Dissertationen zum Thema „H. pylori“

As well as peptic ulcers, Helicobacter pylori is associated with the development of gastritis, gastric adenocarcinoma and lymphoma, and has been classified as a class I carcinogen in humans (International Agency for Research on Cancer Working Group, 1994). Although the bacteria can be eradicated in up to 90% of patients, side effects, poor compliance and the resistance of the bacteria to antibiotics are common causes of frequent treatment failure. Celery seed extracts (CSE) from a unique source in India has been used as herbal medicine since antiquity and found to have anti-inflammatory and gastroprotective properties (Butters et al., 2004; Whitehouse et al., 2001). This study followed on observations that crude extracts exhibited anti-helicobacter activity (Rainsford & Liu, 2006).CSE was selectively fractionated followed by HPLC. Fractions were collected and bio-assayed against different strains of H. pylori using conventional culture methods. The most potent component that was obtained from HPLC and purified was designated celery seed with anti-Helicobacter activity (CAH). This component has strong bactericidal effects against H. pylori; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 3.15 mug ml[-1] and 6.25 - 12.5 mug ml[-1], respectively. This compares favourably with the MIC and MBC of tetracycline, which are in the region of 3.15 mug ml[-1]. The isolated compound has highly specific inhibitory effect on H. pylori, since no inhibitory activity was detected against Campylobacter jejuni or Escherichia coli at these levels. The molecular ion of CAH was measured as 384.23 by mass spectrometry, giving the empirical formula as C[24]H[32]O[4]. The MS and NMR data strongly suggest this compound is a phthalide dimer. From radioactive bioassay, CAH inhibits RNA synthesis by 50% of that seen in a negative control in 3 days, while DNA and protein synthesis were unchanged. These suggested that the new compound may be suitable for further investigation as an agent for treating H. pylori infections.

Helicobacter pylori (H. pylori) infection causes achlorydria, depressed gastric acid barrier, impaired immune response and is suspected in bacterial overgrowth and diarrhoea. These features of the infection are known to cause significant malabsorption of nutrients and impairment of linear growth in children. The prevalence of H. pylori infection in children is known to be much higher in developing countries, especially among the lower socio-economic groups. The true prevalence of infection in urban children in Mexico and its impact on their growth are largely unknown. This study examined the prevalence of H. pylori infection in school children from an urban area in Northwest Mexico and attempted to identify the risk factors that predispose individuals to infection in childhood; as well as to relate the presence of this infection to growth of children. The cross-sectional study was conducted in 1997/98 in the poorest socio-economic sectors of the city of Hermosillo, Sonora, among 178 children aged 9 and 10 years. H. pylori status was determined in children by the 13C-urea breath test. Anthropometric (weight and height) and haemoglobin measurements along with analysis of faecal samples and a 24-hour dietary recall were carried out in each child. Family sociodemographic/socio-economic status and living conditions data were elicited from parents by interview via structured questionnaires. The overall prevalence rate of H. pylori infection for the children in Hermosillo as determined by this study was 47.1%. The findings indicate that rural-born father, number of siblings, the type of main water supply (one tap in the yard) and the sharing of bed by the study child are important risk factors for acquiring the H. pylori infection. A borderline significant but small effect of H. pylori infection on height for-age was observed in this study. H. pylori infection was found to be positively highly associated with Hymenolepis nana. No differences in mean energy, protein and iron intakes between H. pylori positive and negative children were observed. However, significant differences in the mean energy, protein and iron intakes were observed between boys and girls. H. pylori infection and enteric parasites were not significantly correlated with the presence of anaemia.

More than half of all people worldwide are infected with H. pylori. The infection always cause a gastric inflammation that may develop into peptic ulcer disease or gastric cancer. Attachment proteins, adhesins, mediate specific adherence of H. pylori to receptor structures on the human gastric mucosa. The best-characterized H. pylori adhesin-receptor interactions are the BabA adhesin and the binding to the fucosylated blood group antigens ABO/Lewis b (Leb) and the SabA adhesin and its binding to the inflammation associated sialyl-Lewis x antigen. During H. pylori infection the availability of receptor structures on the human gastric mucosa changes as a consequence of the host inflammatory and immune responses. Consequently the bacterial population need to adjust its adherence properties to stay colonized. This thesis describes mechanisms that generate H. pylori populations with variable adherence properties and mechanisms for adjustment of adhesin expression levels.In H. pylori strains devoid of Leb-binding, we found bacterial cells with Leb-binding. Isolation of such H. pylori clones demonstrated that the change in receptor binding phenotype was obtained via the mechanisms of homologous recombination and slipped strand mispairing (SSM). Disease presentation in relation to BabA expression was studied in H. pylori infected Mongolian gerbils. We showed that BabA was not essential for colonization but caused severe injury to the gastric mucosa and was turned off during long-term infection by nucleotide changes within the babA gene. Gerbils infected with BabA-weak-expressing strains maintained BabA expressing clones for a longer period than gerbils that were infected with BabA-high-expressing strains. Studies of the gerbil gastric mucosal glycosylation showed that gerbils respond in a similar way as humans and Rhesus monkeys which support gerbils to be a model suitable for studying H. pylori infection and disease outcome in relation to adherence.We studied the SSM mechanism of SabA phase variation and the cognate shift in sLex-binding phenotype and we show sLex-binding activity to be growth phase dependent. H. pylori vesicles were characterized for the major phosholipid and protein components. Virulence factors e.g., VacA, and CagA were identified and both the BabA and the SabA adhesins was shown to be located on the vesicle surface and to mediate specific binding to their cognate receptors present on the human gastric mucosa. H. pylori generate bacterial cells with different receptor binding phenotypes via the mechanisms of homologous recombination, SSM and nucleotide changes. These mechanisms will probably contribute to bacterial fitness by the generation of quasi species populations where some of the clones will be better adapted to the environmental chances during persistent infection.

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O bacilo Helicobacter pylori é atualmente considerado como o agente etiológico mais importante das gastrites em seres humanos e como um fator de risco para o surgimento das doenças ulcerativas do carcinoma gástrico. Estima-se que 50% da população mundial está infectada por esta bactéria. Atualmente, apenas estudos subjetivos e qualitativos têm sido realizados na análise da colonização do H. pylori. O objetivo deste estudo foi avaliar quantitativamente, através de análise morfométrica, a colonização pelo H. pylori em biópsia de pacientes com diferentes lesões gástricas (gastrites e úlcera). Para o estudo utilizou-se biópsias gástricas de 239 pacientes (95 homens e 144 mulheres) com idade média de 45 anos, provenientes de diferentes municípios do estado de Pernambuco. Os cortes histológicos (4μm) foram corados pelo Giemsa e analisado através de microscopia óptica. O estudo morfométrico foi realizado por meio de um sistema digital de análise de imagens onde as áreas de interesse obtidas foram processadas utilizando-se o software OPTIMAS®. Os resultados indicam uma incidência de 39% de lesões inflamatórias gástricas associadas à infecção por Hp com maior ocorrência (60,3%) no gênero feminino e com faixa etária entre 31-45 anos. Quanto às lesões gástricas mais freqüentes observou-se que a gastrite crônica ativa foi a mais evidente em cerca de 90,2% dos casos estudados. Houve uma correlação positiva entre a análise qualitativa (semi-quantitativa) e a análise morfométrica (número médio de bacilos Hp, por área), principalmente quando se comparou os parâmetros numerosos bacilos com os moderados e raros . A partir destes dados pode-se concluir que os estudos referentes aos aspectos da infecção da mucosa gástrica pelo Hp, e mais especificamente os níveis de infecção quanto ao número de bacilos e extensão das áreas afetadas são de sua importância para que se possa conhecer melhor a evolução das lesões gástricas resultantes deste quadro infeccioso e em que situações e grupos humanos podem representar fatores de risco para o desenvolvimento de lesões mais graves como o câncer

BENIGNO, Tiago Gomes da Silva. Análise da resposta humoral contra H. pylori em pacientes portadores e não portadores de câncer gástrico. 2013. 71 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2013.
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Helicobacter pylori have accompanied humanity in all its migration routes across the planet. Being the cosmopolitan H.pylori bacteria that evolved and adapted the most adverse conditions through mutation and genetic exchange that resulted in favorable changes morph physiological. Such changes contributed to the development of highly virulent strains of bacteria which cause different pathophysiological conditions in the host. The high genetic variability of H. pylori hinders research to uncover associations with certain gastrointestinal conditions. However, bacterial virulence markers have been identified and associated with different gastrointestinal diseases. H. pylori strains that express proteins Cag A and Vac A has been found in patients with gastric cancer, others expressing Dup A and Oip A are found in patients with gastric ulcer were considered protective genes, since they are not related with development of gastric cancer. The methods used to detect H. pylori infection can be divided into invasive and non-invasive. The endoscopy is an invasive method is where the biopsy urease test to detect the presence of bacteria. The problem is that endoscopy can’t identify discrete points of colonization of H. pylori, or else, is indicated when the patient is already symptomatic, where you have a situation of advanced gastric epithelial cell injury. Among the non-invasive methods have serological methods. Among these serological methods have immunoblotting method that searches the presence of specific antibodies (Ig G). The antibodies against virulence factors and specific proteins (Cag A, Vac, Ure A, Ure B, and flagellar protein) serves both to detect infection and measure the type strain of H. pylori present in the infected individual. This method can be used to investigate the humoral response in patients with and without gastric cancer infected with H. pylori.
O Helicobacter pylori tem acompanhado a humanidade em todas as suas rotas de migração pelo planeta. Sendo o H.pylori uma bactéria cosmopolita que evoluiu e se adaptou as condições mais adversas através de mutações e troca de material genético que resultaram em mudanças morfofisiológicas favoráveis. Tais mudanças contribuíram no desenvolvimento de estirpes da bactéria altamente virulentas e que provocam diferentes condições fisiopatológicas no hospedeiro. A grande variabilidade genética do H.pylori dificulta a investigação para se descobrir associações com determinadas patologias gastrointestinais. No entanto, marcadores de virulência da bactéria já foram identificados e associados a diferentes doenças gastrointestinais. Cepas de H.pylori que expressam as proteínas Cag A e Vac A tem sido encontradas em pacientes com câncer gástrico, já outros que expressam Oip A e Dup A são encontrados em pacientes com úlcera gástrica sendo considerados como genes de proteção, pois não estão relacionados com o desenvolvimento de câncer gástrico. Os métodos utilizados para detectar a infecção pelo H.pylori se dividem em invasivos e não invasivos. A endoscopia é um método invasivo onde se faz a biopsia e teste da uréase para detectar a presença da bactéria. O problema é que endoscopia não consegue identificar discretos pontos de colonização do H. pylori, ou então, é indicada quando o paciente já esta sintomático, onde se tem uma situação avançada de injúria das células epiteliais gástricas. Entre os métodos não invasivos temos os métodos sorológicos. Entre esses métodos sorológicos temos o immunoblotting um método que pesquisa a presença de anticorpos específicos (Ig G). A pesquisa de anticorpos contra fatores de virulência e proteínas específicas (Cag A, Vac A, Ure A, Ure B, proteína flagelar), serve tanto para detectar a infecção como mensurar o tipo de cepa de H.pylori presente no individuo infectado. Podendo esse método ser utilizado para investigar a resposta humoral em pacientes com e sem câncer gástrico infectados com H.pylori.

CAVALCANTE, Meyssa Quezado de Figueiredo. Genótipos cagA, vacA e alelos de H. pylori em pacientes portadores de gastrite, úlcera péptica e câncer gástrico. 2010. 87 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará, Faculdade de Medicina, Fortaleza, 2010.
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The bacterial species H. pylori is closely correlated with the development of major diseases and gastric cancer of the stomach. The strains that have more pathogenic genotype are more involved in bacterial virulence. The objective was to genotype strains of H. pylori from patients with gastritis, gastroduodenal ulcer and gastric cancer as the cagA and vacA genes and alleles by PCR technique. The genotyping of strains of H. pylori was performed by PCR technique. We evaluated 134 strains, 76 of patients with gastritis, 28 peptic ulcer and 30 gastric cancer. As a result of this research it was found that all strains subtyped were vacA positive, and 48.5% with the allele s1m1 vacA, vacA s1m2 20.9% and 6.7% s2m1 vacA, vacA s2m2 8.2%; 7 5% were hybrid strains and 8.2% had only the allele of the sequence signal (s). Gastritis in the distribution of vacA alleles were: 46.1% s1m1, s1m2 22.4%, 5.3% s2m1, s2m2 11.8%, 3.9% and 10.5% hybrid strains only with allele s. In cases of ulcer was found: 50% s1m1, s1m2 21.4%, 17.8% s2m1, s2m2 3.6%, 7.2% hybrid strains. In patients with cancer met: s1m1 53.3%, 16.7% s1m2, s2m2 3.3%, 16.7% and 10.0% hybrid strains with allele only s. The cagA gene was observed in 82.8% of strains, where 73.7% had gastritis and cagA positive, 96.4% had peptic ulcer and gastric cancer harbored 96.7% were positive for this gene. Allele vacAs1m1 was the most prevalent observed in the three groups. We have also found strains with multiple alleles of this gene, suggesting a genetic polymorphism arising from infection by more than one type of strain in the same individual. The cagA gene was also highly prevalent in both groups. The presence of cagA was statistically significant in the group of pangastritis and gastric cancer. The relationship of the cagA gene with allele vacAs1m1 was also significant in the groups of pangastritis and gastric cancer, which supports the higher pathogenicity of strains of the studied individuals.
A espécie bacteriana Helicobacter pylori está intimamente correlacionado com o desenvolvimento das principais afecções gástricas e do câncer de estômago. As cepas que apresentam genótipo mais patogênico são as mais implicadas na virulência da bactéria. O objetivo deste trabalho foi caracterizar as cepas de H. pylori provenientes de pacientes portadores de gastrite, úlcera gastroduodenal e câncer gástrico quanto aos genes cagA e vacA e alelos através da técnica de PCR. A genotipagem das cepas de H. pylori foi realizada através da técnica de PCR. Foram avaliadas 134 cepas, sendo 76 de portadores de gastrite, 28 de úlcera péptica e 30 de câncer gástrico. Como resultado desta pesquisa obteve-se que, todas as cepas genotipadas foram vacA positivas, sendo 48,5% com o alelo vacA s1m1; 20,9% vacA s1m2; 6,7% vacA s2m1; 8,2% vacA s2m2; 7,5% eram cepas híbridas e 8,2% apresentaram somente o alelo da sequência sinal(s). Nos portadores de gastrite a distribuição dos alelos vacA foi: 46,1% s1m1, 22,4% s1m2, 5,3% s2m1, 11,8% s2m2, 3,9% cepas híbridas e 10,5% somente com alelo s. Nos casos de úlcera achou-se: 50% s1m1, 21,4% s1m2, 17,8% s2m1, 3,6% s2m2, 7,2% cepas híbridas. Nos portadores de câncer encontrou-se: 53,3% s1m1, 16,7% s1m2, 3,3% s2m2, 16,7% cepas híbridas e 10,0% somente com alelo s. O gene cagA foi observado em 82,8% das cepas, onde 73,7% tinham gastrite e cagA positivo, 96,4% tinham úlcera péptica e 96,7% portavam câncer gástrico com positividade para este gene. O alelo vacAs1m1 foi o mais prevalente observado nos três grupos estudados. Também foram encontradas cepas com alelos múltiplos desse gene, o que sugere um polimorfismo genético advindo de infecção por mais de um tipo de cepa em um mesmo indivíduo. O gene cagA também foi altamente prevalente nos grupos estudados. A presença do cagA foi estatisticamente significante no grupo de pangastrite e câncer gástrico. A relação do gene cagA com o alelo vacAs1m1 também foi significante nos grupos de pangastrite e câncer gástrico, o que corrobora a maior patogenicidade das cepas presentes nos indivíduos estudados.

