaque and 15 of sub-gingival dental plaque which were collected from the patients with periodontitis; H. pylori infection was verified by polymerase chain reaction (PCR) using primers (P1 and P2) that amplify the DNA sequence of a species-specific antigen present in all H. pylori strains, primers Urease A/B and primers for cagA and vacA (m1, m2, s1a, s1b, s2) genotyping. It was not possible to detect H. pylori in any oral samples using P1/P2 and Urease A/B. The genotype cagA was also negative in all samples and vacA genotype could not be characterized (s-m-). In this study, the oral cavity seemed not to be a reservoir for H. pylori in patients with epigastric pain syndrome being detected exclusively in the stomach.

Introdução: O envolvimento gastroduodenal pode ocorrer na doença de Crohn (DC). Seu diagnóstico histológico definitivo é habitualmente realizado através da demonstração do granuloma não caseoso. O achado de gastrite focal H. pylori negativa em biopsias gástricas de pacientes com DC ileal e/ou colônica, apesar de não ser específico, também sugere o envolvimento da doença neste segmento. Objetivos: avaliar a frequência da gastrite focal em pacientes com DC comparada à de pacientes com retocolite ulcerativa (RCU) e controles, assim como as frequências da infecção pelo H. pylori nessas populações e correlacioná-las com a presença de gastrite focal; avaliar a capacidade da imunohistoquímica em diferenciar a gastrite focal nos três grupos; avaliar as associações entre dados demográficos, aspectos clínicos, laboratoriais, uso de medicamentos, presença de sintomas do trato gastrintestinal (TGI) superior e achados endoscópicos com presença de gastrite focal em pacientes com doença inflamatória intestinal (DII); e avaliar a associação entre uso de medicamentos nesses pacientes e infecção pelo H. pylori. Métodos: Foram estudados 62 pacientes com DC, 35 pacientes com RCU e 40 pacientes controles. Todos foram submetidos à endoscopia digestiva alta (EDA) com biopsias para o teste da urease, exame histológico e imunohistoquímico. Resultados: Dos 137 pacientes estudados foram excluídos dois pacientes com DC e um com RCU. Não houve diferença estatisticamente significante entre os grupos com relação à idade (p=0,921) e sexo (p=0,192). A maioria dos pacientes com DC estava em remissão clínica (75%). Cerca de 80% dos pacientes com DC faziam uso de azatioprina. H. pylori foi positivo em 18/60 (30%) pacientes com DC, 12/34 (35%) na RCU e 20/40 (50%) no grupo controle sem diferença estatisticamente significante entre os grupos (p=0,131). Não foram observadas associações estatisticamente significantes entre uso de medicamentos e infecção pelo H. pylori nos pacientes com DII. A gastrite focal H. pylori negativa foi diagnosticada em 7/42 (16,7%) na DC, 3/22 (13,6%) na RCU e 2/20 (10%) no grupo controle, sem diferença estatisticamente significante entre eles (p=0,919). A gastrite focal H. pylori positiva foi diagnosticada em 2/18 (11%) na DC, 3/12 (25%) na RCU e 7/20 (35%) no grupo controle, sem diferença estatisticamente significante (p=0,213). Não foram observadas associações estatisticamente significantes entre características clínicas, laboratoriais, uso de medicamentos, sintomas do TGI superior, achados endoscópicos e gastrite focal. No entanto, foi observado que o uso de azatioprina nos pacientes com DC H. pylori negativos apresentou uma tendência a reduzir a gastrite focal. A imunohistoquímica da gastrite focal dos pacientes com DC e RCU H. pylori negativos foi semelhante e diferiu do grupo controle por este apresentar um maior acúmulo de linfócitos B (CD20). Já a imunohistoquímica da gastrite focal dos pacientes com DC, RCU e controles H. pylori positivos foi indistinguível. Conclusões: Pacientes com DII tendem a ser menos infectados pela bactéria H. pylori. A frequência de gastrite focal H. pylori negativa diagnosticada em nosso estudo foi menor do que a descrita na literatura. O uso de imunossupressor (azatioprina) pode estar relacionado com tal achado / Introduction: Gastroduodenal involvement may occur in Crohns disease (CD). Definitive histological diagnosis of CD in the upper gastrointestinal (GI) tract normally relies on the demonstration of epitheloid granuloma which is considered the histological hallmark of gastric CD. If granulomas are absent, the description of focally enhanced gastritis (FEG) or focal active gastritis in gastric biopsies of patients with known ileal and/or colonic CD, although not exclusive to CD, suggests the involvement of the disease at this site. Objectives: To access the prevalence of FEG in CD patients compared with a group of ulcerative colitis (UC) and CD/UC-free controls, as well as the frequencies of H. pylori infection in those population and