GONÇALVES, Maria Helane Rocha Batista. Genótipos das cepas de H. pylori VacA e alelos, cagA e sítios de fosforilação EPIYA em uma comunidade urbana de Fortaleza utilizando método não endoscópico. 2010. 68 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2010.
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The H. pylori infect currently half of the world’s population and is related to the development of gastric disorders. The Enterotest is a minimally invasive method that can be used for detection of H. pylori through endoscopic not. The objective of this study was to evaluate the sensitivity and specificity of Enterotest opposite the Respiratory Testing and studying the genetic profile of strains of H. pylori individuals resident in the Community College Park. The gastric juice was collected from Enteroteste, and held the culture and the extraction of DNA from H. pylori. The genotyping of strains was carried out by the PCR technique and phosphorylation sites EPIYA sequencing participated in the study 50 individuals, positive and negative 7 43 for infection H. pylori through Respiratory Test. Culture and PCR from Enteroteste showed sensitivity of 86% and 77%, respectively, with 100% specificity in both methods. 33 strains were positive, genotipadas as Cow being 39.4% Cow s1m1; 15.2% Cow s1m2; 18.2% Cow s2m2 27.2% with Cow s profile with absence of allele m. Were positive 66.6% Bran of strains being profiled 54.5% EPIYA-ABC, 41.0% EPIYA-ABCC and 4.5% EPIYA-AB. The Enterotest proved to be a reliable method with great sensitivity and specificity for identification of H. pylori through cultivation and PCR technique. Most strains expressed alelo Cow s1, with a predominance of subtype s1b and allele combination s1m1. More than half of the studied strains expressed gene Shit and most of these strains were 3 or 4, phosphorylation sites profiled EPIYA-ABC or EPIYA-ABCC. The genetic profile presented by these strains is described for South America and nonstandard Asia, most strains circulating in the community have important potential pathogenic. The Enterotest is a safe method, sensitive and specific detection of H. pylori.
O H. pylori infecta atualmente metade da população mundial e é relacionado com o desenvolvimento das afecções gástricas. O Enteroteste é um método minimamente invasivo que pode ser usado para detecção do H. pylori por meio não endoscópico. O objetivo deste estudo foi avaliar a sensibilidade e especificidade do Enteroteste frente ao Teste Respiratório e estudar o perfil genético das cepas de H. pylori em indivíduos residentes na comunidade Parque Universitário. O suco gástrico foi colhido a partir do Enteroteste, e realizada a cultura e a extração do DNA do H. pylori. A genotipagem das cepas foi realizada através da técnica de PCR e os sítios de fosforilação EPIYA de sequenciamento Participaram do estudo 50 indivíduos, 43 positivos e 7 negativos para a infecção pelo H. pylori através do Teste Respiratório. A cultura e o PCR a partir do Enteroteste apresentaram sensibilidade de 86% e 77%, respectivamente, com especificidade de 100% em ambos os métodos 33 cepas foram genotipadas como vacA positivas, sendo 39,4% vacA s1m1; 15,2% vacA s1m2; 18,2% vacA s2m2 27,2% com perfil vacA s com ausência do alelo m. Foram cagA positivas 66,6% das cepas sendo 54,5% com perfil EPIYA-ABC, 41,0% EPIYA-ABCC e 4,5% EPIYA-AB. O Enteroteste mostrou-se um método confiável com boa sensibilidade e especificidade para identificação do H. pylori através do cultivo e da técnica de PCR. A maioria das cepas expressou o alelo vacA s1, com predomínio do subtipo s1b e da combinação alélica s1m1. Mais da metade das cepas estudadas expressaram o gene cagA e a maioria dessas cepas tinham 3 ou 4 sítios de fosforilação, com perfil EPIYA-ABC ou EPIYA-ABCC. O perfil genético apresentado por essas cepas é o descrito para a América do Sul e diferente do padrão Asiático, a maioria das cepas circulantes na comunidade têm importante potencial patogênico. O Enteroteste é um método seguro, sensível e específico para detecção do H. pylori.

BRAGA NETO, Manuel Bonfim. Estresse oxidativo e genótipo do H. pylori em pacientes portadores de câncer gástrico e seus familiares. 2015. 72 f. Tese (Doutorado em Cirurgia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015.
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Gastric cancer is associated with H. pylori infection, environmental factors and genetic predisposition. Relatives of gastric cancer patients are at increased risk to develop cancer, possibly, due to infection by more virulent H. pylori strains and genetic predisposition associated with pro-inflammatory cytokines, which may, in turn, result in an exaggerated inflammatory response and oxidative stress. The aim of this study was to: 1) evaluate the oxidative stress in dispeptic patients without family history of gastric cancer (NRC), patients with gastric cancer (GC) and relatives of gastric cancer (RC); 2) evaluate the prevalence of the H. pylori genotypes homA, homB and cagE in RC and NRC and determine if the cagE gene is associated with GC as well as the pattern of cagA EPYIA phosphorylation in GC. This study was performed at the Hospital Universitário Walter Cantídeo (HUWC) in Fortaleza. Blood samples were collected and centrifuged to obtain serum, in order to measure the concentration of reduced glutathione (GSH), an anti-oxidant, and Malondealdehyde (MDA), a product of lipid peroxidation. DNA extraction was performed in gastric biopsies, followed by PCR and electrophoresis for ureA, vacA, homA, homB, cagA e cagE genes. The concentrations of MDA and GSH were evaluated in 213 patients (54 CG, 91 RC e 68 NRC). The group of CG had higher MDA and lower GSH concentrations when compared to the groups without cancer (p=0.001 and p=0.005, respectively). The H. pylori genotypes were evaluated in 186 patients (48 CG, 74 RC and 64 NRC). The overall cagE prevalence was 54.3% (101/186), 70% (52/74) in the RC group, 52% (25/48) in the GC group and 36% (24/87) in the NRC group. A significant and independent association was found between the cagE genotype and the RC group (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) and between the homB genotype and the RC group (OR (OR 2,357; IC 95%: 1,401 – 3,961; p=0,001). Forty six cagA positive samples from the GC group were included for gene sequencing to evaluate the EPIYA phosphorylation sites of the CagA protein. All of the strains studied had an EPIYA-C pattern and approximately 50% of the strains were mixed. This study demonstrates that patients with gastric cancer are under higher levels of oxidative stress. The group of RC have a higher prevalence of cagE and homB. The group of GC patients had a high prevalence of mixed strains regarding CagA EPIYA-C phosphorylation pattern. Decreasing the lipid peroxidation, increasing the antioxidant reserve and identifying groups at risk of infection with more virulent strains of H. pylori may be important to prevent gastric cancer.
O câncer gástrico está relacionado a infecção por H. pylori, fatores ambientais e predisposição genética. Familiares de pacientes com câncer gástrico (FC) têm maior risco de desenvolvimento da doença, possivelmente, em decorrência da infecção por cepas mais virulentas de H. pylori e predisposição genética relacionado a citocinas pró-inflamatóriase o stress oxidativo. O objetivo do estudo foi: 1) avaliar o estresse oxidativo em pacientes portadores de câncer gástrico (CG), dispépticos sem história familiar de câncer gástrico (NFC), e familiares de câncer gástrico de primeiro grau (FC); 2) averiguar em FC e NFC a prevalência dos genótipos de H. pylori homA, homB, cagE e, em pacientes portadores de CG, avaliar se o gene cagE está associado a essa neoplasia e o padrão dos sítios de fosforilação EPYIA da proteína cagA. Estudo foi realizado no Hospital Universitário Walter Cantídio. O estresse oxidativo foi avaliado no soro através da dosagem de glutationa reduzida (GSH), antioxidante, e malondialdeído (MDA), produto da peroxidação lipídica. Realizou-se extração de DNA da biópsia gástrica, seguida de PCR e eletroforese para os genes ureA, vacA, homA, homB, cagA e cagE. Foi avaliado a concentração sérica de MDA e GSH em 213 indivíduos (54 CG, 91 FC e 68 NFC). O grupo CG apresentou concentrações de MDA mais elevado e de GSH mais baixo quando comparado com o grupo sem neoplasia (p=0.001 e p=0.005, respectivamente). Os genótipos de H. pylori foram avaliados em 186 pacientes (48 CG, 74 FC e 64 NFC). A prevalência geral de cagE na amostra foi de 54,3% (101/186), sendo 70% (52/74) no grupo FC, 52% (25/48) no grupo de CG e 36% (24/87) no grupo NFC. Houve associação significante, mesmo após análise multivariada, entre o genótipo cagE e o grupo de FC (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) e entre homB e o grupo FC (OR 2,357; IC 95%: 1,401 – 3,961; p=0,001). Foram incluídas 46 amostras de CG cagA positivas para avaliação dos sítios EPIYA da proteína CagA. Todas as cepas avaliadas apresentaram padrão EPYIA-C, e aproximadamente 50% de cepas mistas. O presente estudo mostra que pacientes portadores de câncer gástrico possuem estresse oxidativo aumentado. Os indivíduos FC estão infectados com maior prevalência por cepas virulentas de H. pylori, cagE e homB. Além disso, os pacientes portadores de câncer gástrico apresentaram prevalência elevada de cepas mistas de H. pylori quanto ao segmentos EPIYA C da proteína CagA. Possivelmente, diminuir a peroxidação lipídica, aumentar a reserva de antioxidantes e identificar grupos de maior risco quanto ao perfil de cepas de H. pylori poderá ser importante para a prevenção do câncer gástrico.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Helicobacter pylori have accompanied humanity in all its migration routes across the planet. Being the cosmopolitan H.pylori bacteria that evolved and adapted the most adverse conditions through mutation and genetic exchange that resulted in favorable changes morph physiological. Such changes contributed to the development of highly virulent strains of bacteria which cause different pathophysiological conditions in the host. The high genetic variability of H. pylori hinders research to uncover associations with certain gastrointestinal conditions. However, bacterial virulence markers have been identified and associated with different gastrointestinal diseases. H. pylori strains that express proteins Cag A and Vac A has been found in patients with gastric cancer, others expressing Dup A and Oip A are found in patients with gastric ulcer were considered protective genes, since they are not related with development of gastric cancer. The methods used to detect H. pylori infection can be divided into invasive and non-invasive. The endoscopy is an invasive method is where the biopsy urease test to detect the presence of bacteria. The problem is that endoscopy canât identify discrete points of colonization of H. pylori, or else, is indicated when the patient is already symptomatic, where you have a situation of advanced gastric epithelial cell injury. Among the non-invasive methods have serological methods. Among these serological methods have immunoblotting method that searches the presence of specific antibodies (Ig G). The antibodies against virulence factors and specific proteins (Cag A, Vac, Ure A, Ure B, and flagellar protein) serves both to detect infection and measure the type strain of H. pylori present in the infected individual. This method can be used to investigate the humoral response in patients with and without gastric cancer infected with H. pylori.
O Helicobacter pylori tem acompanhado a humanidade em todas as suas rotas de migraÃÃo pelo planeta. Sendo o H.pylori uma bactÃria cosmopolita que evoluiu e se adaptou as condiÃÃes mais adversas atravÃs de mutaÃÃes e troca de material genÃtico que resultaram em mudanÃas morfofisiolÃgicas favorÃveis. Tais mudanÃas contribuÃram no desenvolvimento de estirpes da bactÃria altamente virulentas e que provocam diferentes condiÃÃes fisiopatolÃgicas no hospedeiro. A grande variabilidade genÃtica do H.pylori dificulta a investigaÃÃo para se descobrir associaÃÃes com determinadas patologias gastrointestinais. No entanto, marcadores de virulÃncia da bactÃria jà foram identificados e associados a diferentes doenÃas gastrointestinais. Cepas de H.pylori que expressam as proteÃnas Cag A e Vac A tem sido encontradas em pacientes com cÃncer gÃstrico, jà outros que expressam Oip A e Dup A sÃo encontrados em pacientes com Ãlcera gÃstrica sendo considerados como genes de proteÃÃo, pois nÃo estÃo relacionados com o desenvolvimento de cÃncer gÃstrico. Os mÃtodos utilizados para detectar a infecÃÃo pelo H.pylori se dividem em invasivos e nÃo invasivos. A endoscopia à um mÃtodo invasivo onde se faz a biopsia e teste da urÃase para detectar a presenÃa da bactÃria. O problema à que endoscopia nÃo consegue identificar discretos pontos de colonizaÃÃo do H. pylori, ou entÃo, à indicada quando o paciente jà esta sintomÃtico, onde se tem uma situaÃÃo avanÃada de injÃria das cÃlulas epiteliais gÃstricas. Entre os mÃtodos nÃo invasivos temos os mÃtodos sorolÃgicos. Entre esses mÃtodos sorolÃgicos temos o immunoblotting um mÃtodo que pesquisa a presenÃa de anticorpos especÃficos (Ig G). A pesquisa de anticorpos contra fatores de virulÃncia e proteÃnas especÃficas (Cag A, Vac A, Ure A, Ure B, proteÃna flagelar), serve tanto para detectar a infecÃÃo como mensurar o tipo de cepa de H.pylori presente no individuo infectado. Podendo esse mÃtodo ser utilizado para investigar a resposta humoral em pacientes com e sem cÃncer gÃstrico infectados com H.pylori.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior
Gastric cancer is associated with H. pylori infection, environmental factors and genetic predisposition. Relatives of gastric cancer patients are at increased risk to develop cancer, possibly, due to infection by more virulent H. pylori strains and genetic predisposition associated with pro-inflammatory cytokines, which may, in turn, result in an exaggerated inflammatory response and oxidative stress. The aim of this study was to: 1) evaluate the oxidative stress in dispeptic patients without family history of gastric cancer (NRC), patients with gastric cancer (GC) and relatives of gastric cancer (RC); 2) evaluate the prevalence of the H. pylori genotypes homA, homB and cagE in RC and NRC and determine if the cagE gene is associated with GC as well as the pattern of cagA EPYIA phosphorylation in GC. This study was performed at the Hospital UniversitÃrio Walter CantÃdeo (HUWC) in Fortaleza. Blood samples were collected and centrifuged to obtain serum, in order to measure the concentration of reduced glutathione (GSH), an anti-oxidant, and Malondealdehyde (MDA), a product of lipid peroxidation. DNA extraction was performed in gastric biopsies, followed by PCR and electrophoresis for ureA, vacA, homA, homB, cagA e cagE genes. The concentrations of MDA and GSH were evaluated in 213 patients (54 CG, 91 RC e 68 NRC). The group of CG had higher MDA and lower GSH concentrations when compared to the groups without cancer (p=0.001 and p=0.005, respectively). The H. pylori genotypes were evaluated in 186 patients (48 CG, 74 RC and 64 NRC). The overall cagE prevalence was 54.3% (101/186), 70% (52/74) in the RC group, 52% (25/48) in the GC group and 36% (24/87) in the NRC group. A significant and independent association was found between the cagE genotype and the RC group (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) and between the homB genotype and the RC group (OR (OR 2,357; IC 95%: 1,401 â 3,961; p=0,001). Forty six cagA positive samples from the GC group were included for gene sequencing to evaluate the EPIYA phosphorylation sites of the CagA protein. All of the strains studied had an EPIYA-C pattern and approximately 50% of the strains were mixed. This study demonstrates that patients with gastric cancer are under higher levels of oxidative stress. The group of RC have a higher prevalence of cagE and homB. The group of GC patients had a high prevalence of mixed strains regarding CagA EPIYA-C phosphorylation pattern. Decreasing the lipid peroxidation, increasing the antioxidant reserve and identifying groups at risk of infection with more virulent strains of H. pylori may be important to prevent gastric cancer.
O cÃncer gÃstrico està relacionado a infecÃÃo por H. pylori, fatores ambientais e predisposiÃÃo genÃtica. Familiares de pacientes com cÃncer gÃstrico (FC) tÃm maior risco de desenvolvimento da doenÃa, possivelmente, em decorrÃncia da infecÃÃo por cepas mais virulentas de H. pylori e predisposiÃÃo genÃtica relacionado a citocinas prÃ-inflamatÃriase o stress oxidativo. O objetivo do estudo foi: 1) avaliar o estresse oxidativo em pacientes portadores de cÃncer gÃstrico (CG), dispÃpticos sem histÃria familiar de cÃncer gÃstrico (NFC), e familiares de cÃncer gÃstrico de primeiro grau (FC); 2) averiguar em FC e NFC a prevalÃncia dos genÃtipos de H. pylori homA, homB, cagE e, em pacientes portadores de CG, avaliar se o gene cagE està associado a essa neoplasia e o padrÃo dos sÃtios de fosforilaÃÃo EPYIA da proteÃna cagA. Estudo foi realizado no Hospital UniversitÃrio Walter CantÃdio. O estresse oxidativo foi avaliado no soro atravÃs da dosagem de glutationa reduzida (GSH), antioxidante, e malondialdeÃdo (MDA), produto da peroxidaÃÃo lipÃdica. Realizou-se extraÃÃo de DNA da biÃpsia gÃstrica, seguida de PCR e eletroforese para os genes ureA, vacA, homA, homB, cagA e cagE. Foi avaliado a concentraÃÃo sÃrica de MDA e GSH em 213 indivÃduos (54 CG, 91 FC e 68 NFC). O grupo CG apresentou concentraÃÃes de MDA mais elevado e de GSH mais baixo quando comparado com o grupo sem neoplasia (p=0.001 e p=0.005, respectivamente). Os genÃtipos de H. pylori foram avaliados em 186 pacientes (48 CG, 74 FC e 64 NFC). A prevalÃncia geral de cagE na amostra foi de 54,3% (101/186), sendo 70% (52/74) no grupo FC, 52% (25/48) no grupo de CG e 36% (24/87) no grupo NFC. Houve associaÃÃo significante, mesmo apÃs anÃlise multivariada, entre o genÃtipo cagE e o grupo de FC (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) e entre homB e o grupo FC (OR 2,357; IC 95%: 1,401 â 3,961; p=0,001). Foram incluÃdas 46 amostras de CG cagA positivas para avaliaÃÃo dos sÃtios EPIYA da proteÃna CagA. Todas as cepas avaliadas apresentaram padrÃo EPYIA-C, e aproximadamente 50% de cepas mistas. O presente estudo mostra que pacientes portadores de cÃncer gÃstrico possuem estresse oxidativo aumentado. Os indivÃduos FC estÃo infectados com maior prevalÃncia por cepas virulentas de H. pylori, cagE e homB. AlÃm disso, os pacientes portadores de cÃncer gÃstrico apresentaram prevalÃncia elevada de cepas mistas de H. pylori quanto ao segmentos EPIYA C da proteÃna CagA. Possivelmente, diminuir a peroxidaÃÃo lipÃdica, aumentar a reserva de antioxidantes e identificar grupos de maior risco quanto ao perfil de cepas de H. pylori poderà ser importante para a prevenÃÃo do cÃncer gÃstrico.

An estimated 50% of all people carry the stomach bacterium Helicobacter pylori (H. pylori). This organism is responsible for gastric problems like gastritis and gastric ulcers, and is one of the major causes of gastric cancer worldwide. Large numbers of people carry this organism asymptomatically and many questions remain about why serious symptoms develop in a subset of infected humans. These extremely recombinant bacteria may take different evolutionary trajectories in different people, and some genomic changes may be associated with gastric cancer. To test this, and learn more about the genetics of cancer-associated H. pylori, different approaches were used. First, evolution of H. pylori populations was investigated looking at both core and accessory genomes and revealed traces of the long and complex history of the Americas in the bacterial genomes, as well as a similar evolution in core and accessory genome. This was the first time accessory genome of H. pylori was studied that way. Secondly, evolution occurring in the bacterial genome during colonisation of a single host was studied in mice model. This analysis revealed small changes during the passage from a human host to a mice host, and during the long-term colonisation of mice stomach. Then a Genome Wide Association Study (GWAS) approach was applied to a large isolate collection sampled across Europe comprising strains isolated from cancer patients and strains from asymptomatic or gastritis-suffering patients. This approach identified 11 polymorphisms in 9 genes (3 cagPAI genes, babA, hpaA, 1 outer membrane protein coding gene HP1055 and 3 other core genes (HP0747, HP0709 and HP0468)) associated with cancer and a preliminary risk score was built to identify high risk strains. Finally, variations observed among clinical isolates of H. pylori from European patients with different pathologies in terms of motility and ability to trigger cytokine production in two types of cells were quantified. Motility variations were not associated with the disease type, but a link was observed for cytokine production. This was compared to genomic variations, confirming the role of known genomic factors such as cagPAI genes and sheding light to possible functions of a number of new genes.

La biosynthèse des polysaccahrides de l'enveloppe bactérienne nécessite la translocation, au travers de la membrane cytoplasmique, de sous unités de sucre qui sont produits dans le cytoplasme. A la fin de ces étapes, les sucres doivent se lier à un transporteur lipidique (l'undécaprényl phosphate, C55-P) fixant ces intermédiaires à la membrane, qui sont ensuite transloqué à la face externe de la membrane interne. Finalement, les fragments de glycane sont transférés à un polymère accepteur naissant. Cette étape libère le transporteur dans une forme inactive, l'undécaprényl-pyrophosphate (C55-PP). Le C55-P est synthétisé par la déphosphorylation du C55-PP, lui même provenant d'une synthèse de novo, ou du recyclage du transporteur. Deux types de phosphatases dépendantes du C55-PP ont été décrit : l'enzyme BacA, qui constitue une famille de phosphatases mal caractérisées et la superfamille des enzymes PAP-2 (phosphatases phosphatidique acides de type 2). Le pathogène humain Helicobacter pylori, ne possède pas d'enzyme appartenant à la famille BacA mais 4 protéines appartenant à la famille des PAP-2: HP0021, HP0350, HP1580 et HP0851. Plusieurs études ont montré des caractéristiques étonnantes provenant des enzyme PAP-2 comme une double fonction et des activités secondaires de phosphotransférases. HP0021 et HP1580 de H. pylori modifient le lipide A via des réactions de déphosphorylation, conduisant à une forme déphosphorylée du lipide A qui est nécessaire pour H. pylori afin de survivre dans la muqueuse gastrique, en conférant une résistance aux CAMPS et un échappement au système immunitaire de l'hôte. Ces enzymes sont essentielles pour la synthèse de plusieurs polymères de la paroi bactérienne par la (re)génération du C55-P, mais participe aussi à la biosynthèse d'autres lipides et à différents réseau de modification de l'enveloppe qui peuvent de façon très important modifier la physiologie cellulaire. Le but de ce projet est de mieux comprendre le rôle physiologique de ces 4 phosphatases via des approches multiples et complémentaires telles que des analyses permettant une caractérisation biochimique et structurale ainsi que leurs impacts dans le métabolisme de l'enveloppe bactérienne, et enfin leurs rôle dans l'interaction hôte-pathogène
The biogenesis of all the bacterial cell-envelope polysaccharides requires the translocation, across the plasma membrane, of sugar sub-units that are produced inside the cytoplasm. To this end, the hydrophilic sugars must be anchored to a lipid phosphate carrier (undecaprenyl phosphate (C55-P)), yielding membrane intermediates, which are translocated to the outer face of the membrane. Finally, the glycan moiety is transferred to a nascent acceptor polymer, releasing the carrier in the "inactive" undecaprenyl diphosphate (C55-PP) form. Thus, C55-P is synthesized through the dephosphorylation of C55-PP, itself arising from either de novo synthesis or recycling. Two types of integral membrane C55-PP phosphatases were described: BacA enzymes, which constitute a family of uncharacterized phosphatases, and a sub-group of PAP2 enzymes (type 2 phosphatidic acid phosphatases). The human pathogen Helicobacter pylori, does not contain BacA but has four membrane PAP2 proteins: HP0021, HP0350, HP1580 and HP0851. Further studies have revealed remarkable features of PAP2 enzymes such as dual functions and phosphotransfer side-activities. HP0021 and HP1580 from H. pylori modify lipid A via dephosphorylation reactions, yielding a dephosphorylated form of lipid A that is required for H. pylori to survive in the gastric mucosa, by conferring CAMPS resistance and escape to the host immune system. These enzymes are essential for the synthesis of various cell wall polymers by the (re)generation of C55-P, but they also participate in other lipids biosynthesis and to different networks of cell envelope modifications that can deeply modulate cell physiology. The project aims at deciphering the physiological role of these 4 phosphatases via multiple and complementary approaches ranging from a detailed biochemical and structural characterization to the assessment of their impact on cell envelope metabolism, bacterial fitness and microbe-host interactions

OLIVEIRA, Maria Aparecida Alves de. Sítios de fosforilação de tirosina da proteína caga e genótipos cage do H. pylori em pacientes com gastrite e úlcera péptica. 2014. 77 f. Tese (Doutorado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2014.
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The H. Pylori strains demonstrate a high level of phenotypic and genotypic diversity, the expression of various genes, which confer greater pathogenicity to the bacteria. Most studies have found that the cagE gene is strongly associated with peptic ulcer disease and sites of tyrosine phosphorylation of CagA protei - EPIYA, has not been fully elucidated existing geographical differences. This study aimed to characterize the strains of H. pylori for the sites of tyrosine phosphorylation of CagA protein EPIYA and presence of genes cagE in patients with gastritis and peptic ulcer disease, and to correlate the presence of these genotypes with gastric disorder. Genotyping of H. pylori strains was performed by PCR and sequencing of phosphorylation sites EPIYA. 137 dyspeptic patients from H. pylori-positive, 72 with peptic ulcer and 65 with gastritis were evaluated. Of the 137 strains genotyped 68.6 % were cagA positive, strains cagA studied , 96.8 % had at least 1 C phosphorylation site EPIYA , while 53.2 % of these had only one site , 43.6 % had two or more sites C and 3.2 % had no place C. the mixed strains with two EPIYA patterns were present in 23.4 % of the studied samples . The group of patients with gastritis showed higher prevalence of genotype EPIYA ABCCC , the same was not observed in patients with peptic ulcer disease and this association with gastrics disorders studied significant ( p = 0.001 ). Of the 137 patients studied, 65 were positive for CAGE (47.4%). There was a significant inverse association between genotype and cage group of patients with gastritis (OR = 0.345, p = 0.002). There was a significant association between genotype cagE with peptic ulcer disease (OR = 2.898, p = 0.002) and the subgroup of patients with duodenal ulcer (OR = 3.839, p = 0.001). This study suggests that the population sample studied the presence of genotype Epiya three sites C was associated with gastritis and absent in patients with peptic ulcer. We found association of the cagE gene duodenal ulcer.
As cepas de H. pylori demonstram um alto nível de diversidade fenotípica e genotípica, pela expressão de vários genes, que conferem maior patogenicidade à bactéria. A maioria dos estudos evidenciou forte associação do gene cagE com doença ulcerosa péptica e do aumento dos sítios de fosforilação EPIYA C com doenças gástricas mais graves. No entanto, a associação clínica e a função dos sítios de fosforilação de tirosina da proteína CagA – EPIYA e do gene cagE, ainda não foi completamente elucidada, existindo diferenças geográficas. O objetivo do presente estudo foi caracterizar as cepas de H. pylori quanto à presença dos sítios de fosforilação de tirosina da proteína CagA – EPIYA e do gene cagE em pacientes com gastrite e úlcera péptica, e correlacionar a presença desses genótipos com as afecções gástricas. A genotipagem das cepas de H. pylori foi realizada através da técnica de PCR e do sequenciamento dos sítios de fosforilação EPIYA. Foram avaliados 137 pacientes dispépticos H. pylori positivo, 72 com úlcera péptica e 65 com gastrite. Das 137 cepas genotipadas 68,6% foram cagA positivas. Das cepas cagA estudadas, 96,8% apresentaram pelo menos 1 sítio C de fosforilação EPIYA, sendo que 53,2% destas apresentaram somente um sitio, 43,6% tinham dois ou mais sítios C e 3,2% não apresentaram sitio C. As cepas mistas com dois padrões EPIYA estavam presentes em 23,4% das amostras estudadas. O grupo de pacientes com gastrite apresentou maior prevalência do genótipo EPIYA ABCCC; o mesmo não foi observado no grupo de pacientes com úlcera péptica, sendo essa associação com as afecções gástricas estudadas significante (p= 0,001). Dos 137 pacientes estudados, 65 foram positivos para cagE (47,4%). Houve associação inversa significativa entre o genótipo cagE e o grupo de pacientes com gastrite (O.R=0,345; p=0,002). Houve associação significativa entre o genótipo cagE com doença ulcerosa péptica (O.R=2,898; p=0,002) e com o subgrupo de pacientes com úlcera duodenal (O.R=3,839; p=0,001). Esse estudo sugere que na amostra populacional estudada a presença do genótipo EPIYA com três sítios C estava associada à gastrite e ausente nos pacientes com ulcera péptica. Encontrou-se associação do gene cagE a úlcera duodenal.

Throughout history a diversity of animal species have been used and studied extensively in the development of vaccines for the benefit of humans and animals alike. As mice are a relatively easy species to maintain, handle and manipulate, and have the advantage of being cost effective, they are commonly employed as animal models in the investigation of immunisation strategies against mucosal associated pathogens. Vaccine research against the human gastric pathogen Helicobacter pylori is extensively conducted in a mouse model and typically uses intra-gastric administration for the testing of potential vaccine candidates. An inherent complication with this route, however, is that the vaccine constituents may be inadequately delivered to sites of specific immunity and consequently may not be the optimal method for vaccine delivery. In the present study a mouse model of H. pylori infection was used to determine the efficacy of alternate mucosal routes of immunisation from examination of protective immunity, immune responses and the practical aspects of vaccine administration. Commencing with the optimisation of intra-intestinal immunisation, the direct injection of a H. pylori vaccine to initiator sites of the mucosal immune system established baseline data of dose rates for the comparative analysis of intra-gastric, intra-nasal and intra-rectal immunisation. Following the development of simple administration techniques whilst maintaining the welfare of the animals, intra-nasal immunisation was shown to elicit the highest level of prophylaxis against H. pylori challenge. Effective prophylaxis was also shown to be dependent upon a specific ratio of the vaccine constituents. When using whole cell lysate of H. pylori and the mucosal adjuvant cholera toxin, the ratio of antigen:adjuvant for optimal protective immunity was 10:1. The outcomes of this study have proved conclusively the necessity for optimisation of all aspects of immunisation in an animal model of infection.
Master of Science (Hons)

FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
O H. pylori infecta atualmente metade da populaÃÃo mundial e à relacionado com o desenvolvimento das afecÃÃes gÃstricas. O Enteroteste à um mÃtodo minimamente invasivo que pode ser usado para detecÃÃo do H. pylori por meio nÃo endoscÃpico. O objetivo deste estudo foi avaliar a sensibilidade e especificidade do Enteroteste frente ao Teste RespiratÃrio e estudar o perfil genÃtico das cepas de H. pylori em indivÃduos residentes na comunidade Parque UniversitÃrio. O suco gÃstrico foi colhido a partir do Enteroteste, e realizada a cultura e a extraÃÃo do DNA do H. pylori. A genotipagem das cepas foi realizada atravÃs da tÃcnica de PCR e os sÃtios de fosforilaÃÃo EPIYA de sequenciamento Participaram do estudo 50 indivÃduos, 43 positivos e 7 negativos para a infecÃÃo pelo H. pylori atravÃs do Teste RespiratÃrio. A cultura e o PCR a partir do Enteroteste apresentaram sensibilidade de 86% e 77%, respectivamente, com especificidade de 100% em ambos os mÃtodos 33 cepas foram genotipadas como vacA positivas, sendo 39,4% vacA s1m1; 15,2% vacA s1m2; 18,2% vacA s2m2 27,2% com perfil vacA s com ausÃncia do alelo m. Foram cagA positivas 66,6% das cepas sendo 54,5% com perfil EPIYA-ABC, 41,0% EPIYA-ABCC e 4,5% EPIYA-AB. O Enteroteste mostrou-se um mÃtodo confiÃvel com boa sensibilidade e especificidade para identificaÃÃo do H. pylori atravÃs do cultivo e da tÃcnica de PCR. A maioria das cepas expressou o alelo vacA s1, com predomÃnio do subtipo s1b e da combinaÃÃo alÃlica s1m1. Mais da metade das cepas estudadas expressaram o gene cagA e a maioria dessas cepas tinham 3 ou 4 sÃtios de fosforilaÃÃo, com perfil EPIYA-ABC ou EPIYA-ABCC. O perfil genÃtico apresentado por essas cepas à o descrito para a AmÃrica do Sul e diferente do padrÃo AsiÃtico, a maioria das cepas circulantes na comunidade tÃm importante potencial patogÃnico. O Enteroteste à um mÃtodo seguro, sensÃvel e especÃfico para detecÃÃo do H. pylori.
The H. pylori infect currently half of the worldâs population and is related to the development of gastric disorders. The Enterotest is a minimally invasive method that can be used for detection of H. pylori through endoscopic not. The objective of this study was to evaluate the sensitivity and specificity of Enterotest opposite the Respiratory Testing and studying the genetic profile of strains of H. pylori individuals resident in the Community College Park. The gastric juice was collected from Enteroteste, and held the culture and the extraction of DNA from H. pylori. The genotyping of strains was carried out by the PCR technique and phosphorylation sites EPIYA sequencing participated in the study 50 individuals, positive and negative 7 43 for infection H. pylori through Respiratory Test. Culture and PCR from Enteroteste showed sensitivity of 86% and 77%, respectively, with 100% specificity in both methods. 33 strains were positive, genotipadas as Cow being 39.4% Cow s1m1; 15.2% Cow s1m2; 18.2% Cow s2m2 27.2% with Cow s profile with absence of allele m. Were positive 66.6% Bran of strains being profiled 54.5% EPIYA-ABC, 41.0% EPIYA-ABCC and 4.5% EPIYA-AB. The Enterotest proved to be a reliable method with great sensitivity and specificity for identification of H. pylori through cultivation and PCR technique. Most strains expressed alelo Cow s1, with a predominance of subtype s1b and allele combination s1m1. More than half of the studied strains expressed gene Shit and most of these strains were 3 or 4, phosphorylation sites profiled EPIYA-ABC or EPIYA-ABCC. The genetic profile presented by these strains is described for South America and nonstandard Asia, most strains circulating in the community have important potential pathogenic. The Enterotest is a safe method, sensitive and specific detection of H. pylori.