correlate them to the presence of FEG; evaluate the capacity of immunohistochemistry in differentiating FEG in the three groups; evaluate the correlation with demographic and clinical characteristics, laboratory findings, current medical therapy as well as the presence of forgut symptoms and mucosal lesions at endoscopy with the presence or absence of FEG in patients with inflammatory bowel disease (IBD) and evaluate the association between medical therapy and H. pylori infection in IBD patients. Methods: We studied 62 patients with CD, 35 patients with UC and 40 patients from control group. All underwent upper GI endoscopy. Biopsy specimens taken from angulus, antrum and gastric body were evaluated by urease test, histology and immunohistochemistry. Results: Of the 137 patients studied we excluded 2 patients with CD and 1 with UC. There was no statistically significant difference among the groups in terms of age (p=0.921) and gender (p=0.192). The majority of CD patients were in clinical remission (75%). Around 80% of CD patients were taking azathioprine. H. pylori was positive in 18/60 (30%) CD patients, in 12/34 (35%) UC and in 20/40 (50%) controls with no statistically significance difference among the groups (p=0.131). No association was found between use of medications and H. pylori infection in IBD patients. In H. pylori negative patients, FEG was diagnosed in 16.7% cases (7/42) of CD, compared with 13.6% (3/22) of UC patients and 10% (2/20) of controls, with no statistically significance difference among them (p=0.919). In H.pylori positive patients, FEG was diagnosed in 11% cases (2/18) of DC, 25% (3/12) in UC and 35% (7/20) of controls with no significant difference among them (p=0.213). There was no statistical interrelationship between FEG and demographic and clinical characteristics, laboratory findings, use of medications, upper GI symptoms and endoscopic findings. However, it was observed that use of azathioprine in H. pylori negative CD patients presented a tendency to reduce FEG. In H. pylori negative patients, immunohistochemistry of FEG of CD and UC was similar and differed from controls as it presented a higher accumulation of B lymphocytes (CD20). On the other hand in H. pylori positive IBD patients, immunohistochemistry of FEG was indistinguishable from controls. Conclusions: IBD patients tend to be less infected by H. pylori. The frequency of H. pylori negative FEG diagnosed in our study was lower than described in literature. The use of immunossupressants (azathioprine) may be related to such findings

Le développement d’un lymphome gastrique du MALT (LGM) émane d’un processus inflammatoire chronique initié par Helicobacter pylori.A partir du matériel issu d’un modèle animal de LGM, préalablement développé au laboratoire, basé sur des infections chez des souris thymectomisées à la naissance, la réponse inflammatoire gastrique favorable à l’émergence de LGM a été étudiée. Une dérégulation de cytokines et chimiokines au stade LGM a été identifiée permettant de recruter, faire proliférer et faire émerger des infiltrats lymphoïdes. La susceptibilité des souris thymectomisées à développer des lymphomes n’est pas liée à un déficit en lymphocytes T régulateurs. Cinq microARNs ont été retrouvés dérégulés au stade lymphome agissant probablement en synergie pour favoriser la prolifération lymphocytaire en particulier via un mécanisme anti-apoptotique. Enfin, nous décrivons un modèle original de LGM basé sur l’utilisation de souris C57BL6 exprimant la chimiokine APRIL humaine au niveau des lymphocytes T infectées par des espèces du genre Helicobacter. Ce modèle est prometteur pour une meilleure compréhension de la lymphomagénèse gastrique. / The development of gastric MALT lymphoma (GML) originates from a chronic inflammatory process initiated by Helicobacter pylori.The gastric inflammatory response was investigated in a mouse model of GML previously described by the laboratory using BALB/c mice thymectomized at day 3 post-birth and infected by H. pylori. A deregulation of numerous cytokines and chemokines at GML stage was identified which explained the recruitment, proliferation and emergence of lymphoid infiltrates. The susceptibility of thymectomized mice to develop lymphoma was not linked to a deficiency in regulatory T cells. A deregulation of 5 microRNAs was observed at lymphoma stage. These microRNAs may be involved in cell survival and lymphocyte proliferation and act in synergy to promote the development of GML. Finally, we described an original model of GML based on infection by Helicobacter species of transgenic C57BL6 mice expressing the human form of the cytokine APRIL in T cells. This model is promising for a better understanding of gastric lymphomagenesis.