Three H. pylori proteins contributing to stomach colonization have been chosen to be structurally characterized: Fur, Hp1454, and HypB. The first one, which is a Fe-dependent transcription regulator was expressed and purified. Unfortunately. after the crystallization test started, the crystal structure of Fur was published by another group. Hp1454 is a secreted protein of unknown function. It has been purified, expressed and crystallized. Since it lacks any sequence similarity with other proteins of known structure, Se-Met derivatives were produced and the structure determined. A function for the protein has been also proposed, based on the study of Hp1454 interaction network. The GTPase HypB is an essential factor for maturation of H. pylori’s hydrogenase and urease. It was cloned, expressed and purified and preliminary crystallization tests performed.
Tre proteine di H. pylori che rivestono un importante ruolo nella colonizzazione dello stomaco sono state scelte per essere caratterizzate strutturalmente: Fur, Hp1454 e HypB. La prima, un regolatore trascrizionale ferro-dipendente, è stata espressa e purificata. Purtroppo, dopo i primi test di cristallizzazione, la struttura di Fur è stata pubblicata da un altro gruppo. Hp1454 è una proteina secreta la cui funzione è sconosciuta. Hp1454 é stata purificata, espressa e cristallizzata. Poichè la proteina non presenta omologia di sequenza con altre proteine di struttura nota, sono stati prodotti derivati di Se-Met e la struttura è stata quindi determinata. Una funzione per la proteina è stata inoltre proposta sulla base degli studi di interazione tra Hp1454 e altre proteine. La GTPasi HypB è un essenziale fattore nella maturazione dell' idrogenasi e dell' ureasi di pylori. La proteina è stata clonata e purificata e sono stati effettuate delle prove preliminari di cristallizzazione.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
The bacterial species H. pylori is closely correlated with the development of major diseases and gastric cancer of the stomach. The strains that have more pathogenic genotype are more involved in bacterial virulence. The objective was to genotype strains of H. pylori from patients with gastritis, gastroduodenal ulcer and gastric cancer as the cagA and vacA genes and alleles by PCR technique. The genotyping of strains of H. pylori was performed by PCR technique. We evaluated 134 strains, 76 of patients with gastritis, 28 peptic ulcer and 30 gastric cancer. As a result of this research it was found that all strains subtyped were vacA positive, and 48.5% with the allele s1m1 vacA, vacA s1m2 20.9% and 6.7% s2m1 vacA, vacA s2m2 8.2%; 7 5% were hybrid strains and 8.2% had only the allele of the sequence signal (s). Gastritis in the distribution of vacA alleles were: 46.1% s1m1, s1m2 22.4%, 5.3% s2m1, s2m2 11.8%, 3.9% and 10.5% hybrid strains only with allele s. In cases of ulcer was found: 50% s1m1, s1m2 21.4%, 17.8% s2m1, s2m2 3.6%, 7.2% hybrid strains. In patients with cancer met: s1m1 53.3%, 16.7% s1m2, s2m2 3.3%, 16.7% and 10.0% hybrid strains with allele only s. The cagA gene was observed in 82.8% of strains, where 73.7% had gastritis and cagA positive, 96.4% had peptic ulcer and gastric cancer harbored 96.7% were positive for this gene. Allele vacAs1m1 was the most prevalent observed in the three groups. We have also found strains with multiple alleles of this gene, suggesting a genetic polymorphism arising from infection by more than one type of strain in the same individual. The cagA gene was also highly prevalent in both groups. The presence of cagA was statistically significant in the group of pangastritis and gastric cancer. The relationship of the cagA gene with allele vacAs1m1 was also significant in the groups of pangastritis and gastric cancer, which supports the higher pathogenicity of strains of the studied individuals.
A espÃcie bacteriana Helicobacter pylori està intimamente correlacionado com o desenvolvimento das principais afecÃÃes gÃstricas e do cÃncer de estÃmago. As cepas que apresentam genÃtipo mais patogÃnico sÃo as mais implicadas na virulÃncia da bactÃria. O objetivo deste trabalho foi caracterizar as cepas de H. pylori provenientes de pacientes portadores de gastrite, Ãlcera gastroduodenal e cÃncer gÃstrico quanto aos genes cagA e vacA e alelos atravÃs da tÃcnica de PCR. A genotipagem das cepas de H. pylori foi realizada atravÃs da tÃcnica de PCR. Foram avaliadas 134 cepas, sendo 76 de portadores de gastrite, 28 de Ãlcera pÃptica e 30 de cÃncer gÃstrico. Como resultado desta pesquisa obteve-se que, todas as cepas genotipadas foram vacA positivas, sendo 48,5% com o alelo vacA s1m1; 20,9% vacA s1m2; 6,7% vacA s2m1; 8,2% vacA s2m2; 7,5% eram cepas hÃbridas e 8,2% apresentaram somente o alelo da sequÃncia sinal(s). Nos portadores de gastrite a distribuiÃÃo dos alelos vacA foi: 46,1% s1m1, 22,4% s1m2, 5,3% s2m1, 11,8% s2m2, 3,9% cepas hÃbridas e 10,5% somente com alelo s. Nos casos de Ãlcera achou-se: 50% s1m1, 21,4% s1m2, 17,8% s2m1, 3,6% s2m2, 7,2% cepas hÃbridas. Nos portadores de cÃncer encontrou-se: 53,3% s1m1, 16,7% s1m2, 3,3% s2m2, 16,7% cepas hÃbridas e 10,0% somente com alelo s. O gene cagA foi observado em 82,8% das cepas, onde 73,7% tinham gastrite e cagA positivo, 96,4% tinham Ãlcera pÃptica e 96,7% portavam cÃncer gÃstrico com positividade para este gene. O alelo vacAs1m1 foi o mais prevalente observado nos trÃs grupos estudados. TambÃm foram encontradas cepas com alelos mÃltiplos desse gene, o que sugere um polimorfismo genÃtico advindo de infecÃÃo por mais de um tipo de cepa em um mesmo indivÃduo. O gene cagA tambÃm foi altamente prevalente nos grupos estudados. A presenÃa do cagA foi estatisticamente significante no grupo de pangastrite e cÃncer gÃstrico. A relaÃÃo do gene cagA com o alelo vacAs1m1 tambÃm foi significante nos grupos de pangastrite e cÃncer gÃstrico, o que corrobora a maior patogenicidade das cepas presentes nos indivÃduos estudados.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Infecção persistente pela Helicobacter pylori (H. pylori) promove inflamação crônica que resulta em danos na mucosa e o consequente desenvolvimento de lesões gástricas, como a gastrite crônica. Esta bactéria e seus fatores de virulência vacA e cagA influenciam na resposta imune do hospedeiro, desregulando a expressão de mediadores inflamatórios e microRNAs (miRNAs), assim ativando vias relacionadas ao processo carcinogênico. Portanto, a erradicação da H. pylori pode ter um papel importante para reverter lesões precursoras, prevenindo a transformação maligna. O presente estudo avaliou a influência da terapia de erradicação da bactéria na expressão do RNAm e da proteína de mediadores inflamatórios (TNFA, IL6, IL1B, IL12A, IL2 e TGFBRII) e de miRNAs (hsa-miR-103a-3p, hsa-miR-181c-5p, hsa-miR-370-3p, hsa-miR-375 e miR-223-3p) em pacientes com gastrite crônica H. pylori positivos (Hp+), em comparação com pacientes com gastrite não infectados (Hp-). Também avaliou a relação entre os níveis de expressão dos miRNAs e os dos RNAm e proteínas e a participação em redes de interação, assim como a influência dos fatores de virulência bacteriano cagA e vacA sobre estes mediadores. A quantificação relativa (RQ) do RNAm e dos miRNAs foi realizada por PCR quantitativa em tempo real (qPCR) usando ensaio TaqMan® em 20 pacientes Hp- e 31 Hp+, os quais foram também avaliados três meses após o tratamento de erradicação da bactéria. A genotipagem de cagA e vacA foi realizada pela técnica de PCR, enquanto que a expressão protéica foi avaliada por imuno-histoquímica. Pacientes Hp+ apresentaram níveis aumentados do RNAm de TNFA, IL6, IL1B e IL12A, enquanto que IL2 e TGFBRII bem como os miRNAs miR-103, miR-181c, miR-370 e miR- 375 mostraram expressão reduzida. Após o tratamento, o RNAm de TNFA e IL6 apresentouse significantemente reduzido e de TGFBRII aumentado em pacientes...
Persistent infection by Helicobacter pylori (H. pylori) promotes chronic inflammation resulting in damage to the mucosa and the consequent development of gastric lesions as chronic gastritis. This bacterium and its virulence factors vacA and cagA influence the host immune response, deregulating the expression of inflammatory mediators and microRNAs (miRNAs), and activate pathways related to carcinogenic process. Thus, H. pylori eradication may have a key role to reverse precursor lesions, preventing malignant transformation. In the present study evaluated whether the eradication therapy of bacterium influences the mRNA and protein expression of inflammatory mediators (TNFA, IL6, IL1B, IL12A, IL2 and TGFBRII) and miRNAs (hsa-miR-103a-3p, hsa-miR-181c-5p, hsa-miR-370-3p, hsa-miR-375 and miR-223-3p) in H. pylori-positive chronic gastritis patients (Hp+) and compared with non-infected gastritis patients (Hp-). We also evaluated the relationship between the expression levels of miRNAs with of mRNA and protein and participation in interaction networks, as well as the influence of bacterial virulence factors cagA and vacA on these mediators. Relative quantification (RQ) of mRNA and miRNAs was performed by qPCR-real time using TaqMan® assay in 20 patients Hp- and 31 Hp+, which were also evaluated three months after eradication treatment of the bacterium. Genotyping of cagA and vacA was performed by PCR, while the protein expression was assessed by immunohistochemistry. Hp+ patients showed increased mRNA levels for TNFA, IL6, IL1B and IL12A, while for IL2 and TGFBRII as well as for the miRNAs miR-103, miR-181c, miR-370 and miR-375 it were decreased. After treatment, only TNFA and IL6 mRNA were significantly reduced and TGFBRII increased in eradicated patients (p<0.05). On the other hand, all miRNAs, except to miR-223, were significantly increased after bacterium eradication (p<0.05). In general, the...

Eines der prävalentesten humanen Pathogene ist das Magenbakterium Helicobacter pylori, welches ca. die Hälfte der Weltbevölkerung infiziert. Die Persistenz geht mit einer chronischen Gastritis einher, welche bis zu Magenkrebs fortschreiten kann. H.pylori bedient sich diverser Mechanismen um sich der Erkennung des Immunsystems zu entziehen und somit eine chronische Infektion zu ermöglichen. Erhöhte Expression des Immunzellinhibitors PD-L1 wurde in Magenepithelzellen gefunden, welche mit diesem Gram-negativen Erreger infiziert wurden. In dieser Arbeit wurde die Regulation auf in vitro Ebene untersucht, wobei zwei unterschiedliche Mechanismen identifiziert wurden. Ursächlich für die frühe PD-L1-Induktion ist die ADP-heptose/ALPK1 Signalkaskade. Der bakterielle Metabolit ADP-heptose, welcher für die Bildung von LPS benötigt wird, wurde als PAMP identifiziert, welcher durch das Sekretionssystems cagT4SS in die infizierte transportiert und anschließend von der Host Kinase ALPK1 erkannt wird. Gegensätzlich hierzu, wurde festgestellt, dass die zweite PD-L1-Hochregulation auf der metabolischen Reprogrammierung des Wirts beruht. Ein Merkmal von H. pylori ist dessen Bedarf an Cholesterin, welches es aus dem Medium oder aus membranösen Lipidregionen des Wirts extrahiert wird. Es konnte bewiesen werden, dass dieser Sterol-Abbauprozess zu einer erhöhten Stoffwechselaktivität führt, die spezifisch mit einer Zunahme der Glykolyse verbunden ist und mit einer Expressionsverschiebung des ersten Glykolyseenzyms Hexokinase von der Isoform 1 zu 2 einhergeht. Knockdown und Knockout- Experimente wiesen auf einen Zusammenhang mit der Regulation des Immunzellinhibitoren PD-L1 hin.
One of the most prevalent bacteria is the gastric bacterium Helicobacter pylori, which infects half of the world’s population. Persistence is accompanied with chronic gastritis which can progress towards gastric cancer. Several strategies are used by H.pylori to evade the immune system, enabling chronic infection. Heightened expression of the immune cell inhibitor PD-L1 was found in gastric epithelial cells, infected with this Gram-negative pathogen. Within this thesis, upregulation was studied in in vitro models, revealing two distinct mechanism. Causative for early PD-L1 induction is the ADP heptose/ALPK1 signaling axis. The bacterial metabolite ADP heptose, which is needed for LPS synthesis, was identified as PAMP, which is transported through the secretion system cagT4SS into the infected cell and is recognized by the host kinase ALPK1. In contrast, late upregulation of PD-L1 was found to be linked to metabolic reprogramming upon infection. Characteristic to H.pylori is its need of cholesterol, which it has to extract from the surrounding medium or lipid-rich regions within the host membrane. It could be shown that this sterol extraction process is accompanied with an increased metabolic activity which is linked with enhanced glycolysis and an expression shift of the glycolytic enzyme hexokinase isoform 1 to 2. Knockdown and knockout experiments showed a link between HK2 and regulation of the immune checkpoint PD-L1.

Orientador: Ana Elizabete Silva
Banca: Cláudia Aparecida Rainho
Banca: Marília de Freitas Calmon Saiki
Resumo: Infecção persistente pela Helicobacter pylori (H. pylori) promove inflamação crônica que resulta em danos na mucosa e o consequente desenvolvimento de lesões gástricas, como a gastrite crônica. Esta bactéria e seus fatores de virulência vacA e cagA influenciam na resposta imune do hospedeiro, desregulando a expressão de mediadores inflamatórios e microRNAs (miRNAs), assim ativando vias relacionadas ao processo carcinogênico. Portanto, a erradicação da H. pylori pode ter um papel importante para reverter lesões precursoras, prevenindo a transformação maligna. O presente estudo avaliou a influência da terapia de erradicação da bactéria na expressão do RNAm e da proteína de mediadores inflamatórios (TNFA, IL6, IL1B, IL12A, IL2 e TGFBRII) e de miRNAs (hsa-miR-103a-3p, hsa-miR-181c-5p, hsa-miR-370-3p, hsa-miR-375 e miR-223-3p) em pacientes com gastrite crônica H. pylori positivos (Hp+), em comparação com pacientes com gastrite não infectados (Hp-). Também avaliou a relação entre os níveis de expressão dos miRNAs e os dos RNAm e proteínas e a participação em redes de interação, assim como a influência dos fatores de virulência bacteriano cagA e vacA sobre estes mediadores. A quantificação relativa (RQ) do RNAm e dos miRNAs foi realizada por PCR quantitativa em tempo real (qPCR) usando ensaio TaqMan® em 20 pacientes Hp- e 31 Hp+, os quais foram também avaliados três meses após o tratamento de erradicação da bactéria. A genotipagem de cagA e vacA foi realizada pela técnica de PCR, enquanto que a expressão protéica foi avaliada por imuno-histoquímica. Pacientes Hp+ apresentaram níveis aumentados do RNAm de TNFA, IL6, IL1B e IL12A, enquanto que IL2 e TGFBRII bem como os miRNAs miR-103, miR-181c, miR-370 e miR- 375 mostraram expressão reduzida. Após o tratamento, o RNAm de TNFA e IL6 apresentouse significantemente reduzido e de TGFBRII aumentado em pacientes...
Abstract: Persistent infection by Helicobacter pylori (H. pylori) promotes chronic inflammation resulting in damage to the mucosa and the consequent development of gastric lesions as chronic gastritis. This bacterium and its virulence factors vacA and cagA influence the host immune response, deregulating the expression of inflammatory mediators and microRNAs (miRNAs), and activate pathways related to carcinogenic process. Thus, H. pylori eradication may have a key role to reverse precursor lesions, preventing malignant transformation. In the present study evaluated whether the eradication therapy of bacterium influences the mRNA and protein expression of inflammatory mediators (TNFA, IL6, IL1B, IL12A, IL2 and TGFBRII) and miRNAs (hsa-miR-103a-3p, hsa-miR-181c-5p, hsa-miR-370-3p, hsa-miR-375 and miR-223-3p) in H. pylori-positive chronic gastritis patients (Hp+) and compared with non-infected gastritis patients (Hp-). We also evaluated the relationship between the expression levels of miRNAs with of mRNA and protein and participation in interaction networks, as well as the influence of bacterial virulence factors cagA and vacA on these mediators. Relative quantification (RQ) of mRNA and miRNAs was performed by qPCR-real time using TaqMan® assay in 20 patients Hp- and 31 Hp+, which were also evaluated three months after eradication treatment of the bacterium. Genotyping of cagA and vacA was performed by PCR, while the protein expression was assessed by immunohistochemistry. Hp+ patients showed increased mRNA levels for TNFA, IL6, IL1B and IL12A, while for IL2 and TGFBRII as well as for the miRNAs miR-103, miR-181c, miR-370 and miR-375 it were decreased. After treatment, only TNFA and IL6 mRNA were significantly reduced and TGFBRII increased in eradicated patients (p<0.05). On the other hand, all miRNAs, except to miR-223, were significantly increased after bacterium eradication (p<0.05). In general, the...
Mestre

The cytokine APRIL (A proliferation inducing ligand) and the cytokine BLyS (B lymphocyte simulator) are two recently discovered factors that belong to the TNF-(tumor necrosis factor) family. Since their discovery, these cytokines have been intensively studied for their role in B cells biology. Indeed, APRIL and BLyS are involved in the maturation of B cells, but, most importantly, they increase the survival and proliferation of these cells. However, the effect of these cytokines is amplified in neoplastic B cells in lymphomas, and self-reactive B cells in autoimmune diseases. In particular, it has been demonstrated that APRIL is over-expressed in different types of lymphoma, whereas BLyS is very abundant in serum and tissues of patients with different types of autoimmune diseases, such as, rheumatoid arthritis or systemic lupus erythematous. In our study, we evaluated the role of these cytokines in two diseases caused by the bacterium Helicobacter pylori (H. pylori): MALT lymphoma (I) and autoimmune gastritis (AIG) (II). (I) the development of lymphoid follicles with germinal centers and an acquired MALT (mucosa-associated lymphoid tissue) are characteristic of H. pylori-induced gastritis. These MALT represents a pre-neoplastic condition associated with infection, which can evolve into a B-cell lymphoma. It is currently unclear which cytokines or soluble factors promote B cell activation and the genesis of lymphoma during H. pylori infection. In our study, we have demonstrated that gastric MALT lymphoma is characterized by high expression of APRIL. Furthermore, APRIL is over-expressed also in a pre-MALT situation, suggesting that this cytokine may be involved in the induction, as well as in the proliferation of the lymphoma. Further analysis allowed us to identify the macrophages as the main source of APRIL in this type of cancer. We confirmed this observation by in vitro experiments, suggesting that H. pylori is able to induce the secretion of APRIL by macrophages. We have also shown that the contribution to APRIL secretion is supported also by tumour infiltrating T cells specific for H. pylori. Thus, we have demonstrated that H. pylori is able to induce APRIL secretion by macrophage in a direct way, following in vitro infection, but even in an undirect way, mediated by H. pylori-specific T lymphocytes. Our data represent the first evidence of the involvement of APRIL in the development of MALT lymphoma in H. pylori infected patients. Moreover, we identified macrophages as key cells in this process. (II) Among the diseases caused by H. pylori there is also AIG. It has recently been reported that the cytokine BLyS plays a pivotal role in the pathogenesis of two different models of autoimmune disease (collagen induced arthritis and experimental autoimmun encephalomyelitis); in these works, it has been demonstrated that BLyS induce naive T cells to differentiate into the Th17 phenotype. These effector T cells, produce IL-17 and they play a crucial role in the pathogenesis and maintenance of several autoimmune diseases. Thus, given that it is established that H. pylori infection is associated with a Th17 response, we wondered to investigate whether the BLyS/Th17 axis is involved in the development of AIG following H. pylori-infection. To test this hypothesis, we have considered H. pylori-induced chronic gastritis as a model for our analysis because it is the typical inflammatory context that might evolve AIG in susceptible patients. We have demonstrated that patients with chronic gastritis not only present, as expected, a Th17-enriched cellular infiltrate in gastric mucosa, but also that in these biopses there is a significant expression of BLyS. Noteworthy, after the eradication of the bacterium there is a marked reduction of both BLyS and IL-17 cytokines in gastric mucosa, underlining the crucial role of H. pylori in the axis BLyS/Th17. We have also shown that BLyS is able to activate in vitro monocytes and macrophages, inducing the secretion of IL-1β, IL-6, TGF-β and IL-23 by these cells: all these cytokines are fundamental for Th17 cells differentiation. In addition, we identified neutrophils as a possible source of BLyS in gastric mucosa. Indeed, H. pylori-stimulated neutrophils secrete a great amount of BLyS in vitro. Accordingly, there are evidence that neutrophils represent a great percentage of cellular infiltrate during H. pylori-sustained inflammation. Therefore, we hypothesized a model for AIG development of following H. pylori-infection: the bacterium stimulates gastric mucosa-infiltrating neutrophils to release BLyS. BLyS, in turn could on one hand lead to the survival of autoreactive B cells (characterized by auto-antibodies production), but on the other hand, it might also acts on monocytes/macrophages (which are also abundant in the inflammatory infiltrate) for the induction of pro-Th17 cytokines. The Th17-pro-inflammatory profile may increase inflammation and worsen the tissue damage which is typically found in patients with AIG. However, this study is still in progress and many things remain to be understood to validate this model. We first of all need to identify BLyS-expressing cells in chronic gastritis; to this aim different double-immunohistochemistry staining with specific markers for different immune cell types are currently underway. In addition, the results of immunohistochemical analysis will be corroborated with in vitro experiments aimed to: i) confirm the ability of cells, identified by immunohistochemistry, to secrete BLyS following stimulation with H. pylori, ii) confirm the role of BLyS in the differentiation of Th17 cells. Although this second study is currently ongoing, we have demonstrated that H. pylori might create a cytokine microenvironment which can drive the development of MALT lymphoma or AIG. In particular, we have shown that H. pylori is able to induce the production and release of BLyS and APRIL by macrophages and neutrophils, respectively, in two diseases characterized by dysfunction of B cells. These data may suggest that targeting these cytokines may be a new additional therapeutic approach for the treatment of AIG and MALT lymphoma
La citochina APRIL (A proliferation inducing ligand) e la citochina BLyS (B lymphocyte simulator) sono due fattori, scoperti recentemente, facenti parte della famiglia dei TNF (tumour necrosis factor). Dalla loro scoperta entrambe queste citochine sono state intensivamente studiate per il loro ruolo nello sviluppo dei linfociti B. Infatti sia APRIL che BLyS sono coinvolte nella maturazione delle cellule B, ma è stato anche dimostrato che ne aumentano la sopravvivenza e la proliferazione. L’effetto di queste citochine risulta però amplificato su cellule B neoplastiche, che caratterizzano i linfomi, e sulle cellule B auto-reattive, che giocano un ruolo cruciale nelle patologie autoimmuni. In particolare, è stato dimostrato che APRIL risulta sovraespressa specificamente in diversi tipi di linfoma, mentre BLyS è molto abbondante nel siero e nei tessuti di pazienti affetti da diversi tipi di patologie autoimmuni, come ad esempio, l’artrite reumatoide, o il lupus eritematoso sistemico. Nel nostro studio abbiamo valutato il ruolo di queste citochine in due patologie causate dal batterio Helicobacter pylori (H. pylori): i linfomi tipo MALT (I) e la gastrite autoimmune (II). (I) L’infiammazione causata da H. pylori è caratterizzata da una massiva infiltrazione di cellule, che però non riescono ad contrastare efficacemente la crescita batterica. Questo porta molto facilmente alla cronicizzazione dell’infezione che porta allo sviluppo di follicoli linfatici con centri germinativi. Questo follicoli costituiscono una sorta di organo linfoide associato alla mucosa gastrica detto MALT (tessuto linfatico mucosa-associato). L’acquisizione del MALT rappresenta una condizione pre-neoplastica associata all’infezione da H. pylori, che può evolvere verso un linfoma a cellule B. Attualmente non è noto quali citochine o fattori solubili promuovano l’attivazione delle cellule B e la genesi del linfoma a seguito dell’infezione da H. pylori. Nel nostro studio, abbiamo dimostrato che nel linfoma gastrico di tipo MALT sono espressi alti livelli di APRIL. Inoltre, APRIL risulta sovraepresso anche in una situazione pre-MALT, suggerendo che questa citochina possa essere coinvolta anche nell’induzione, oltre che nel mantenimento del linfoma. Un’analisi approfondita ci ha in seguito permesso di identificare la popolazione macrofagica come fonte principale di APRIL in questo tipo di tumore. Abbiamo poi confermato con esperimenti in vitro che H. pylori è in grado di indurre la secrezione di APRIL da parte dei macrofagi. Abbiamo infine dimostrato che anche le cellule T, specifiche per H. pylori, possono indurre i macrofagi a secernere APRIL. I nostri dati rappresentano la prima evidenza di un coinvolgimento della citochina APRIL nello sviluppo del linfoma MALT in pazienti con infezione da H. pylori e identificano nei macrofagi le cellule chiave nel processo. (II) Tra le malattie che conseguono all’infezione da H. pylori c’è la gastrite autoimmune. E’ stato recentemente dimostrato il ruolo determinante della citochina BLyS in due differenti modelli di malattia autoimmune; BLyS infatti indurrebbe i linfociti T naive a differenziare verso il fenotipo Th17. Queste cellule T effettrici, producono IL-17 e giocano un ruolo cruciale nella genesi e nel mantenimento di numerose patologie autoimmuni. Con queste premesse, abbiamo voluto verificare se nella gastrite autoimmune da H. pylori fosse coinvolto l’asse BLyS/Th17, anche alla luce del fatto che era già noto che l’infezione da H. pylori si associa ad una risposta Th17. Per valutare questa possibilità abbiamo utilizzato come modello di studio la gastrite cronica indotta da H. pylori, in quanto essa può costituire il contesto da cui evolve la gastrite autoimmune. Abbiamo dimostrato che pazienti con gastrite cronica non solo presentano, come atteso, una mucosa riccamente infiltrata di linfociti Th17, ma quest’ultima è anche caratterizzata da una significativa espressione della citochina BLyS. Degno di nota il fatto che l’eradicazione del batterio si accompagna ad una riduzione di entrambe le citochine, BLyS e IL-17, nella mucosa dei pazienti, a sottolineare il ruolo determinante del batterio nel sostenere l’asse BLyS/Th17. Inoltre, abbiamo dimostrato che BLyS è in grado attivare in vitro monociti e macrofagi e di indurre la secrezione da parte di queste cellule di IL-1β, IL-6, TGF-β and Il23, tutte citochine coinvolte nel differenziamento delle cellule Th17. In aggiunta abbiamo identificato i neutrofili come potenziale fonte di BLyS nella mucosa gastrica. Infatti, neutrofili stimolati in vitro con H. pylori secernono grandi quantitativi di BLyS ed inoltre sono dimostrati essere la maggior componente dell’infiltrato cellulare nell’infezione di H. pylori. Quindi, abbiamo ipotizzato un modello di sviluppo della gastrite autoimmune a seguito dell’infezione di H. pylori: il batterio stimolerebbe i neutrofili infiltranti la mucosa gastrtica a rilasciare BLyS. BLyS, potrebbe da un lato indurre la sopravvivenza delle cellule B autoreattive (con relativa produzione di auto-anticorpi), ma anche agire sui monociti/macrofagi (anch’essi abbondanti nell’infilatrato infiammatorio) inducendo la secrezione di citochine che portano al differenziamento delle cellule Th17. Il profilo pro-infiammatorio delle cellule Th17, potrebbe aumentare l’infiammazione e peggiorare il danno tissutale tipicamente riscontrato nei pazienti con gastrite autoimmune. Tuttavia, questo studio è tuttora in corso d’opera e molte cose restano da capire per validare questo modello. Bisogna innanzitutto identificare le cellule esprimenti BLyS nella gastrite cronica; a questo scopo,diverse prove di co-immunoistochimica con marcatori specifici per i diversi tipi di cellule del sistema immunitario sono attualmente in corso. Inoltre, i risultati delle immunoistochimiche saranno corroborati con esperimenti in vitro mirati a: i) confermare la capacità delle cellule, identificate per immunoistochimica, di secernere BLyS a seguito di stimolazione con H. pylori; ii) confermare il ruolo di BLyS nel differenziamento delle cellule Th17. Ancorché questo secondo studio sia attualmente in corso, riteniamo plausibile proporre l’idea generale che H. pylori possa creare un microambiente citochinico favorevole allo sviluppo sia del linfoma tipo MALT che della gastrite autoimmune. In particolare, abbiamo dimostrato che questo batterio è in grado di indurre la produzione ed il rilascio di APRIL e BLyS da parte di macrofagi e neutrofili rispettivamente, in due patologie caratterizzate da una disfunzione delle cellule B. Questi dati potrebbero suggerire che terapie mirate verso queste citochine potrebbero diventare un nuovo approccio terapeutico per la cura del MALT linfoma e della gastrite autoimmune

L’objectif de ma thèse a été d’étudier les réponses transcriptionnelles suite à l’infection par Statphylococcus aureus (S. Aureus) et Helicobacter pylori (H. Pylori) grâce aux puces à ADN. Pour identifier les mécanismes sous-jacents à une plus forte sensibilité de l’épithélium respiratoire à S. Aureus lors de la mucoviscidose, j’ai étudié les réponses transcriptionnelles des cellules épithéliales respiratoires MM-39 (CFTR non muté) après contact avec S. Aureus ou avec le surnageant bactérien qui contient les produits de sécrétion. Les facteurs de transcription comme HIF ou NF-kB sont surexprimés et activés, expliquant la surexpression de molécules inflammatoires. Une comparaison entre les cellules MM-39 et les cellules CF-KM4 (mutation delta F508 du CFTR) après contact avec le surnageant de S. Aureus a révélé une surexpression d’IL-1 et de prostaglandines dans les MM-39 alors que l’IL-6, les leucotriènes, et les calgranulines sont surexprimées dans les CF-KM4. J’ai ensuite analysé le transcriptome de biopsies stomacales infectées par H. Pylori, caractérisée par une activation du complément, et une réponse induite par le CMH-2. Alors que les biopsies ont été extraites au stade gastrite, deux gènes trouvés, surexprimés dans certains cancers, sont surexprimés, résultant probablement d’une réponse non appropriée de type CD4+ Th1, associée à la production d’interféron. Après avoir développé une puce double brin à ADNc, utilisable pour l’étude de nombreuses espèces mammifères, j’ai participé à la création et a la validation d’une collection d’oligonucléotides correspondant à 95% des transcrits humains. J’ai également validé plusieurs méthodes d’analyse
The aim of my thesis was to study the host transcriptional responses to infection by Staphylococcus aureus (S. Aureus) and Helicobacter pylori (H. Pylori) with DNA microarray. To identify the mechanisms underlying the stronger sensibility to S. Aureus of the cystic fibrosis respiratory epithelium, I first studied the transcriptional response to epithelial respiratory MM-39 cells (WT) after a contact with S. Aureus or to bacterial supernatant containing bacterial secreted products. Transcription factors such as HIF and NF-kB, were over-expressed and activated, explaining the up-regulation of inflammatory molecules. A comparison between MM-39 cells and CF-KM4 cells (DF 508 mutated form of CFTR) after a contact with S. Aureus supernatant revealed that IL-1 and prostaglandins were up-regulated mainly in MM-39 cells, whereas IL-6, leukotrienes, and calgranulins were up-regulated mainly in CF-KM4 cells. I then analysed the transcriptome of stomach biopsies infected by H. Pylori, characterized by an activation of the complement cascade, along with a MHC-II elicited response. Whereas biopsies were extracted at gastritis stage, two genes previously found over-expressed in cancers were up-regulated, probably resulting from a non appropriate Th1 type CD4+ response associated with interferon gamma production. The microarray technology requires numerous interacting steps. After having developed a double strand cDNA microarray that can be used for most mammal species studies, I participated to the creation and validation of a human oligonucleotide collection corresponding to 95 % of all known human transcripts. I also set up and validated many analysis methods