La traduction implique l’utilisation d’aminoacyl-ARNt produits par les aminoacyl-ARNt synthétases (aaRS). Il devrait exister 20 aaRS, une spécifique de chaque acide aminé. Or, les données actuelles montrent qu’une grande majorité des organismes ne possèdent pas l’asparaginyl- (AsnRS) et/ou la glutaminyl-ARNt synthétase (GlnRS). Ils ne peuvent synthétiser l’Asn-ARNtAsn et le Gln-ARNtGln que par l’utilisation de voies impliquant la formation préalable d’aspartyl-ARNtAsn et/ou de glutamyl-ARNtGln. Ces précurseurs « mésacylés » sont synthétisés par une aspartyl-ARNt synthétase et/ou une glutamyl-ARNt synthétase non-discriminantes (AspRS-ND ou GluRS-ND). Ils sont ensuite amidés par une amidotransférase (AdT), pour fournir à la cellule l’Asn-ARNtAsn et/ou le Gln-ARNtGln nécessaires à la traduction des codons Asn et Gln.Ce travail de thèse, effectué dans le contexte biologique de deux organismes différents, Thermus thermophilus et Helicobacter pylori, a permis de montrer que les étapes enzymatiques – formation du précurseur, et amidation par l’AdT – sont réalisées au sein de complexes ribonucléoprotéiques, réunissant l’aaRS-ND, l’ARNtAsn ou l’ARNtGln, et l’AdT : l’Asn-transamidosome ou le Gln-transamidosome. Selon leur origine ou la voie à laquelle ils appartiennent (asparaginylation ou glutaminylation), ces complexes possèdent des particularités mécanistiques et structurales très différentes, mais sont tous adaptés pour éviter la libération des intermédiaires mésacylés toxiques par des stratégies spécifiques. Ce travail permet de mieux comprendre les mécanismes évolutifs qui ont conduit à l’incorporation de l’Asn et de la Gln dans le code génétique. / Protein synthesis requires the biosynthesis of aminoacyl-tRNAs by aminoacyl-tRNA synthétases (aaRS). Since 20 amino acids are présent within the genetic code, 20 aaRS should be used by a single organism. However, the vast majority of organisms found today are deprived of asparaginyl- and/or glutaminyl-tRNA synthetases (Asn- or GlnRS). They can only synthesize Asn-tRNAAsn and/or Gln-tRNAGln through biosynthesis pathways involving the preliminary formation of aspartyl-tRNAAsn and /or glutamyl-tRNAGln. Those « misacylated » precursors are synthesized by so called non-discriminating aspartyl- or glutamyl-tRNA synthetases (ND-AspRS or –GluRS). Then, they are transferred to an amidotransferase (AdT) to provide the Asn-tRNAAsn and/or Gln-tRNAGln species (necessary to fuel protein synthesis) through amidation.This work was performed in the context of two organisms – Thermus thermophilus and Helicobacter pylori. It showed that the two enzymatic steps of asparaginylation and glutaminylation – biosynthesis of the misacylated precursor and amidation by AdT – are carried out within a single ribonucleoprotein complex, namely the (Asn- or Gln-) transamidosome, gathering the ND-aaRS necessary for the misacylation, the tRNA substrate (Asn or Gln) and the AdT. According to their origin or the pathway they originate from (asparaginylation or glutaminylation), those complexes display significant mechanistical and structural peculiarities, but they are all adapted to prevent libération of the toxic misacylated species through specific strategies. This work shed new light on the évolutive mechanisms that led to the incorporation of Asn or Gln into the genetic code.