Introduction: Helicobater pylori interferes with vitamin C homeostasis and low levels of vitamin C may be a risk factor for H. pylori infection. Objective: to investigate the vitamin C serum and gastric juice levels of patients infected and non-infected by H. pylori. Additionally assess the association of vitamin C levels with H.pylori status, gastric pH and histological changes of gastric mucosa of dyspeptic patients with normal upper GI endoscopy. Methods: We studied 42 dyspeptic patients undergoing upper GI endoscopy had biopsies for diagnosis of H. pylori infection and gastric mucosa changes. We also collected 5 ml of blood (for dosage of serum vitamin C) and 10 ml of gastric juice. We measure pH of gastric juice. We used high-performance liquid chromatography to assess Vitamin C in serum and gastric juice. Results: The average age was 52 years (SD±11.8), 84.4% female, overweight BMI median 27,7) with adequate dietary intake of vitamin C. Twenty four (53.3%) patients had H. pylori infection. The median serum vitamin C levels in infected and non-infected was 3.9 and 3.4 μg (p = 0.59). The median gastric juice levels of vitamin C was 9,8 μg in infected and 18.4 μg in non-infected (p = 0.03). The histology showed normal mucosa in eight 8 (20%), chronic non-atrophic gastritis in 23 (55%) and chronic atrophic gastritis in 11 (26%) patients. There was no significant association of histology with Vitamin C serum levels (p = 0.26) or gastric (p = 0.29). Conclusion: Vitamin C serum levels were within the normal range, and most patients had adequate food ingestion of vitamin C. Serum levels of vitamin C were similar in individuals infected and non-infected while vitamin C levels in the gastric juice of patients infected were reduced compared with patients non-infected. Serum levels of vitamin C were higher in the gastric juice than in serum, regardless the patients were infected or not.
Introdução: A infecção por Helicobater pylori influencia na homeostase da vitamina C e níveis baixos da mesma podem determinar risco para a infecção. Objetivo: Investigar os níveis de vitamina C no soro e suco gástrico de pacientes infectados e não infectados por H. pylori e estudar sua associação com a infecção, com o pH gástrico e com as alterações anatomopatológicas da mucosa gástrica de pacientes dispépticos funcionais. Métodos: Foram estudados 42 pacientes dispépticos submetidos a endoscopia digestiva alta e biópsia para diagnóstico da infecção por H. pylori e de alterações anatomopatológicas da mucosa gástrica, classificadas de acordo com o Sistema Sidney. Aspirado suco gástrico com mensuração do pH e coleta de 5 ml de sangue para dosagem de vitamina C no soro. A dosagem da Vitamina C no soro e no suco gástrico foi realizada por cromatografia líquida de alta performance. Resultados: A idade média foi de 52 anos (DP±11,8), 84,4% do sexo feminino, com sobrepeso (IMC mediana=27,7), com adequada ingestão alimentar de vitamina C. Vinte e quatro (53,3%) pacientes apresentavam infecção por H. pylori. A mediana dos níveis de vitamina C no soro dos infectados foi 3,9 μg/ml e nos não infectados 3,4 μg/ml (p=0,59). A mediana dos níveis de vitamina C no suco gástrico foi 9,8 μg/ml nos infectados e 18,4 μg/ml nos não infectados (p=0,03). A histologia demonstrou mucosa normal em 8 (20%), gastrite crônica não atrófica em 23 (55%) e gastrite crônica atrófica em 11 (26%) pacientes, sem associação significativa com os níveis séricos (p=0,26) ou gástricos (p=0,29) da vitamina C. Conclusões: O nível sérico da vitamina C nos pacientes estudados esteve dentro dos valores de referência normais e o consumo de vitamina C foi adequado na maioria. Os níveis séricos da vitamina C foram semelhantes em indivíduos infectados e não infectados enquanto os níveis de vitamina C apresentaram-se reduzidos no suco gástrico dos pacientes infectados por H. pylori em comparação com os não infectados. Os níveis séricos de vitamina C foram mais altos no suco gástrico do que no soro, independentemente da condição de infectado ou não.

Reaction of BiPh3 or Bi(OtBu)3 with benzohydroxamic acid (H2-BHA) results in formation of novel mono- and di-anionic hydroxamato complexes; [Bi2(BHA)3]∞1, [Bi(H-BHA)3] 2, [Bi(BHA)(H-BHA)] 3, all of which display nM activity against Helicobacter pylori. Subsequent dissolution of [Bi2(BHA)3]∞ in DMSO/toluene results in hydrolysis to the first structurally authenticated {Bi34} oxido-cluster [Bi34O22(BHA)22(H-BHA)14(DMSO)6] 4
Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Being among the most prevalent of persistent infectious agents in humans worldwide, Helicobacter pylori induces chronic inflammation (gastritis), which may progress to peptic ulceration and stomach cancer. The ability to adhere to the gastric mucosa is considered to be both a colonization and virulence property of H. pylori. For adherence, H. pylori expresses surface-located attachment proteins (adhesins) that bind to specific receptors in the gastric mucosa. The best characterized H. pylori adhesin-receptor interaction is that between the blood group antigen binding adhesin (BabA) and the fucosylated blood group antigens, which are glycans highly expressed in the gastric mucosa. Our recent results have changed the view of the blood group antigen-specific binding mode of H. pylori. We have tested clinical isolates of H. pylori from human populations worldwide for their ability to bind to ABO blood group antigens. The results revealed that more than 95% of isolates from Sweden, Germany, Spain, Japan and Alaska that bind fucosylated blood group antigens, bind both the Lewis b antigen (Leb) (of blood group O) and the blood group A-related antigen A-Lewis b, i.e. they exhibit a generalist type of binding mode. In contrast, the majority of strains (62%) from South American Amerindians bound best to Leb, i.e. they exhibit a specialist blood group “O antigen” binding mode. This specialization in binding coincides with the unique predominance of blood group O in the South American Amerindian populations. Furthermore, we also showed that H. pylori could switch from specialist to generalist binding modes by chromosomal integration of foreign babA gene fragments. A mutant strain lacking the babA gene turned out to adhere to inflamed gastric epithelium, despite the fact that it did not bind Leb. We identified the receptor to which the mutant binds to as the sialyl-dimeric-Lewis x antigen (sdiLex) and found its expression to be associated with persistent H. pylori infection and chronic inflammation, both in humans and Rhesus monkeys. The cognate sialic acid binding adhesin (SabA) was identified by our ReTagging technique. Deletion of sabA caused loss of H. pylori binding to sialylated glycans, and screening of single colony isolates revealed a high frequency of spontaneous on⇒off phase variation in sLex binding. Using erythrocytes as a model for sialyl dependent cell adhesion, we could show that SabA is the sought-after H. pylori sialyl-dependent hemagglutinin. Swedish clinical H. pylori isolates were analyzed for sialyl-dependent hemagglutination (sia-HA), and the sia-HA titers were found to be highly correlated to the levels of sLex binding. Clinical isolates were shown to exhibit several distinct binding modes for sialylated glycans, which suggest that SabA exhibit polymorphism in binding. We also found that SabA binds to sialylated glycans on neutrophil surfaces by mechanisms involving “selectin mimicry”, and that SabA plays an important role in nonopsonic activation of neutrophils. In the human stomach, H. pylori is exposed to selective pressures such as immune and inflammatory responses, and this is reflected by changes in mucosal glycosylation patterns. The high mutation and recombination rates of H. pylori in combination with bio selection will continuously generate clones that are adapted to changes in individual gastric mucosa. Such adaptive selection contributes to the remarkable diversity in binding modes and to the extraordinary chronicity of H. pylori infections worldwide.

Campylobacter jejuni and Helicobacter pylori are pathogens which cause gastrointestinal diseases and are therefore of significant importance. However, their metabolism and physiology is relatively poorly understood. It had been noted that the genome of these pathogens lack open reading frames for some glycolytic enzymes. Notably, both pathogens lack the gene encoding phosphofructokinase (6-PFK) and thus regulation of the complementary gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) might be different to that in the majority of organisms which retain 6-PFK. In order to further understand the metabolism of C. jejuni and H. pylori, the structure and function of FBPase and the key gluconeogenic/glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated. Specifically, the regulation of FBPase in both pathogens was investigated using kinetic and biophysical techniques. The results suggested that the enzymes are insensitive to AMP inhibition, unlike mammalian and E. coli FBPases. The FBPases were also insensitive to the other compounds of glycolytic and gluconeogenic pathways. The essentiality of fbp in C. jejuni was also tested and confirmed with gene complementation methodology. The essentiality and difference in regulation of these enzymes suggests they have potential as drug targets. The crystal structures of C. jejuni GAPDH (cjGAPDH) with bound NAD+ and NADP+ showed dual coenzyme specificity, revealing similarities with plant GAPDHs and suggesting a gluconeogenic role. The mechanism of inhibition of cjGAPDH was investigated further through covalent modification of the active site cysteine by iodoacetamide, this blocked NAD(P)+ binding. A competitive inhibitor-bound structure of cjGAPDH, in which the coenzyme was replaced by ADP, was also solved. Comparison of the crystal structures of cjGAPDH-ADP and cjGAPDH-NAD(P)+ complexes highlighted specific conformational changes linked to interactions with the ribose 2’-phosphate. The interactions of this 2’-phosphate might also be utilised to inform the design of lead compounds for inhibitory drug development.

H. pylori treatment failure is a growing problem in daily practice. Aim: to evaluate and compare the efficacy of two new regimes as second-line options in a randomized, prospective study. Methods: Patients previously submitted, without success, to clarithromycin-based regimes were included. 13C-urea breath test (UBT) was performed in all patients. The patients were randomized to receive a combination of rabeprazole 20mg, levofloxacin 500mg and furazolidone 200mg (2 tablets) administered in a single dose for 10 days (RLF) or the association of rabeprazole 20mg, bismuth subcitrate 120mg (2 tablets), doxiciclyn 100 mg and furazolidone 200 mg, twice daily, for 10 days (RBDF). Clinical examination and a new UBT was carried out 60 days after therapy. Results: 60 patients were included (mean age, 46 years, 43% males, 62% peptic ulcer, 38% functional dyspepsia). Two patients were excluded: one due to adverse effects on 4th day of treatment and another due the need to use other antimicrobial before control UBT. Compliance was similar in both groups (90% took correctly all medications). Sideeffects, mostly mild, were also comparable between groups, except diarrhea in group RLF (p=0.0025). Per-protocol cure rates were 80% (95% CI, 65-95%) in the RLF group and 82% (95% CI 67-97%) in the RBDF (p=1.0). Intention-to-treat cure rates were, respectively, 77% (95% CI 62-93%) and 83% (95% CI 68-97%) (p=0.750). Conclusions: Both once-daily triple (rabeprazole, levofloxacin, furazolidone) and twice-daily quadruple therapy (rabeprazole, bismuth subcitrate, doxiciclyn and furazolidone) for ten days constitute effective alternatives in patients with clarithromycin-based regime failure.
O retratamento da infecção por Helicobacter pylori é cada vez mais freqüente devido à crescente falha dos esquemas de erradicação de primeira linha. Os regimes tradicionalmente recomendados para o retratamento da infecção são complexos e com efeitos adversos importantes. A diretriz nacional para o tratamento da infecção - Consenso Brasileiro sobre Helicobacter pylori sugeriu a adoção do regime quádruplo composto por sal de bismuto, doxiciclina ou amoxicilina, IBP e furazolidona, com o ajuste posológico adotado em outros países, embora tal regime careça de validação nacional. A associação de levofloxacina e furazolidona apresenta-se como alternativa promissora no retratamento de H. pylori. OBJETIVOS: Comparar a eficácia da associação de levofloxacina (500 mg), furazolidona (400 mg) e rabeprazol (20 mg) - esquema LFR -, em tomada única diária, durante 10 dias, com o esquema quádruplo composto de subcitrato de bismuto (120 mg), doxiciclina (100 mg), furazolidona (200 mg) e rabeprazol (20 mg) - esquema BDFR -, em duas tomadas diárias, por 10 dias, no retratamento de H. pylori. MÉTODOS: 37 pacientes ulcerosos e 23 dispépticos funcionais, previamente tratados, sem sucesso, da infecção por H. pylori, foram alocados aleatoriamente nos dois grupos. A avaliação dos efeitos adversos foi realizada imediatamente após o término da medicação. Teste respiratório de controle com uréia marcada com C13 foi realizado oito semanas após o retratamento. RESULTADOS: dois pacientes foram excluídos; um por interromper o tratamento no quarto dia devido a efeitos adversos e outro pelo uso repetido de antimicrobianos logo após o regime anti-H. pylori. Os efeitos adversos foram leves e moderados na maioria dos casos e sem diferença entre os grupos, exceto por diarréia, maior no grupo LFR (p=0,0025). As taxas de erradicação dos esquemas LFR e BDFR foram, respectivamente, por intenção de tratamento, de 77 e 83% (p=0,750) e por protocolo de 80 e 82% (p=1). CONCLUSÕES: a associação de levofloxacina, furazolidona e rabeprazol em dose única diária por 10 dias é comparável ao esquema quádruplo administrado em duas tomadas diárias por 10 dias no retratamento da infecção por H. pylori.

SILVA, Cicero Igor Simoes Moura. Genótipos cagA , vacA e alelos e sítios de fosfolilação de tirosina da proteína caga do h. pylori em pacientes com e sem história familiar de câncer gástrico. 2009. 73 f. Dissertação (Mestrado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009.
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The H. Pylori strains demonstrate a high level of phenotypic and genotypic diversity, the expression of various genes, which confer greater pathogenicity to the bacteria. Relatives of patients with gastric cancer have a higher risk of developing gastric diseases, especially if they are infected with more virulent strains of H. pylori. The objective was to characterize the strains of H. pylori, the presence of genes vacA, cagA, and sites of tyrosine phosphorylation of CagA protein - EPIYA in patients with and without family history of gastric cancer, and correlate the presence of the cagA gene to the level of activity and inflammation chronic gastritis. The genotyping of strains of H. pylori was performed by PCR and sequencing of the phosphorylation sites EPIYA. We evaluated 94 biopsy samples of gastric mucosa from dyspeptic patients H. pylori positive, 55 with a family history of gastric cancer and 39 without family history. All genotyped strains were vacA positive, and 45.7% with the vacA allele s1m1; s1m2 vacA 26.6%, 4.3% were vacA s2m1; 10.6% vacA s2m2; 3 hybrid strains and 9 (9.6 %) showed only the allele sequence signal (s). Of the 94 strains genotyped 87.2% were cagA positive, with no statistical difference between groups of familiar and unfamiliar (92.7% vs 82.0%, p = 0.058). The cagA gene was present in most patients with gastritis, with statistical significance in the group of relatives of cancer (p = 0.004). The degree of activity and inflammation of gastritis, was more significant in the group of patients with cagA positive strains, with pangastritis Corpal and gastritis. In the antrum, there were significant only in relation to the activity of gastritis. The studed strains, 93.9% showed EPIYA phosphorylation sites, with 59.2% of these had only one site, 38.2% with two sites and 2.6% with three sites. There was no statistical difference in the number of sites EPIYA between the groups. It is important to genetically characterize the strains of H. pylori to establish clearly its role in different clinical conditions related to it. Keywords: H. pylori.
Cepas de H. pylori demonstram um alto nível de diversidade fenotípica e genotípica, pela expressão de vários genes, que conferem maior patogenicidade à bactéria. Familiares de pacientes com câncer gástrico têm maior risco de desenvolver doenças gástricas, principalmente se forem infectados por cepas mais virulentas de H. pylori. O objetivo foi caracterizar as cepas de H. pylori, quanto à presença dos genes vacA, cagA e dos sítios de fosforilação de tirosina da proteína CagA – EPIYA, em pacientes com e sem história familiar de câncer gástrico, e correlacionar a presença do gene cagA com o grau de atividade e de inflamação da gastrite crônica. A genotipagem das cepas de H. pylori foi realizada através da técnica de PCR e do sequenciamento dos sítios de fosforilação EPIYA. Foram avaliados 94 fragmentos de biópsia de mucosa gástrica provenientes de pacientes dispépticos H. pylori positivo, 55 com história familiar de câncer gástrico e 39 sem historia familiar. Todas as cepas genotipadas foram vacA positivas, sendo 45,7% com o alelo vacA s1m1; 26,6% vacA s1m2; 4,3% eram vacA s2m1; 10,6% vacA s2m2; 3 cepas híbridas e 9 (9,6%) apresentaram somente o alelo da sequência sinal(s). Das 94 cepas genotipadas 87,2% foram cagA positivas, não havendo diferença estatística entre os grupos de familiares e não familiares (92,7% vs 82,0%; p= 0.058). O gene cagA estava presente na maioria dos pacientes portadores de gastrite, havendo significância estatística no grupo dos familiares de câncer (p= 0,004). O grau de atividade e inflamação da gastrite, foi mais significante no grupo de pacientes com cepas cagA positivo, com pangastrite e gastrite corpal. No antro, somente houve significância com relação à atividade da gastrite. Das cepas estudadas, 93,9% apresentaram sítios de fosforilação EPIYA, sendo que 59,2% destas apresentaram somente um sitio, 38,2% com dois sítios e 2,6% com três sítios. Não houve diferença estatística com relação ao número de sítios EPIYA, entre os grupos estudados. É importante a caracterização genética das cepas de H. pylori para se estabelecer com clareza o seu papel nos diversos quadros clínicos a ele relacionado.

Introdução e objetivos: Eosinofilia duodenal está associada com parasitoses intestinais e com alergias alimentares, e tem sido sugerida como possível fator etiológico da dispepsia funcional, pela capacidade de causar alterações na motilidade e na sensibilidade do aparelho digestivo. Sua relação com o Helicobacter pylori é pouco conhecida, tendo sido avaliada apenas como achado secundário em alguns estudos, com resultados controversos. Esse estudo tem como objetivos avaliar o papel da infecção gástrica pelo H. pylori no número de eosinófilos duodenais e avaliar a relação dos eosinófilos duodenais com os sintomas da dispepsia funcional. Métodos: foram avaliados 100 pacientes dispépticos funcionais, de acordo com os critérios de Roma III, dos quais 50 foram H. pylori positivos e 50 negativos. Os pacientes foram submetidos à endoscopia digestiva alta com biópsias gástricas e duodenais. A positividade do H. pylori foi avaliada pelo teste de urease e pelo exame histológico (Hematoxilina-eosina e Giemsa). As biópsias duodenais foram avaliadas com hematoxilina-eosina e a número de eosinófilos duodenais foi quantificada pela média de eosinófilos por 5 campos de grande aumento (CGA) aleatórios e não sobrepostos. Eosinofilia duodenal foi definida pela presença de >22 eosinófilos/CGA. As medianas das médias aritméticas dos eosinófilos duodenais por cinco CGA foram comparadas entre os pacientes H. pylori positivos e negativos. Também foi avaliada a relação do número de eosinófilos duodenais com a intensidade e tipo de sintomas dispépticos, determinados por questionário validado (PADYQ). Os eosinófilos duodenais foram avaliados para variáveis demográficas e endoscópicas. Resultados: Pacientes do sexo feminino representaram 88% da amostra e a idade média foi de 41,7 anos As características basais dos pacientes H. pylori positivos e H. pylori negativos foram semelhantes. Apenas um paciente, no grupo H. pylori positivo, apresentou eosinofilia duodenal. As medianas dos eosinófilos duodenais/CGA foram 4,6 [P25-75: 2,8-7,2] nos pacientes H. pylori negativos e 4,7 [P25-75: 3,4-8,4] nos H. pylori positivos (p= 0,403). O número de eosinófilos 8 duodenais foi significativamente maior em pacientes com sintomas mais intensos: pacientes com escore do PADYQ >22 (>50% da pontuação máxima) apresentaram mediana de eosinófilos duodenais/CGA de 5,4 [P25-75: 3,4–7,6] e pacientes com escore ≤22 de 3,4 [P25-75: 2,2–6,0] (p= 0,018). Os pacientes foram divididos em tercis, de acordo com a intensidade dos sintomas: grupo 1 com 31 pacientes (sintomas leves); grupo 2 com 30 pacientes (sintomas moderados); e grupo 3 com 31 pacientes (sintomas acentuados). A mediana dos eosinófilos duodenais/CGA no grupo 1 foi de 3,4 [P25-75: 2,2 -6,0]; no grupo 2 de 4,7 [P25-75: 3,2-6,4]; e o grupo 3 de 5,8 [P25-75: 3,6-8,2] (P=0,033). Houve diferença estatisticamente significativa no número de eosinófilos duodenais entre fumantes e não fumantes (p= 0,030) e entre pacientes com índice de massa corporal (IMC) <25 kg/m2 e IMC ≥ 25 kg/m2 (p= 0,035). Na análise multivariada por regressão linear, os fatores que tiveram influência sobre o número de eosinófilos duodenais foram o tabagismo (p= 0,026) e a intensidade dos sintomas dispépticos (p= 0,039). Conclusões: Esse estudo não mostrou associação entre a infecção pelo H. pylori e a contagem de eosinófilos duodenais, nessa população de pacientes dispépticos funcionais. Entretanto, foi demonstrada uma relação diretamente proporcional e estatisticamente significativa entre o número de eosinófilos duodenais e a intensidade dos sintomas dispépticos.
Background and Aims: Duodenal eosinophilia is associated with intestinal parasitosis and food allergies. It has also been implicated as a potential factor on the etiology of functional dyspepsia, probably by causing changes in digestive tract motility and sensitivity. The association with Helicobacter pylori is poorly understood, and has been only evaluated as a secondary finding in 9 previous studies, with conflicting results. This study aims to evaluate the potential role of gastric H. pylori infection in the duodenal eosinophil count, and the influence of duodenal eosinophils on symptoms in functional dyspeptic subjects. Methods: One hundred functional dyspeptic subjects, according to Rome III criteria, were evaluated, and 50 were H. pylori positive and 50 H. pylori negative. Patients were submitted to upper gastrointestinal endoscopy with gastric and duodenal biopsies. H. pylori positivity was evaluated by urease test and gastric histology (Hematoxylin-eosin and Giemsa). Duodenal biopsies were evaluated with Hematoxylin-Eosin staining, and the duodenal eosinophil count was determined by the mean of eosinophil by 5 random nonoverlapping high power fields (HPF). Duodenal eosinophilia was defined as >22 eosinophils/HPF. The median of the arithmetic means of the duodenal eosinophils counts per high power field were compared between H. pylori positive and H. pylori negative subjects. The relationship between the number of duodenal eosinophils and the intensity and type of dyspeptic symptoms was determined by validated questionnaire (PADYQ). Duodenal eosinophils counts were also evaluated by demographic variables and endoscopic findings. Results: 88% of the subjects were female and the mean age was 41.7 years. Baseline characteristics were similar between H. pylori positive and H. pylori negative subjects. Only one patient, in the H. pylori positive group, had duodenal eosinophilia. The median duodenal eosinophils/HPF were 4.6 [Percentiles 25-75(P25-75): 2.8-7.2] in H. pylori negative and 4.7 [P25-75: 3.4-8.4] in H. pylori positive subjects (p= 0.403). The duodenal eosinophil count was greater in subjects with higher symptoms severity: patients with PADYQ score more than 22 (>50% of the maximum score) had median duodenal eosinophil/HPF of 5.4 [P25-75: 3,4–7,6] and subjects with PADYQ score ≤22 of 3.4 [P25-75: 2.2–6.0] (p= 0.018). The patients were divided into terciles, according to symptoms severity: group 1 with 31 subjects (mild symptoms); group 2 with 30 subjects (moderate symptoms); and group 3 with 31 subjects (severe symptoms). 10 The median duodenal eosinophils/HPF was 3.4 [P25-75: 2.2-6.0] in group 1; 4.7 [P25-75: 3.2-6.4] in group 2; and 5.8 [P25-75: 3.6-8.2] in group 3 (p=0.033). There was a higher duodenal eosinophils count in smokers (current or former) (p=0.030), and subjects with BMI ≥ 25 kg/m2 (p=0.035). In the multivariate analysis by linear regression, the duodenal eosinophil count were influenced by smoking (p = 0.026) and dyspeptic symptoms severity (p= 0.039). Conclusion: This study did not show an association between H. pylori infection and the number of duodenal eosinophils, in this population of functional dyspeptic patients. However, a directly proportional and statistically significant relationship between the number of duodenal eosinophils and the intensity of dyspeptic symptoms has been demonstrated.

Diversos estudios señalan que varias especies pertenecientes a las Bacterias Acido Lácticas (BAL) poseen propiedades probióticas y se encuentran comúnmente en los derivados lácteos. Actualmente, en la Comunidad Valenciana se comercializan quesos frescos y madurados elaborados a partir de la leche de oveja guirra, autóctona de esta comunidad e incluida dentro del catálogo de razas protegidas en España. Sin embargo, hasta el momento no se han realizado ensayos para determinar el potencial probiótico de cepas aisladas de esta leche. En esta tesis, se han aislado 131 BAL de leche de oveja Guirra. Siguiendo los criterios establecidos por la FAO, estas cepas se han identificado inicialmente con métodos fenotípicos a nivel de género y especie. Posteriormente, se ha evaluado, en condiciones in vitro, la actividad antimicrobiana de las cepas lácticas frente a patógenos implicados en enfermedades del hombre como H. pylori y Salmonella spp. En detalle, se ha estudiado el efecto de las sustancias producidas por dos cepas BAL frente a la viabilidad de H. pylori. Las cepas BAL destacadas en la actividad antimicrobiana han sido identificadas y caracterizadas por medio de técnicas moleculares, perfiles de resistencia a antibióticos, habilidad de adhesión a la mucina de cerdo y finalmente, se ha evaluado la resistencia frente a las condiciones gastrointestinales. Los resultados muestran que las propiedades probióticas se pueden atribuir a una cepa y no es posible generalizarlo a una especie o género. Algunas de las cepas lácticas de oveja y de la CECT resultaron de especial interés porque en condiciones in vitro demostraron características probióticas que deberían ser confirmadas en posteriores estudios in vivo. Cabe resaltar que algunas de estas cepas se han aislado de productos que son actualmente comercializados y por tanto las propiedades probióticas de estas cepas representarían un valor añadido para los mismos.
Amorocho Cruz, CM. (2011). Caracterización y potencial probiótico de bacterias lácticas aisladas de leche de oveja guirra [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/13830
Palancia

To elucidate the molecular mechanisms involved in persistency/latency of the H. pylori infection or in its progression towards serious diseases, it is necessary to analyse the host pathogen interaction in vivo. The circulating antibody repertoire represents an important source of diagnostic information, serving as biomarker to provide a “disease signature”. The aim of this work is the identification of H. pylori epitopes responsible for host immuno­response modulation through: a discovery­driven approach that couples “phage display” and deep sequencing (interactome-sequencing) and the development of a specific webtool for interactome-sequencing data analysis. We used this approach to identify novel antigens by screening gDNA libraries created from the pathogen’s genome, directly with sera from infected patients. Two genomic phage display libraries from 26695 and B128 H. pylori strains have been constructed by using ß­lactamase ORF selection vector. Genomic DNA was sonicated, fragments cloned into the filtering vector, after transformation libraries of 1x106 clones were obtained and sequenced by Illumina technology. More than 93% of Hp CDSs were represented in the phage genomic libraries therefore being representative of the whole H. pylori antigenic ORFeome. A webtool for interactome-sequencing data analysis was developed and used to identify the H. pylori antigens/epitopes which could be considered specific for infection progression towards three different pathological outcomes. Putative antigens were selected from libraries using sera from patients affected by: i) gastric adenocarcinoma; ii) autoimmune gastritis; iii) MALT lymphoma. The results, obtained thanks to the new interactome sequencing pipeline developed, show that the diversity of the libraries after selection is significantly reduced. Furthermore, individual ranks, for each infection condition, have been compared highlighting the pattern of putative antigens, shared by all the conditions, and some that can distinguish the different stages of infection. One of this new antigens, that seems to be specific for infection progression towards more serious diseases, has been successfully validated through ELISA assay on a wide number of sera from patients. Other more specific antigens identified by our approach and by the application of the new data analysis pipeline here described are in validation.

L'infection par Helicobacter pylori provoque une inflammation qui peut persister de façon asymptomatique ou évoluer vers de nombreuses pathologies gastroduodénales sévères telles que les ulcères gastroduodénaux, le lymphome du MALT et le cancer gastrique. L'îlot de pathogénicité cag est l'un des facteurs de virulence majeurs de cette bactérie. Plusieurs cytokines et peptides antimicrobiens sont impliqués dans la modulation de la réponse inflammatoire de la muqueuse épithéliale gastrique lors de l'infection par H. pylori. Ce travail a porté sur l'étude des interactions entre H. pylori et les cellules épithéliales gastriques. L'étude in vivo sur un modèle murin d'infection a permis de décrire un profil de la réponse antimicrobienne liée à cette bactérie. Une production accrue de S100A9 pourrait être impliquée dans l'échec d'implantation et de colonisation de la bactérie dans la muqueuse gastrique murine. Nous avons également établi un protocole de culture primaire de cellules épithéliales gastriques humaines à partir d'estomacs obtenus après gastrectomie partielle chez des sujets atteints d'obésité morbide. Cette étude a permis de modéliser in vitro la réponse inflammatoire et antimicrobienne de la muqueuse gastrique humaine après infection par H. pylori. L'induction cag-dépendante d'un panel de médiateurs inflammatoires et antimicrobiens par les cellules épithéliales gastriques exposées à H. pylori confirme l'implication de ce facteur au cours de la réponse inflammatoire précoce. Des cellules gastriques à phénotype de cellules souches ont également été isolées et des caractérisations phénotypiques et moléculaires ont été initiées pour déterminer leur nature. Les modèles cellulaires développés au cours de cette étude permettent une meilleure compréhension des interactions entre H. pylori et les cellules épithéliales gastriques.