Infection with Helicobacter pylori has been associated with the pathogenesis of numerous stomach and gastroduodenal diseases that pose threats to public health. Eradicaftion of this pathogen is a global challenge due to its alarming rate of multidrug resistance. Consequently, to find an alternative treatment, the search is increasingly focused on new antimicrobial product from natural sources including honey. Honey has been used as medicine in several cultures since ancient time due to its enormous biomedical activities. Its beneficial qualities have been endorsed to its antimicrobial, antioxidant, anti-inflammatory properties added to its phytocomponents. In this study, the anti-H. pylori activity of South African honeys and their solvent extracts as well as the phytochemicals present in the two most active honeys were evaluated. Agar well diffusion test was used to investigate the antimicrobial activity of six honey varieties obtained from different locations in the country. Subsequently, the honeys were extracted with four organic solvents viz n-hexane, diethyl ether, chloroform and ethyl acetate employed in order of increasing polarity. The antibacterial activity of the different solvent extracts of each honey was evaluated by agar well diffusion; broth micro dilution and time kill assays. Different chromatographic techniques (Thin layer & column chromatography) were employed to enumerate the phytochemical constituents in the most active solvent extracts of Pure Honey (PH) and Champagne Royal Train (CRT); and were identified by gas-chromatography linked mass-spectrometry. Linalool pure compound was equally evaluated for anti-H. pylori activity in a bid to trace the antibacterial agent among the variety of compounds identified. Data were analyzed by One-way ANOVA test at 95% confidence interval. Crude honeys and their solvent extracts demonstrated potent anti-H. pylori activity with zone diameter that ranged from [16.0mm (crude) to 22.2mm (extract)] and percentage susceptibilities of test isolates between 73.3% (crude) and 93.3% (extract). The chloroform extracts of PH and CRT were most active with MIC50 in the ranges 0.01- viii 10%v/v and 0.625-10%v/v respectively, not significantly different from amoxicillin (P> 0.05); and efficient bactericidal activity (100% bacterial cells killed) at 1/2MIC and 4xMIC over different time intervals, 36-72hrs and 18-72hrs respectively. The appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts of PH and CRT; and developed with hexane: ethyl acetate: acetic acid (HEA) and methanol: acetic acid: water (MAAW) solvent systems indicated the presence of compounds. Purification of the compounds contained in these extracts over silica gel column yielded numerous fractions which were evaluated for antibacterial activity and purity. PHF5 was the most active fraction with a mean MIC50 value of 1.25mg/mL. Volatile compounds belonging to different known chemical families in honey were identified in all the active fractions obtained from PH. Conversely, only four compounds were identified in the active fractions obtained from CRT hence the non volatile constituents could be of prime relevance with respect to antibacterial activity of this honey. Of novelty was the presence of thiophene and N-methyl-D3-azirdine compounds, essential precursors used for the synthesis of natural products and pharmaceuticals with vital biomedical properties. Linalool demonstrated potent inhibitory (MIC95, 0.002- 0.0313mg/mL) and bactericidal activity (0.0039-0.313mg/mL) against the test isolates. On the other hand, a significant difference was recorded (P < 0.05) in comparing the activity of linalool compound to the fractions. PH could serve as a good economic source of bioactive compounds which could be employed as template for the synthesis of novel anti-H. pylori drugs. However, further studies are needed to determine the non volatile active ingredients in PH and CRT as well as toxicological testing