Helicobacter pylori (H.pylori) had been confirmed by the World Health Organization (WHO) as Group 1 carcinogens, in which it has been identified to be related with the development of gastric carcinoma. Gastroesophageal reflux disease (GERD) is less commonly found in Asia, while the number of H.pylori infection is considerably to be higher than that of the Western population. The relationship between H.pylori and GERD still remains ambiguous nowadays. One of the contributing factors affecting the level of gastric secretion might be due to the genetic cause. The aim of this review is to assess whether the current first-line therapy on GERD would be effective or not in relieving the symptoms of the patients with H.pylori infection.
published_or_final_version
Community Medicine
Master
Master of Public Health

The H. Pylori strains demonstrate a high level of phenotypic and genotypic diversity, the expression of various genes, which confer greater pathogenicity to the bacteria. Most studies have found that the cagE gene is strongly associated with peptic ulcer disease and sites of tyrosine phosphorylation of CagA protei - EPIYA, has not been fully elucidated existing geographical differences. This study aimed to characterize the strains of H. pylori for the sites of tyrosine phosphorylation of CagA protein EPIYA and presence of genes cagE in patients with gastritis and peptic ulcer disease, and to correlate the presence of these genotypes with gastric disorder. Genotyping of H. pylori strains was performed by PCR and sequencing of phosphorylation sites EPIYA. 137 dyspeptic patients from H. pylori-positive, 72 with peptic ulcer and 65 with gastritis were evaluated. Of the 137 strains genotyped 68.6 % were cagA positive, strains cagA studied , 96.8 % had at least 1 C phosphorylation site EPIYA , while 53.2 % of these had only one site , 43.6 % had two or more sites C and 3.2 % had no place C. the mixed strains with two EPIYA patterns were present in 23.4 % of the studied samples . The group of patients with gastritis showed higher prevalence of genotype EPIYA ABCCC , the same was not observed in patients with peptic ulcer disease and this association with gastrics disorders studied significant ( p = 0.001 ). Of the 137 patients studied, 65 were positive for CAGE (47.4%). There was a significant inverse association between genotype and cage group of patients with gastritis (OR = 0.345, p = 0.002). There was a significant association between genotype cagE with peptic ulcer disease (OR = 2.898, p = 0.002) and the subgroup of patients with duodenal ulcer (OR = 3.839, p = 0.001). This study suggests that the population sample studied the presence of genotype Epiya three sites C was associated with gastritis and absent in patients with peptic ulcer. We found association of the cagE gene duodenal ulcer.
As cepas de H. pylori demonstram um alto nÃvel de diversidade fenotÃpica e genotÃpica, pela expressÃo de vÃrios genes, que conferem maior patogenicidade à bactÃria. A maioria dos estudos evidenciou forte associaÃÃo do gene cagE com doenÃa ulcerosa pÃptica e do aumento dos sÃtios de fosforilaÃÃo EPIYA C com doenÃas gÃstricas mais graves. No entanto, a associaÃÃo clÃnica e a funÃÃo dos sÃtios de fosforilaÃÃo de tirosina da proteÃna CagA â EPIYA e do gene cagE, ainda nÃo foi completamente elucidada, existindo diferenÃas geogrÃficas. O objetivo do presente estudo foi caracterizar as cepas de H. pylori quanto à presenÃa dos sÃtios de fosforilaÃÃo de tirosina da proteÃna CagA â EPIYA e do gene cagE em pacientes com gastrite e Ãlcera pÃptica, e correlacionar a presenÃa desses genÃtipos com as afecÃÃes gÃstricas. A genotipagem das cepas de H. pylori foi realizada atravÃs da tÃcnica de PCR e do sequenciamento dos sÃtios de fosforilaÃÃo EPIYA. Foram avaliados 137 pacientes dispÃpticos H. pylori positivo, 72 com Ãlcera pÃptica e 65 com gastrite. Das 137 cepas genotipadas 68,6% foram cagA positivas. Das cepas cagA estudadas, 96,8% apresentaram pelo menos 1 sÃtio C de fosforilaÃÃo EPIYA, sendo que 53,2% destas apresentaram somente um sitio, 43,6% tinham dois ou mais sÃtios C e 3,2% nÃo apresentaram sitio C. As cepas mistas com dois padrÃes EPIYA estavam presentes em 23,4% das amostras estudadas. O grupo de pacientes com gastrite apresentou maior prevalÃncia do genÃtipo EPIYA ABCCC; o mesmo nÃo foi observado no grupo de pacientes com Ãlcera pÃptica, sendo essa associaÃÃo com as afecÃÃes gÃstricas estudadas significante (p= 0,001). Dos 137 pacientes estudados, 65 foram positivos para cagE (47,4%). Houve associaÃÃo inversa significativa entre o genÃtipo cagE e o grupo de pacientes com gastrite (O.R=0,345; p=0,002). Houve associaÃÃo significativa entre o genÃtipo cagE com doenÃa ulcerosa pÃptica (O.R=2,898; p=0,002) e com o subgrupo de pacientes com Ãlcera duodenal (O.R=3,839; p=0,001). Esse estudo sugere que na amostra populacional estudada a presenÃa do genÃtipo EPIYA com trÃs sÃtios C estava associada à gastrite e ausente nos pacientes com ulcera pÃptica. Encontrou-se associaÃÃo do gene cagE a Ãlcera duodenal.

Le rôle majeur de l’Helicobacter pylori dans l’étiopathogénie des maladies gastroduodénales (gastrite, ulcère gastrique et duodénal, lymphome gastrique) est bien établi aujourd’hui. L’OMS l’a reconnue comme jouant un rôle important dans la survenue des lésions cancéreuses gastriques. La prévalence de l’infection à H. pylori varie selon les pays de 20% à 90% avec des taux supérieurs à 60% dans les pays en développement, dont le Bénin. Les méthodes usuelles de diagnostic sont soit invasives nécessitant une endoscopie gastrique avec biopsies (test rapide à l’urée, histologie, culture et PCR), soit non-invasives (test respiratoire à l’urée marquée au carbone, sérologie, et détection de l’antigène dans les selles). La trithérapie associant un inhibiteur de la pompe à protons (IPP) et deux antibiotiques choisis parmi l’amoxicilline, la clarithromycine et le métronidazole est recommandée pour son traitement. La survenue de résistance des souches H. pylori aux différents antibiotiques devient une cause majeure de l’échec des régimes d’éradication.

Afin d’évaluer l’applicabilité et l’efficacité des régimes thérapeutiques recommandés en pratique courante, et à partir d’une étude de cohorte prospective, nous avons étudié la prévalence de l’infection à H. pylori chez les patients consultant à la clinique de Gastroentérologie de l’hôpital universitaire Erasme à Bruxelles, déterminé son taux de résistance primaire aux antibiotiques, et évalué le taux d’éradication d’H. pylori par la trithérapie. Nous avons aussi évalué la performance du test de détection de l’antigène d’H. pylori dans les selles pour le diagnostic chez l’adulte (avant traitement) comparé avec les méthodes de référence (culture, histologie), également dans le contrôle de l’éradication.

Au Bénin, nous avons évalué à partir d’une étude transversale prospective, la prévalence de l’infection à H. pylori dans une population en milieu urbain et rural. Nous avons déterminé la distribution par famille des sujets infectés, ainsi que l’influence des variables démographiques individuelles, et les caractéristiques socio-économiques familiales sur le risque de l’infection.

La prévalence de résistance primaire à la clarithromycine et au métronidazole fut observée respectivement dans 3% et 31% des souches isolées. Aucune résistance primaire à l’amoxicilline et à la tétracycline n’a été observée.

Les analyses en intention de traiter, ont montré que H. pylori a été éradiqué chez 80% des patients inclus dans l’étude thérapeutique. Le taux d’échec d’éradication fut de 20%. Comparé au 14C-TRU, le test HpSA avait une sensibilité de 100%, une spécificité de 91%, VPP de 69%, VPN de 100%. De même, la sensibilité du test HpSA par rapport aux deux méthodes usuelles (culture et histologie) est de 96.5% pour une spécificité de 91.2%, une VPP de 90.3% et une VPN de 96.8%.

Au Bénin, la prévalence de H. pylori était de 75.4% en ville et de 72.3% dans le village (p = 0.459). Aucune association n’a été observée avec l’âge, le sexe, le niveau d’instruction, la taille du ménage, l’activité économique ou le mode d’approvisionnement en eau potable. Le taux d’infection était plus élevé chez les enfants dont les parents étaient infectés et chez ceux ayant une mère H. pylori positive (p < 0.001). L’analyse multivariée par régression logistique a montré que la densité d’occupation des dortoirs [OR (95%) = 9.82 (4.13-23.31)] p < 0.001), et le statut des mères dans le ménage ([OR (95%) = 3.85 (1.53-9.67)] p < 0.001) étaient les prédicteurs indépendants de l’infection par H. pylori. Le risque de l’infection chez les enfants était 13 fois plus élevé quand les deux parents sont simultanément positifs OR (95% CI) = 13.6 (3.63-51.22), il l’était respectivement de 5.3 (1.52-18.45); 2.7 (0.47-15.44), quand la mère et le père sont positifs p < 0.001. Aussi le risque d’infection à H. pylori comparé aux enfants qui dorment seul dans leur chambre, était élevé pour ceux qui dorment avec un ou deux personnes OR (95% CI) = 5.2 (1.08-25.16), p < 0.05, et plus élevé chez les enfants qui dorment à 4 ou plus OR (95% CI) = 16.6 (2.66-103.44), p < 0.005, comparé à ceux qui dorme seuls. Donc, le contact avec des personnes infectées au sein de la famille et la vie en promiscuité, étaient associés avec un risque d’infection plus élevé indiquant une transmission intrafamiliale de l’infection par H. pylori.

En conclusion, nos résultats montrent une séroprévalence encore élevée de l’infection à H. pylori dans la population béninoise. Une surveillance de l’épidémiologie accompagnée de mesures de prévention ciblées sur les facteurs potentiels de risque de l’infection doit être poursuivie. La validation du test de détection de l’antigène dans les selles avant traitement et dans le contrôle de l’éradication de la bactérie pour le suivi thérapeutique des patients infectés, est une alternative intéressante notamment au Bénin. Le taux de résistance primaire pour le métronidazole est actuellement stable en Belgique, alors que la prévalence de la résistance à la clarithromycine mérite d’être précisée par d’autres études multicentriques. La trithérapie classique à base d’inhibiteur de la pompe à protons–amoxicilline-clarithromycine reste recommandable en première intention. La surveillance épidémiologique de l’infection basée sur la prévalence locale des souches clarithro-résistantes et métronidazole-résistantes devrait être poursuivie.

SUMMARY OF THE THESIS

The major role of H. pylori in the etiopathogeny of various gastroduodenal diseases (gastritis, gastric and duodenal ulcers, gastric lymphoma) is well established today. The World Health Organization concluded that H. pylori plays a causal role in the chain of events leading to cancer of the stomach.

The prevalence of H. pylori infection varies by country from 20% to 90%, with higher prevalence rates over 60% observed in developing countries, including Bénin. The usual methods allowing the diagnosis of the gastric infection by H. pylori are either invasive, requiring a gastric endoscopy and biopsies (fast urease test, anatomopathological examination, culture and PCR), or noninvasive (breath test with 13C or 14C marked urea, serology and stool antigen detection). Triple therapy associating a proton pump inhibitor (PPI) with two antibiotics, chosen between amoxicillin, clarithromycin and metronidazole, is currently recommended. Resistance of H. pylori strains to antibiotics becomes a major determinant in the failure of eradication of regimens.

To evaluate the applicability and efficacy of the therapeutic recommendations in our pratice, based on a prospective study, we studied the prevalence of H. pylori infection in the outpatient population of the Gastroenterology clinic at the Erasme University hospital in Brussels, determined its rate of primary resistance to antimicrobial agents and evaluated the rate of eradication of H. pylori by triple therapy. We also evaluated the performance of a stool antigen detection test for the diagnosis of H. pylori infection in adults (before treatment) compared with reference methods (culture and histology) as well as in control of eradication.

In Benin, we evaluated by a cross-sectional study the prevalence of the infection with H. pylori in the population living in urban and rural environment. We determined the family distribution of infected subjects as well as the influence of individual demographic variables and of the socio-economic family characteristics on the risk of infection.

In Brussels, primary resistance to clarithromycin and metronidazole was observed in 3% and 31% of the isolates, respectively. No primary resistance to amoxicillin and tetracycline was observed. By intention to treat analysis, H. pylori was eradicated in 80% of patients included in the therapeutic study. The rate of eradication failure was 20%. In comparison with 14C-Urea breath test, the H. pylori Stool Antigen test showed a sensitivity of 100%, a specificity of 91 %, PPV of 69%, and NPV of 100%. Compared to the reference methods (culture and histology), the HpSA test had a sensitivity of 96.5% and a specificity of 91.2%. PPV of 90.3% and NPV of 96.8%.

In Benin, the prevalence of H. pylori antibodies was 75.4% in town and 72.3% in the village (P= 0.459). No association was found between infection and age, sex, education level, size of the household, economic activity or source of drinking water. The infection rate was higher in children of parents who were both infected and also in those whose mother was infected (p < 0.001). By logistic regression analysis, the density of occupation of dormitories (more than three persons sharing dormitory, [OR (95%) = 9.82 (4.13-23.31)] p < 0.001), and mother status within the household ( [OR (95%) = 3.85 (1.53-9.67) ] p < 0.001), were independent predictors for H. pylori infection. The risk of H. pylori infection in children was 13 times higher when the two parents were simultaneously positive: OR (95% CI) = 13.6 (3.63-51.22) and it was respectively of 5.3 (1.52-18.45); 2.7 (0.47-15.44), when mother and father were positive p < 0.001. H. pylori infection risk in children was higher for a sharing a dormitory with one or two persons, OR (95% CI) = 5.2 (1.08-25.16), p < 0.05 and was even higher if a dormitory of 4 persons or more, OR (95% CI) = 16.6 (2.66-103.44), p < 0.005 as compared to sleeping alone. Family contact with infected persons and crowded living conditions were associated with increased risk of infection consistent with intrafamilial H. pylori transmission.

In conclusion, our results confirm a still high H. pylori seroprevalence in population in Benin. An epidemiolgic survey with prevention mesures targeted on potential risk predictors should be going on. Validation of antigen detection test in patients stools before treatment and for eradication control could be an interested alternative, notably in Benin. Primary resistance rate on metronidazole is stable today in Belgium, though the resistance prevalence on clarithromycin should be determined by other multicentric studies. Standard triple therapy by (PPI)-amoxicillin-clarithromycin is still recommended in first intention to treat. Epidemiological survey of infection based on local prevalence of claritromycin-resistant and metronidazole-resistant strains should be continued.

Doctorat en Santé Publique
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Cancer is a disease with a high mortality rate in Brazil, including, stomach cancer is currently the fourth most common type of cancer worldwide, responsible for countless deaths. Its etiology is multifactorial, because studies suggest associations to various factors such as dietary habits, environmental factors, genetic and epigenetic factors and gastric infection by Helicobacter pylori. Epigenetic changes such as methylation of the promoter regions of genes involved in cellular homeostasis may contribute to gastric carcinogenesis. To verify the methylation status of THBS1, GPX3 and COX2 genes and to evaluate their association with H. pylori in gastric adenocarcinomas, Methylation-Sensitive Restriction Enzyme PCR (MSRE-PCR) assay was performed in 39 gastric carcinomas (intestinal and diffuse types) and 15 normal stomach tissue samples. The presence of H. pylori was performed by amplification of the fragment of the 16S rRNA. Statistical analysies were performed using Fisher s exact test. The hypermethylation of GPX3, THBS1 and COX2 occurred in 18% (n = 7), 5% (n = 2) 36% (n = 14) of gastric cancer samples, respectively, whereas in normal samples was found in 13%, 7% and 67%. The presence of H. pylori was detected in 67% of gastric cancer samples and 67% in normal gastric samples. No correlation was found between the methylation profile of the studied samples and clinicopathological variables and the presence of H. pylori (P ≥ 0,05). The presence of H. pylori in gastric cancer samples and normal was not associated with clinicopathologic variables analyzed (P> 0.05).
O câncer é uma doença com alta taxa de mortalidade no Brasil, dentre eles, o câncer de estômago constitui atualmente, o quarto tipo de câncer mais comum a nível mundial. No ano de 2012, estimam-se, para o Brasil, 12.670 casos novos de câncer do estômago em homens e 7.420 em mulheres. A sua etiologia é multifatorial, pois estudos sugerem associações a diversos fatores como: hábitos alimentares, fatores ambientais, fatores genéticos e epigenéticos e a infecção gástrica por Helicobacter pylori. Alterações epigenéticas tais como a metilação das regiões promotoras de genes envolvidos na homeostase celular podem contribuir para carcinogênese gástrica. Para verificar o estado de metilação de genes THBS1, GPX3 e COX2 e avaliar a sua associação com a Helicobacter pylori (H. pylori) em adenocarcinomas gástricos, Methylation-Sensitive Restriction Enzyme PCR (MSRE-PCR) foi realizada em 39 carcinomas gástricos (intestinal e tipo difusa) e 15 amostras de tecido normal do estômago. A presença de H. pylori foi realizada por amplificação de um fragmento de rRNA 16S. Analysies estatísticas foram realizadas utilizando o teste exato de Fisher. A hipermetilação de GPX3, THBS1 e COX2 ocorreu em 18% (n = 7), 5% (n = 2) 36% (n = 14) das amostras de câncer gástrico, respectivamente, ao passo que em amostras normais foi encontrada em 13%, 7 % e 67%. A presença de H. pylorifoi detectada em 67% das amostras de câncer gástrico e 67% em amostras gástricas normais. Não foi encontrada correlação entre o perfil de metilação das amostras estudadas com variáveis clínico-patológicas e com presença de H. pylori (P > 0,05). A presença de H. pylori nas amostras de câncer gástrico e normais não foi associada com as variáveis clínico-patológicas analisadas (P > 0.05).