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative motile curved rod that inhabits the gastric mucosa of the human stomach. The organism chronically infects billions of people worldwide and is one of the most genetically diverse of bacterial species. Infection with the organism potentially induces chronic gastritis and peptic ulcer disease. In addition, H. pylori plays a role in the etiology of gastric cancer and gastric MALT lymphoma. The risk of infection is increased in those living in the developing world, which has been ascribed to precarious hygiene standards, crowded households, and deficient sanitation common in this part of the world. Thus, the aim of this study was to identify the risk factors in the transmission of H. pylori in our environment, i.e. in Nkonkobe Municipality in the Eastern Cape Province, South Africa. Faecal samples were collected from 356 apparently healthy subjects, consisting of 168 males and 188 females aged from 3 months to 60 years (Mean = 31 years). A standardized questionnaire was applied, it described demographic characteristics including age, sex, household hygiene, socioeconomic status, area of residence, duration of stay in the area, sharing bath water, sharing tooth brush, habit of sucking thumb, medication currently being taken or medication taken within the past three months, source of water, type of toilet used, education and occupation. A sandwich-type enzyme immunoassay amplification technology (Amplified IDEIA TM Hp StAR TM , Oxoid, UK) was used to analyze the faecal samples for the detection of H. pylori antigens using monoclonal antibodies specific for H. pylori antigens. To assess the possibility of faecal oral route with tap water as an intermediary link, PCR targeting the ureC (glmM), a highly conserved gene in H. pylori ii was carried out to detect H. pylori DNA in faecal samples of already positive samples by HpSA test as well as in direct tap water used by the H. pylori positive subjects. QIAamp DNA stool mini kit was used to extract DNA from faecal samples. Tap water samples were then obtained using sterile bottles from areas inhabited by H. pylori positive subjects as determined by HpSA test and PCR. DNA extraction from water samples was done using UltraCleanTM Water DNA Isolation Kit (0.22μm) according to the manufacturer’s instructions. PCR with primers specific for H. pylori glmM gene was carried out with both positive and negative controls incorporated. Fisher’s exact test was used to assess the univariate association between H. pylori infection and the possible risk factors. Odds ratio (OR) and the corresponding 95 percent confidence interval (CI) were calculated to measure the strength of association using EPI INFO 3.41 package. P values of < .05 were required for significance. The precision rate of the diagnostic tests used was also determined. H. pylori antigen was detected in 316 of the 356 subjects giving an overall prevalence of 88.8 percent. Prevalence increased with age from 75.9 percent in children < 12 years age to 100 percent in the age group from 13 years to 24 years, also 100 percent prevalence of H. pylori was recorded in young adults aged 25-47 years and subjects aged 60 years (P < .05). H. pylori prevalence was higher in females than in males. Of 188 females who participated in the study, H. pylori antigen was detected in 172 (91.5 percent) versus 144 (85.7 percent) of 168 males (P > .05). Interestingly, H pylori antigen was detected more often (100 percent) in the high socioeconomic group than in those of low socioeconomic group (85.9 percent). Sixteen (66.7 percent) of twenty four faecal samples that had previously tested positive for the organism by HpSA test were confirmed positive by PCR. However none of the treated tap water samples tested positive for the organism by PCR. The present iii study revealed a high prevalence of H. pylori in faecal samples of asymptomatic individuals in the Nkonkobe Municipality, an indication of active infection. The obtained results also revealed that direct treated tap water might not be playing a crucial role in the oral transmission of H. pylori in the studied population.

Enzyme DNA methylation is an important biochemical process that imprints DNA with additional information. DNA methylation is catalyzed by S-adenosyl-L-methionine (AdoMet)-dependent methyltraferases (MTases). Prokaryotic DNA MTases are usually components of restriction-modification(R-M) systems that enable cells to resist propagation of foreign genomes that would otherwise kill them. Based on the position methyl group transfer on the bases in DNA, MTases are classified into two groups-exocyclic or amino MTases and endocyclic or ring MTases. The amino MTases methylate exocyclic amino nitrogen to form either N6-methyladenine or n4-methycytosine. N6-methyaladenine is mostly found in the genomes of bacteria, archaea protists and fungi. Helicobacter pylori is a gram-negative, flagellated, fastidious bacterium that colonizes the highly acidic environment of the gastric mucosa. Frequently and persistence of H.paylori infection in humans make it attractive model for studying the host- pathogen interaction mechanisms. Analysis of the genome sequence of H.pylori strains 26695, J99.HPAGI, and G27 revealed an abundance of restriction-modification (R-M) systems. Most of the R-M system genes are either conserved among the strains or specific to each strain. Strain specific genes are responsible for different phenotypes in several host adapted pathogens such as H.pylori. Many of the R-M gene homologues exhibit different usages of condon bias and lower G+C content from the average genes suggesting horizontal transfer of the R-M system genes in H. Pylori. Genome analysis of strain 26695 showed the presence of three putative type III R-M systems and hp0592-hp0593 constitutes one such type III R-M system. Based on the conserved motif arrangements, HP0593 MTases belongs to the subgroups of MTases. The amino acid sequence of HP0593 MTases has 38% sequence identity to Ecop11 MTases and EcoP151 MTase, both of which belongs to type IIIR-M systems therefore, it was important to study in detail previously unexplored role of this putative type III DNA MTase (HP0593) in H. Pylori. Investigation of methyltransferease activity and sequence specifically of putative DNA adenine MTase (HP0593) HP0593 (N6-adenine) - DNA MTase is a member of a type III R-M system in H. pylori strain 26695. HP0593 MTase has been cloned, over expressed and purified heterologously in Escherichia coli. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was carried out with purified HP0593 and profile showed a single peak with expected molecular mass of 70.6kDa. The protein was determined as-5.8. HP0593 MTase exits predominantly as monomer and a small fraction as dimer in solution as determined by size exclusion chromatography and glutaraldehyde cross-linking studies. The recognition sequence of the purified MTase was determined as 5’GCAG-3’ and the target base of methylation is adenine. Dot-blot assay using antibodies that reacted specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine specific MTase. Exocyclic MTase have a conserved catalytic motif (D/N/S/SPPY/F/W). Most interestingly, the amino acid sequence analysis of HP0593 MTase revealed the presence of a PCQ-like motif, which is the catalytic motif for C5-cytosine MTase in addition to DPPY motif. In order to check the role of both these MTase by glycine. HP0593 –Y107G and C54G mutant proteins were purified to near homogeneity. It was found that the Y107G mutant protein was catalytically inactive as compared to wild-type HP0593 MTase. On the other hand the C54G mutant protein was found to be as active as the wild-type HP0593 MTase indicating that HP0593 MTase is an adenine MTase and not a C5- cytosine MTase. Kinetic and catalytic properties of HP0593 DNA adenine methyltransferase DNA binding studies were carried out by electrophoretic mobility shift assay using DNA having cognate site and either in absence or presence of AdoHcy or sinefungin. In all the three cases two different DNA-protein complexes were observed-a fast running complex I and a slow running complex 2. It can be surmised that the fast running complex could be HP0593 monomer-DNA and the slow running complex could be a HP0593 dimer-DNA complex. With non specific DNA (lacking 5’-GCAG-3’ sequences) no complexes were formed even in the presence of cofactors. Based on the above observations it is suggested that a specific interactions of HP0593 MTase with DNA occurs on cognate recognition site. The activity of HP0593 MTase is optional at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. When initial velocities were plotted against varying concentrations of duplex DNA having a single 5’GCAG-3’ site a rectangular hyperbola was obtained confirming that HP0593 MTase obeys michaelis menten kinetics. From non-linear regression analysis of the plot of initial velocity versus DNA concentration Km (DNA) and kcat were calculated. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of methylation activity on enzyme concentration indicated that more than one molecule of enzyme is required for its activity. Metal ion cofactors such as CO 2, Mn2+ and Mg2+ stimulated the HP09593 MTase activity. As Mn2+ showed maximum stimulation of methyaltion activity compared to other metal ions, surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by HP0593 MTase in the absence and presence of Mn2+. In the presence of Mn2+, HP0593 MTase showed~1000-fold increase in affinity to duplex DNA. DNA MTase bind substrates in random or sequential order. Preincubation study demonstrated that the preformed enzyme-DNA complex is competent than the preformed enzyme-AdoMet complex. This suggests that MTase binds to DNA first followed by AdoMet. Isotope partitioning analysis indicated that HP0593 MTase shows a distributive mechanism of methylation DNA having more than one recognition site. Effects of inactivation of HP0593 DNA MTase in Helicobacter pylori 26695 strain and its functional role. DNA dot-blot assay using hp0593 gene specific primer showed that this gene is present in 25.15% of the clinical strains checked suggesting that hp0593 is strain-specific gene. Strain-specific genes in many host-adapted pathogene impart strain specific phenotype. Wild-type 26695 strain grew slightly faster at the initial phase of growth in PH 4.5 compared to pH 7.4. A~5-fold enhanced level of hp0593 mRNA expression was growth under acidic condition HP0593 MTase could play an important role in H. pylori physiology through methylation. To elucidate the possible role(s) played by the MTase in H.pylori physiology, an hp0593 knock-out in 26695 strain was generated by chloramphenecol cassette mediated insertional gene inactivation. Growth kinetic study was carried out with both wild-type and hp0593 knock-out strain at pH7.4, the growth of the hp0593 strain. At pH 4.5 no major differences were observed in the growth compared to the wild-type hp0593 knock-out strain. To further investigate the effect of the knock-out, cell-morphology study was carried out after growing the strains at pH 7.4 till mid-exponential phase. Transmission electron microscopy studies reveled changes in cell shape, presence of sheathed structure and production of outer membrane vesicles (OMVs) in the hp0593 knock out strain. OMVs contain effectors molecules during infection helps in pathogenicity caused by H.pylori.This is the first report where inactivation of DNA MTase causes shedding of vesicles. OMVs are also known to modulate the production of IL-8 by gastic epitheial cells. To check weather H.pylori strains could produce IL-8, both wild-type and hp0593 knock-out strains were co-cultured with AGS cell infected with the hp0593 knock out strain. This was further confirmed by semi-quantitative RT-PCR analysis. To analyze the different phenotypes observed in the hp0593 knock-out strain, transcriptome profile were compared by microarray and RT-PCR analysis. In thehp0593 knock-out strain peptidologlycan and murein synthesis genes like pbp2, murC and neu4 showed upregulation which could be responsible for the changes in cell shape presence of sheathed structure and OMVs production. The RT-PCR data showed ~9-fold down-regulation of dank chaperone which might play a key role in slow growth phenotype in the hp0593 knock-out strain. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes such as dank, neuA, murC, fliH, filP and cag5, the results presented in this study provide impetus for exploring the role of HP0593 DNA MTase in the cellular processes of H.pylori. However, R-M systems are not absolutely essential, but different methylation patterns may contribute to strain-specific epigenetic gene regulation and may contribute to variability among the strains.

Krebserkrankungen sind das Resultat komplexer Vorgänge und Veränderungen. Eine wichtige Variable in der Initialisierung ist die Interaktion zwischen Genotyp und Umwelt. Da Magenkarzinome zu den wenigen Krebserkrankungen gehören, bei denen im letzten Jahrhundert ein deutlicher Rückgang in der Inzidenz beobachtet werden konnte (Becker 2006), stellt sich die Frage, ob es Unterschiede in der genetischen Ausstattung von historischen Tumorgenomen im Vergleich zu rezenten gibt. Als typisches Zielgen für Tumormutationen wurde das Tumorsuppressorgen p53 untersucht. Au¬ßerdem wurde Helicobacter pylori (H. pylori) als Risikofaktor für die Entstehung von Magenkarzinomen in den historischen Proben identifiziert. Insgesamt standen 51 Gewebeproben von Magenkarzinompatienten aus den Medizinhistori¬schen Museen Berlin und Zürich, dem Pathologisch-anatomischen Bundesmuseum Wien sowie der anatomischen Sammlung der Universität Tartu zur Verfügung. Bei 47 Proben handelte es sich nach histologischer Diagnose um Magenkarzinome. Die Proben sind zwischen 50 und 180 Jahre alt und wurden in Konservierungsflüssigkeiten unbekannter Zusammensetzung gelagert. Von 45% der Proben konnten vollständige Sequenzen der Exons 5 bis 8 des p53-Gens generiert werden. Es konnten Veränderungen im Mutationsspektrum und in der Lokalisation der Mutationen in den historischen Proben im Vergleich zu rezenten Tumormutationen festgestellt werden. Die Mutationsfrequenz betrug 52%. Insgesamt wurden zehn verschiedene Mutationsereignisse in p53 bei elf verschiedenen Proben detektiert, wobei drei Proben von zwei Mutationen betroffen waren. Zwei Mutationen führen direkt (Q144X) bzw. indirekt (S166fs) zu der Entstehung von Stoppcodons. Zu einem Verlust des p53-Proteins bei gleichzeitiger Akquirierung onkogener Funktionen kommt es durch die Hotspot-Mutation R248W (Wang & El-Deiry 2007). Zu einer Zunahme der Funktionsfähigkeit (gain-of-function) kommt es außerdem durch die Mutation K291E. Die Mutation E271K ist an einer splice site lokalisiert, so dass das Exon nicht in die mRNA übernommen werden kann. Die Konsequenzen der anderen Mutationsereignisse sind nicht eindeutig belegt, scheinen aber einen geringeren Einfluss auf die Funktionalität von p53 zu haben. Abweichend zu rezenten Magenkarzinomen konnte die Mutation M246K als Hotspot der historischen Proben identifiziert werden. Insgesamt waren nur vier historische Mutationen bisher aus rezentem Magenkarzinomgewebe beschrieben, davon die Mehrheit in Studien mit asiatischen Patienten. Die veränderten Mutationen der historischen Magenkarzinome können als Hinweis auf Veränderungen in den karzinogen wirkenden Einflussfaktoren dienen, etwa eine veränderte Lebens- und Ernährungsweise durch die Einführung von Kühlmöglichkeiten für Lebensmittel. Der Risikofaktor H. pylori konnte in 70% der Gewebeproben sicher nachgewiesen werden. Der Anteil der Träger des Virulenzfaktors cag betrug 44%. 73% der Proben mit p53-Mutation waren nachweislich mit H. pylori und davon wiederum 63% mit einem cag-positiven Stamm infiziert. Die Infektion ermöglicht eine Teilerklärung für die untypische Lokalisation der Mutationen, da H. pylori-induzierte Mutationen seltener an Magenkarzinom-Hotspots vorkommen (Murakami et al. 1999). Es konnten keine resistenztypischen Mutationen gegen das Antibiotikum Clarithromycin in den historischen Geweben identifiziert werden. Die vorliegende Arbeit konnte p53-Mutationen und das Bakterium H. pylori in den historischen Gewebeproben identifizieren. Ein verändertes Mutationsspektrum und die veränderte Lokalisation der Mutationen geben den Hinweis, dass sich die Einflüsse auf die Entstehung der Magenkarzinome im letzten Jahrhundert verändert haben.

Seit 1983 ist das Konzept des Mukosa-assoziierten lymphatischen Gewebes (MALT) im Magen auf dem Boden einer chronischen Helicobacter(H.)-pylori-Infektion bekannt. Viele epidemiologische, biologische und molekulargenetische Studien haben die Rolle von H. pylori in der Lymphomgenese unterstützt. Bis heute wurden weltweit mehr als 650 Patienten mit gastralem MALT-Lymphom und H.-pylori-Infektion antibiotisch behandelt. Bei etwa 75% der Fälle kann mit Hilfe dieser Therapie eine komplette Lymphomremission induziert werden. Klinische prädiktive Faktoren helfen dabei, Patienten bezüglich ihres Risikos besser zu stratifizieren und damit die Probabilität des Ansprechens zu verbessern. Neue zytogenetische Erkenntnisse haben zudem dazu beigetragen, ein besseres Verständnis der Lymphomgenese zu erlangen. Mit der kürzlich beschrieben Translokation t(11;18) (q21;q21) könnte in Zukunft ein prädiktiver genetischer Faktor verfügbar sein. / The Role of Helicobacter pylori Infection for the Development and Treatment of Gastric MALT Lymphomas Since 1983, it is well known that mucosa-associated lymphoid tissue (MALT)-type lymphoma of the stomach is due to chronic Helicobacter pylori (H. pylori) infection. Many epidemiological, biological, and moleculargenetic studies have implicated the role of H. pylori in lymphomagenesis. Nowadays, more than 650 patients with gastric MALT lymphoma worldwide have been treated with antibiotics for H. pylori infection, achieving a complete remission in about 75% of cases. Clinical predictive factors help to stratify patients into risk groups, and help to predict the probability of lymphoma remission. New insights into cytogenetics have also contributed to the understanding of lymphomagenesis, and with the newly identified translocation t(11;18)(q21;q21) we might have also a genetic factor at hand to predict treatment response. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.